Somaclonal variation in insect‐resistant transgenic sugarcane (Saccharum hybrid) plants produced by cell electroporation

A population of 42 transgenic sugarcane ( hybrid, cv. Ja605) clones expressing a truncated cryIA(b) gene from Bacillus thuringiensis was evaluated in field trials under artificial borer (Diatraea saccharalis Fab.) infection. Five clones displaying the highest borer tolerance were selected and analysed with molecular tools (RAPD, AFLP and RAMP) to verify genomic changes. Results of field trials provided evidence both for the expression of the resistance trait and for the occurrence of limited but consistent morphological, physiological and phytopathological variation, as compared with control plants regenerated from dedifferentiated culture without transformation (C1control) or with plants that were clonally propagated in the field (C2control). The five elite transgenic clones, selected for consistent borerresistance and good agronomic traits, were further evaluated in a large scale field trial. It was found that the majority of agronomic and industrial traits were those of the original cv. Ja605, but that a small number of qualitative traits was different. DNA changes were verified in the five selected clones. A total of 51 polymorphic DNA bands (out of the 1237 analysed bands) was identified by extensive AFLP and RAMP analysis, thus showing rare but consistent genomic changes in the transgenic plants, as compared with C1 and C2control plants. It is proposed that the increased variability verified in transgenic plants by field trials and DNA analysis is essentially correlated with cell growth in the dedifferentiated state during the transformation procedure. The results, which are consistent with those published in the case of other transgenic plant populations, are discussed in the context of selecting approaches to gene transfer that minimize somaclonal variation. This is important especially in cases, such as that of sugarcane, where success of backcrosses to restore the original genotype is made difficult by the complex ploidy state of the plant.     

Proposal of a ‘hot lock’ as a barrier to fish invasion: an afterthought

Environmental Biology of Fishes -     

‘Minority’ as a sociological concept

This article considers how a Muslim cultural discourse of ‘propriety’ has influenced Muslim Arab Sudanese ethnic identity in two locations and time periods in an expanding diaspora. Focusing in particular on women and their embodied practices of whitening and propriety in Egypt in the nineties and the United Kingdom a decade later, I argue that the recent turn towards Muslim expressions of Sudaneseness is a form of resistance to racial labelling. While Sudanese have rejected being labelled ‘black’ in Egypt and in the UK, their renegotiation of a Muslim religious identity in the diaspora nevertheless confirms a racialized Sudanese ethnicity. This study contributes to the rethinking of ethnicity in a transnational space where ethnic nationalism and globalized Islamic discourse intersect with local histories and hierarchies of race and gender.     

Root development of ‘Red Lady’ papaya plants grown on a hillside

Red Lady papaya (Carica papaya L.) seedlings were grown for 7 weeks in one experiment and 17 weeks in a second experiment on a 60% to 70% sloped hillside. Depth and lateral root developmental characteristics were determined to establish a more informed basis for developing management procedures during hillside production of papaya. A trench was dug perpendicular to the contour line, and positioned 10 cm from the stem base of each plant. A 1 cm layer of soil was removed from the profile wall, and the roots on the trench profile were mapped. Thereafter, the intact half of each root system was excavated to determine the dry mass and length distribution. Roots were separated into the taproot system, and the lateral roots of the uphill and downhill sides. The downhill portion of lateral roots accounted for 56% of total length and 64% of total mass after 7 weeks, and 71% of total length and 69% of total mass after 17 weeks. Some lateral roots on the uphill side of the plants developed with a negative gravitropic orientation to maintain a portion of the root system, close to the surface of the slope. After 17 weeks, 34% of the roots on the uphill side of the trench profile walls were located above a horizontal plane positioned at the stem base. Root growth of Red Lady papaya plants growing on hillsides was maintained in all directions, including up the slope. The results signify that there may be no need to modify fertilizer placement practices for hillside production of papaya. However, the higher concentration of roots on the downhill side of these plants indicates a need for placement of soil moisture sensors for irrigation scheduling in uphill and downhill sites.     

Overlapping synthetic peptides as a tool to map protein-protein interactions ̶ FSH as a model system of nonadditive interactions

In earlier work, we used partially overlapped synthetic peptides as a tool to find regions of interaction between the human FSH hormone and its receptor, aiming to find possible antagonists or agonists. Years later, the FSH and FSH receptor 3D structures were reported by other laboratories. The 3D results were in close agreement with the interacting regions predicted by using synthetic peptides. These earlier studies are reviewed here, and the predicted regions of interaction compared to the FSH and FSH receptor 3D structures to illustrate the usefulness of the synthetic peptide strategy to find binding regions. Different contact regions contribute multiplicatively to the high affinity of the entire ligand; thus, peptides covering a fraction of the anchor sites and with low free energy density cannot reach the affinity of the entire molecule. The earlier use of multiple linear regression to find the relevant predictors for effective binding, and a new way to estimate ΔG° and nonadditive interactions for the synthetic peptides in solution, by using the buried surface area (BSA), will be discussed.     

Identifying ‘climate keystone species’ as a tool for conserving ecological communities under climate change

Aim

Climate change affects ecological communities via impacts on species. The community's response to climate change can be represented as the temporal trend in a climate-related functional property that is quantified using a relevant functional trait. Noteworthy, some species influence this response in the community more strongly than others.

Innovation

Leveraging on the concept of keystone species, we propose that species with a strong effect on the community's functional response to climate change beyond their relative abundance can be considered as ‘climate keystone species’. We develop a stepwise tool to determine species' effects on a community's climate response and identify climate keystone species. We quantify the species-specific effect by measuring the difference in the community's climate response with and without the species. Next, we identify climate keystone species as those with a strong residual effect after weighting with their relative abundances in the community.

Main Conclusions

To illustrate the use of the stepwise tool with empirical data, we identify climate keystone species that have a strong effect on the change in the average temperature niche in North American bird communities over time and find the identification tool ecologically relevant. Identification of climate keystone species can serve as an additional conservation method to efficiently protect ecological communities and, in turn, the ecosystem functions they provide.     

Evans Blue as a Simple Method to Discriminate Mosquitoes’ Feeding Choice on Small Laboratory Animals

Background

Temperature, humidity, vision, and particularly odor, are external cues that play essential roles to mosquito blood feeding and oviposition. Entomological and behavioral studies employ well-established methods to evaluate mosquito attraction or repellency and to identify the source of the blood meal. Despite the efficacy of such methods, the costs involved in the production or acquisition of all parts, components and the chemical reagents involved are unaffordable for most researchers from poor countries. Thus, a simple and relatively low-cost method capable of evaluating mosquito preferences and the blood volume ingested is desirable.

Principal Findings

By using Evans blue (EB) vital dye and few standard laboratory supplies, we developed and validated a system capable of evaluating mosquito’s choice between two different host sources of blood. EB-injected and PBS-injected mice submitted to a number of situations were placed side by side on the top of a rounded recipient covered with tulle fabric and containing Aedes aegypti mosquitoes. Homogenates from engorged mosquitoes clearly revealed the blood source (EB- or PBS-injected host), either visually or spectrometrically. This method was able to estimate the number of engorded mosquitoes, the volume of blood ingested, the efficacy of a commercial repellent and the attractant effects of black color and human sweat.

Significance

Despite the obvious limitations due to its simplicity and to the dependence of a live source of blood, the present method can be used to assess a number of host variables (diet, aging, immunity, etc) and optimized for several aspects of mosquito blood feeding and vector-host interactions. Thus, it is proposed as an alternative to field studies, and it could be used for initial screenings of chemical compound candidates for repellents or attractants, since it replicates natural conditions of exposure to mosquitoes in a laboratory environment.     

An updated review of the toxicology and ecotoxicology of sodium fluoroacetate (1080) in relation to its use as a pest control tool in New?Zealand

Sodium fluoroacetate (1080) is a vertebrate pesticide, originally developed in the 1940s and principally used for the control of unwanted introduced animals in New?Zealand and Australia. Fluoroacetate is also a toxic component of poisonous plants found in Australia, Africa, South America, and India. In relation to its use as a pesticide, recent research has focused on further elucidation of its potential sub-lethal effects, on animal welfare issues, on understanding and reducing its risk to non-target species, on its ecotoxicology, and fate in the environment following use in baits. 1080 acts by interfering with cellular energy production through inhibition of the tricarboxylic acid cycle and lethal doses can kill animal pests within 6?48 h of eating baits. Exposure to sub-lethal doses has been shown to have harmful effects on the heart and testes in animal studies, and strict safety precautions are enforced to protect contractors and workers in the pest control industry. Considerable care must be taken when using 1080 for the control of animal pests. Primary poisoning of non-target birds and secondary poisoning of dogs must be minimised to ensure that benefits in terms of conservation outcomes and pest and disease control significantly outweigh the risks associated with its use. Despite over 60 years of research and practical experience, the use of 1080 is still embroiled in controversy, while research efforts continue to improve its target specificity when it is used as a conservation tool or for Tb vector control.     

Chromosome ‘painting’ in plants — a feasible technique?

It is shown that chromosome painting is as yet not possible for plants with very complex genomes, neither intra- nor interspecific. The reasons are inefficient blocking of dispersed repetitive sequences and insufficient signal intensity of short unique sequences. Future perspective are indicated.     

Non-invasive microelectrode potassium flux measurements as a potential tool for early recognition of virus–host compatibility in plants

Diseases caused by plant viruses are widespread, resulting in severe economic losses worldwide. Understanding the cellular basis of defense responses and developing efficient diagnostic tools for early recognition of host specificity to viral infection is, therefore, of great importance. In this work, non-invasive ion selective microelectrodes (the MIFE technique) were used to measure net ion fluxes in mesophyll tissue of host (potato, tomato, tobacco) and non-host (sugar beet and periwinkle) plants in response to infection with Potato virus X (PVX). These results were complemented by FLIM (Fluorescence Lifetime Imaging) measurements of PVX-induced changes in intracellular Ca²⁺ concentrations. Our results demonstrate that, unlike in other plant–pathogen interactions, Ca²⁺ signaling appears to be non-essential in recognition of the early stages of viral infection. Instead, we observed significant changes in K⁺ fluxes as early as 10 min after inoculation. Results of pharmacological experiments and membrane potential measurements pointed out that a significant part of these fluxes may be mediated by depolarization-activated outward-rectifying K⁺ channels. This may suggest that viral infections trigger a different mechanism of plant defense signaling as compared to signals derived from other microbial pathogens; hence, altered Ca²⁺ fluxes across the plasma membrane may not be a prerequisite for all elicitor-activated defense reactions. Clearly pronounced host specificity in K⁺ flux responses suggests that the MIFE technique can be effectively used as a screening tool for the early diagnostics of virus–host compatibility.     

Selection and characterization of in vitro–induced mutants of Dendrobium ‘Earsakul’ resistant to black rot

In Vitro Cellular & Developmental Biology - Plant - The development of Dendrobium ‘Earsakul’ with improved resistance to black rot is essential for sustainable orchid production. In...     

Garlic extract as a novel strategy to hasten dormancy release in buds of ‘Anna’ apple trees

A 2-season orchard trial was carried out to verify the effects of garlic extract (GE), as a novel dormancy-breaking substance, at different concentrations; 0, 50, 100, 150 and 200 ml l^− 1 on bud break dormancy and the metabolic alterations in buds of ‘Anna’ apple trees. Water content, total carbohydrates, reducing and total sugars, anthocyanins, total free amino acids, free proline, total indoles and free phenols were analyzed after the application of GE. The obtained results showed that, GE treatments hastened date of floral bud break and increased percentage of bud break, fruit set, total number of fruits and fruit yield per tree. Except for free phenols, GE also increased the abovementioned chemical components in buds. The best results were obtained from the treatment of 150 ml l^− 1 GE. Therefore, it is concluded that the use of GE at 150 ml l^− 1 could be recommended for improving the bud break, growth and yield of apple trees cv. “Anna”.     

It’s a wrap: encapsulation as a management tool for marine biofouling

Encapsulation of fouled structures is an effective tool for countering incursions by non-indigenous biofoulers. However, guidelines for the implementation of encapsulation treatments are yet to be established. This study evaluated the effects of temperature, biomass, community composition, treatment duration and the biocide acetic acid on biofoulers. In laboratory trials using the model organisms Ciona spp. and Mytilus galloprovincialis, increasing the temperature or biomass speeded up the development of a toxic environment. Total mortality for Ciona spp. occurred within 72 and 24 h at 10 and 19°C, respectively. M. galloprovincialis survived up to 18 days, with high biomass increasing mortality at 10°C only. In a field study, three-month-old and four-year-old communities were encapsulated with and without acetic acid. Mortality took up to 10 days for communities encapsulated without acetic acid, compared to 48 h with acetic acid. The insights gained from this study will be useful in developing standardised encapsulation protocols.     

Enzymatic resolution of racemates with a ‘remote’ stereogenic center as an efficient tool in drug,flavor and vitamin synthesis

The enantioselective recognition of ‘remote’ stereogenic centers represents a scientific task in organic chemistry being also of current interest in the pharmaceutical industry. This is due to a range of pharmaceutically relevant molecules or intermediates thereof bearing a stereogenic center, which is separated from the functional group by a larger non-chiral moiety such as, for example, a longer sequence of bonds of at least three carbon or hetero-atoms or by a planar aromatic moiety. Notably, biocatalysis turned out to provide an excellent solution for a range of challenging syntheses in this field. For example, efficient enzymatic resolution processes of racemates with such a ‘remote’ stereogenic center were developed for the synthesis of pelitrexol, lasofoxifene and (S)-monastrol. In general, good yields accompanied by high enantioselectivities were obtained, thus underlining the tremendous potential of enzymes to recognize and enantioselectively transform enantiomers of racemates with ‘remote’ stereogenic centers. Such or similar types of stereoselective recognitions of ‘remote’ stereogenic centers by means of enzymes have been also reported in the field of flavor and vitamin synthesis. Thus, biocatalysis represents a promising solution for the efficient approach to enantiomerically pure complex chiral molecules with stereogenic centers being located apart from the functional group, and it can be expected that enzymatic resolution will be increasingly applied when searching for an efficient and also technically feasible process for also novel complex chiral molecules bearing a ‘remote’ stereogenic center.     

Identifying ‘firebreaks’ to fragment dispersal networks of a marine parasite

Marine ecosystems are beset by disease outbreaks, and efficient strategies to control dispersal of pathogens are scarce. We tested whether introducing no-farming areas or ‘firebreaks’ could disconnect dispersal networks of a parasitic disease affecting the world’s largest marine fish farming industry (～1000 farms). Larval salmon lice (Lepeophtheirus salmonis) are released from and transported among salmon farms by ocean currents, creating inter-farm networks of louse dispersal. We used a state-of-the-art biophysical model to predict louse movement along the Norwegian coastline and network analysis to identify firebreaks to dispersal. At least one firebreak that fragmented the network into two large unconnected groups of farms was identified for all seasons. During spring, when wild salmon migrate out into the ocean, and louse levels per fish at farms must be minimised, two effective firebreaks were created by removing 13 and 21 farms (1.3% and 2.2% of all farms in the system) at ～61°N and 67°N, respectively. We have demonstrated that dispersal models coupled with network analysis can identify no-farming zones that fragment dispersal networks. Reduced dispersal pathways should lower infection pressure at farms, slow the evolution of resistance to parasite control measures, and alleviate infection pressure on wild salmon populations.     

‘Phytoantibodies’: a general vector for the expression of immunoglobulin domains in transgenic plants

Sequences encoding the immunoglobulin heavy-chain variable (VH) domains were engineered in a new general purpose vector to transform plants via Agrobacterium. The expression of an isolated VH domain (IVD) after introduction into the plant genome has been monitored by northern, western and immuno-histochemical analysis. Immunoblotting showed that the polypeptide was stably expressed and accounted for up to 1% of the soluble protein fraction. It is therefore proposed that single immunoglobulin domains of suitable specificity expressed in plants may constitute an effective system to inhibit the activity of molecules involved in plant pathology or plant development.     

CRISPR/Cas9: a promising way to exploit genetic variation in plants

Creation of variation in existing gene pool of crop plants is the foremost requirement in crop improvement programmes. Genome editing is a tool to produce knock out of target genes either by introduction of insertion or by deletion that disrupts the function of a specific gene. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system is the most recent addition to the toolbox of sequence-specific nucleases that includes ZFNs and TALENs. The CRISPR/Cas9 system allows targeted cleavage of genomic DNA guided by a small noncoding RNA, resulting in gene modifications by both non-homologous end joining and homology-directed repair mechanisms. Here, we present an overview of mechanisms of CRISPR, its potential roles in creating variation in germplasm and applications of this novel interference pathway in crop improvement. The availability of the CRISPR/Cas9 system holds promise in facilitating both forward and reverse genetics and will enhance research in crops that lack genetic resources.     

Implementation of a ‘standard’ LIMS

This article is an extract of a presentation given at the LIMS conference in the Hague June 4, 1997 [S.M. Fransen et al., Presentation given at the conference LIMS 97, June 4 1997 in the Hague.]. It describes the project steps for the LIMS project in the Danish company Novo Nordisk, with focus on vendor evaluation, validation and implementation of a ‘Standard’ LIMS software. The main project steps are described and experiences gained are discussed.     

Repurposed Automated Handheld Counter as a Point-of-Care Tool to Identify Individuals ‘At Risk’ of Serious Post-Ivermectin Encephalopathy

Introduction

Administration of ivermectin (IVM) as part of mass drug administration (MDA) campaigns for onchocerciasis and/or lymphatic filariasis (LF) has been suspended in areas co-endemic for Loa loa due to severe post-treatment adverse events (SAEs) associated with high-burden of infection (>30,000 mf/ml). One simple approach for preventing SAEs is to identify and exclude individuals at risk from MDA. Here, we describe a repurposed hand-held automated cell counter (Scepter 2.0; HHAC) as a rapid, point-of-care method for quantifying microfilariae (mf) in the blood of infected individuals.

Methodology/Principal Findings

The quantification of microfilarial levels in blood of naturally infected humans, experimentally infected baboons, or mf-spiked human blood was tested using a microfluidic-based automated counter and compared to traditional calibrated thick-smears. We demonstrate that mf can be quantified in 20 µl of whole blood following lysis with 10% saponin within a minute of obtaining blood. There was a highly significant concordance between the counts obtained by the HHAC and those by microscopy for mf densities of >5,000 (p<0.0001, r_c = 0.97) or >30,000 per ml (p<0.0001, r_c = 0.90). Preliminary proof of concept field studies in Cameroon with 20 µl of blood from L. loa infected humans (n = 22) and baboons (n = 4) also demonstrated a significantly high concordance (p<0.0001, r_c = 0.89) with calibrated thick blood smears counts.

Conclusions/Significance

A repurposed HHAC is a portable, sensitive, rapid, point-of-care and quantitative tool to identify individuals with high levels of L. loa mf that put them at risk for SAEs following MDA. In addition, it provides ease of data storage and accessibility.