Investigating the molecular mechanisms of organophosphate and pyrethroid resistance in the fall armyworm Spodoptera frugiperda.

Published onlineJournal ArticleResearch Support, Non-U.S. Gov'tThe fall armyworm Spodoptera frugiperda is an economically important pest of small grain crops that occurs in all maize growing regions of the Americas. The intensive use of chemical pesticides for its control has led to the selection of resistant populations, however, to date, the molecular mechanisms underlying resistance have not been characterised. In this study the mechanisms involved in the resistance of two S. frugiperda strains collected in Brazil to chlorpyrifos (OP strain) or lambda-cyhalothrin (PYR strain) were investigated using molecular and genomic approaches. To examine the possible role of target-site insensitivity the genes encoding the organophosphate (acetylcholinesterase, AChE) and pyrethroid (voltage-gated sodium channel, VGSC) target-site proteins were PCR amplified. Sequencing of the S. frugiperda ace-1 gene identified several nucleotide changes in the OP strain when compared to a susceptible reference strain (SUS). These result in three amino acid substitutions, A201S, G227A and F290V, that have all been shown previously to confer organophosphate resistance in several other insect species. Sequencing of the gene encoding the VGSC in the PYR strain, identified mutations that result in three amino acid substitutions, T929I, L932F and L1014F, all of which have been shown previously to confer knockdown/super knockdown-type resistance in several arthropod species. To investigate the possible role of metabolic detoxification in the resistant phenotype of the OP and PYR stains all EST sequences available for S. frugiperda were used to design a gene-expression microarray. This was then used to compare gene expression in the resistant strains with the susceptible reference strain. Members of several gene families, previously implicated in metabolic resistance in other insects were found to be overexpressed in the resistant strains including glutathione S-transferases, cytochrome P450s and carboxylesterases. Taken together these results provide evidence that both target-site and metabolic mechanisms underlie the resistance of S. frugiperda to pyrethroids and organophosphates.BBSRCNational Council for Scientific and Technological Development of Brazi

Identification of the main malaria vectors in the Anopheles gambiae species complex using a TaqMan real-time PCR assay

Background: The Anopheles gambiae sensu lato species complex comprises seven sibling species of mosquitoes that are morphologically indistinguishable. Rapid identification of the two main species which vector malaria, Anopheles arabiensis and An. gambiae sensu stricto, from the non-vector species Anopheles quadriannulatus is often required as part of vector control programmes. Currently the most widely used method for species identification is a multiplex PCR protocol that targets species specific differences in ribosomal DNA sequences. While this assay has proved to be reasonably robust in many studies, additional steps are required post-PCR making it time consuming. Recently, a high-throughput assay based on TaqMan single nucleotide polymorphism genotyping that detects and discriminates An. gambiae s.s and An. arabiensis has been reported. Methods: A new TaqMan assay was developed that distinguishes between the main malaria vectors (An. Arabiensis and An. gambiae s.s.) and the non-vector An. quadriannulatus after it was found that the existing TaqMan assay incorrectly identified An. quadriannulatus, An. merus and An. melas as An. gambiae s.s. The performance of this new TaqMan assay was compared against the existing TaqMan assay and the standard PCR method in a blind species identification trial of over 450 samples using field collected specimens from a total of 13 countries in Sub-Saharan Africa. Results: The standard PCR method was found to be specific with a low number of incorrect scores (<1%), however when compared to the TaqMan assays it showed a significantly higher number of failed reactions (15%). Both the new vector-specific TaqMan assay and the exisiting TaqMan showed a very low number of incorrectly identified samples (0 and 0.54%) and failed reactions (1.25% and 2.96%). In tests of analytical sensitivity the new TaqMan assay showed a very low detection threshold and can consequently be used on a single leg from a fresh or silica-dried mosquito without the need to first extract DNA. Conclusion: This study describes a rapid and sensitive assay that very effectively identifies the two main malaria vectors of the An. gambiae species complex from the non-vector sibling species. The method is based on TaqMan SNP genotyping and can be used to screen single legs from dried specimens. In regions where An. merus/melas/ bwambae, vectors with restricted distributions, are not present it can be used alone to discriminate vector from non-vector or in combination with the Walker TaqMan assay to distinguish An. arabiensis and An. gambiae s.s

PCR-based detection of Plasmodium in Anopheles mosquitoes: a comparison of a new high-throughput assay with existing methods.

Published onlineComparative StudyEvaluation StudiesJournal ArticleResearch Support, Non-U.S. Gov'tBACKGROUND: Detection of the four malaria-causing Plasmodium species (Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae) within their mosquito hosts is an essential component of vector control programmes. Several PCR protocols have been developed for this purpose. Many of these methods, while sensitive, require multiple PCR reactions to detect and discriminate all four Plasmodium species. In this study a new high-throughput assay was developed and compared with three previously described PCR techniques. METHODS: A new assay based on TaqMan SNP genotyping was developed to detect all four Plasmodium species and discriminate P. falciparum from P. vivax, P. ovale and P. malariae. The sensitivity and the specificity of the new assay was compared to three alternative PCR approaches and to microscopic dissection of salivary glands in a blind trial of 96 single insect samples that included artificially infected Anopheles stephensi mosquitoes. The performance of the assays was then compared using more than 450 field-collected specimens that had been stored on silica gel, in ethanol or in isopropanol. RESULTS: The TaqMan assay was found to be highly specific when using Plasmodium genomic DNA as template. Tests of analytical sensitivity and the results of the blind trial showed the TaqMan assay to be the most sensitive of the four methods followed by the 'gold standard' nested PCR approach and the results generated using these two methods were in good concordance. The sensitivity of the other two methods and their agreement with the nested PCR and TaqMan approaches varied considerably. In trials using field collected specimens two of the methods (including the nested protocol) showed a high degree of non-specific amplification when using DNA derived from mosquitoes stored in ethanol or isopropanol. The TaqMan method appeared unaffected when using the same samples. CONCLUSION: This study describes a new high-throughput TaqMan assay that very effectively detects the four Plasmodium species that cause malaria in humans and discriminates the most deadly species, P. falciparum, from the others. This method is at least as sensitive and specific as the gold standard nested PCR approach and because it has no requirement for post-PCR processing is cheaper, simpler and more rapid to run. In addition this method is not inhibited by the storage of mosquito specimens by drying or in ethanol or isopropanol.BBSRCInnovative Vector Control Consortiu

Mutation (G275E) of the nicotinic acetylcholine receptor α6 subunit is associated with high levels of resistance to spinosyns in Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae).

PublishedJournal ArticleThe tomato leafminer, Tuta absoluta, now a major pest of tomato crops worldwide, is primarily controlled using chemical insecticides. Recently, high levels of resistance to the insecticide spinosad have been described in T. absoluta populations in Brazil. Selection of a resistant field-collected strain led to very high levels of resistance to spinosad and cross-resistance to spinetoram, but not to other insecticides that target the nicotinic acetylcholine receptor (nAChR). In this study the mechanisms underlying resistance to spinosad were investigated using toxicological, biochemical and molecular approaches. Inhibition of metabolic enzymes using synergists and biochemical assessment of detoxification enzyme activity provided little evidence of metabolic resistance in the selected strain. Cloning and sequencing of the nAChR α6 subunit from T. absoluta, the spinosad target-site, from susceptible and spinosad-resistant strains were done to investigate the role of a target-site mechanism in resistance. A single nucleotide change was identified in exon 9 of the α6 subunit of the resistant strain, resulting in the replacement of the glycine (G) residue at position 275 observed in susceptible T. absoluta strains with a glutamic acid (E). A high-throughput DNA-based diagnostic assay was developed and used to assess the prevalence of the G275E mutation in 17 field populations collected from different geographical regions of Brazil. The resistant allele was found at low frequency, and in the heterozygous form, in seven of these populations but at much higher frequency and in the homozygous form in a population collected in the Iraquara municipality. The frequency of the mutation was significantly correlated with the mortality of these populations in discriminating dose bioassays. In summary our results provide evidence that the G275E mutation is an important mechanism of resistance to spinosyns in T. absoluta, and may be used as a marker for resistance monitoring in field populations.Thanks to CAPES for the scholarship granted to the first author and to Conselho Nacional de Desenvolvimento Científico e Tecnológico — CNPq for the financial support to the project (Universal 484240/2011-0, H.A.A.S.). The research leading to these results has received funding from the People Programme (Marie Curie Actions) of the European Union Seventh Framework Programme FP7/2007-2013/ under REA grant agreement PIRSES-GA-2012 – 318246. This work was in part funded by a fellowship grant (BB/G023352/1) from the Biotechnology and Biological Sciences Research Council of the UK to Dr. Chris Bass and a PhD studentship award from the BBSRC which funded Madeleine Berger

Genomic insights into neonicotinoid sensitivity in the solitary bee Osmia bicornis

This is the final version. Available from the publisher via the DOI in this record.The Osmia bicornis whole genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession MPJT00000000. The RNAseq data generated in this study has been deposited in the Sequence Read Archive (SRA) under accession SRP065762. Accession numbers of the bee P450 genes manually curated in this study are shown in S5 Table. All other relevant data are within the paper and its Supporting Information files.The impact of pesticides on the health of bee pollinators is determined in part by the capacity of bee detoxification systems to convert these compounds to less toxic forms. For example, recent work has shown that cytochrome P450s of the CYP9Q subfamily are critically important in defining the sensitivity of honey bees and bumblebees to pesticides, including neonicotinoid insecticides. However, it is currently unclear if solitary bees have functional equivalents of these enzymes with potentially serious implications in relation to their capacity to metabolise certain insecticides. To address this question, we sequenced the genome of the red mason bee, Osmia bicornis, the most abundant and economically important solitary bee species in Central Europe. We show that O. bicornis lacks the CYP9Q subfamily of P450s but, despite this, exhibits low acute toxicity to the N-cyanoamidine neonicotinoid thiacloprid. Functional studies revealed that variation in the sensitivity of O. bicornis to N-cyanoamidine and N-nitroguanidine neonicotinoids does not reside in differences in their affinity for the nicotinic acetylcholine receptor or speed of cuticular penetration. Rather, a P450 within the CYP9BU subfamily, with recent shared ancestry to the Apidae CYP9Q subfamily, metabolises thiacloprid in vitro and confers tolerance in vivo. Our data reveal conserved detoxification pathways in model solitary and eusocial bees despite key differences in the evolution of specific pesticide-metabolising enzymes in the two species groups. The discovery that P450 enzymes of solitary bees can act as metabolic defence systems against certain pesticides can be leveraged to avoid negative pesticide impacts on these important pollinators.Biotechnology and Biological Science Research Council (BBSRC)Bayer AGEuropean Research Council (ERC

Assessing the acute toxicity of insecticides to the buff-tailed bumblebee (Bombus terrestris audax)

This is the final version. Available from Elsevier / Academic Press via the DOI in this record. The buff-tailed bumblebee, Bombus terrestris audax is an important pollinator within both landscape ecosystems and agricultural crops. During their lifetime bumblebees are regularly challenged by various environmental stressors including insecticides. Historically the honey bee (Apis mellifera spp.) has been used as an ‘indicator’ species for ‘standard’ ecotoxicological testing, but it has been suggested that it is not always a good proxy for other eusocial or solitary bees. To investigate this, the susceptibility of B. terrestris to selected pesticides within the neonicotinoid, pyrethroid and organophosphate classes was examined using acute insecticide bioassays. Acute oral and topical LD values for B. terrestris against these insecticides were broadly consistent with published results for A. mellifera. For the neonicotinoids, imidacloprid was highly toxic, but thiacloprid and acetamiprid were practically non-toxic. For pyrethroids, deltamethrin was highly toxic, but tau-fluvalinate only slightly toxic. For the organophosphates, chlorpyrifos was highly toxic, but coumaphos practically non-toxic. Bioassays using insecticides with common synergists enhanced the sensitivity of B. terrestris to several insecticides, suggesting detoxification enzymes may provide a level of protection against these compounds. The sensitivity of B. terrestris to compounds within three different insecticide classes is similar to that reported for honey bees, with marked variation in sensitivity to different insecticides within the same insecticide class observed in both species. This finding highlights the need to consider each compound within an insecticide class in isolation rather than extrapolating between different insecticides in the same class or sharing the same mode of action.European Union Horizon 2020Biotechnology and Biological Sciences Research Council (BBSRC

Overfeeding, Autonomic Regulation and Metabolic Consequences

The autonomic nervous system plays an important role in the regulation of body processes in health and disease. Overfeeding and obesity (a disproportional increase of the fat mass of the body) are often accompanied by alterations in both sympathetic and parasympathetic autonomic functions. The overfeeding-induced changes in autonomic outflow occur with typical symptoms such as adiposity and hyperinsulinemia. There might be a causal relationship between autonomic disturbances and the consequences of overfeeding and obesity. Therefore studies were designed to investigate autonomic functioning in experimentally and genetically hyperphagic rats. Special emphasis was given to the processes that are involved in the regulation of peripheral energy substrate homeostasis. The data revealed that overfeeding is accompanied by increased parasympathetic outflow. Typical indices of vagal activity (such as the cephalic insulin release during food ingestion) were increased in all our rat models for hyperphagia. Overfeeding was also accompanied by increased sympathetic tone, reflected by enhanced baseline plasma norepinephrine (NE) levels in both VMH-lesioned animals and rats rendered obese by hyperalimentation. Plasma levels of NE during exercise were, however, reduced in these two groups of animals. This diminished increase in the exercise-induced NE outflow could be normalized by prior food deprivation. It was concluded from these experiments that overfeeding is associated with increased parasympathetic and sympathetic tone. In models for hyperphagia that display a continuously elevated nutrient intake such as the VMH-lesioned and the overfed rat, this increased sympathetic tone was accompanied by a diminished NE response to exercise. This attenuated outflow of NE was directly related to the size of the fat reserves, indicating that the feedback mechanism from the periphery to the central nervous system is altered in the overfed state.

Effective-Range Expansion of the Neutron-Deuteron Scattering Studied by a Quark-Model Nonlocal Gaussian Potential

The S-wave effective range parameters of the neutron-deuteron (nd) scattering are derived in the Faddeev formalism, using a nonlocal Gaussian potential based on the quark-model baryon-baryon interaction fss2. The spin-doublet low-energy eigenphase shift is sufficiently attractive to reproduce predictions by the AV18 plus Urbana three-nucleon force, yielding the observed value of the doublet scattering length and the correct differential cross sections below the deuteron breakup threshold. This conclusion is consistent with the previous result for the triton binding energy, which is nearly reproduced by fss2 without reinforcing it with the three-nucleon force.Comment: 21 pages, 6 figures and 6 tables, submitted to Prog. Theor. Phy

The Role of Individual Variables, Organizational Variables and Moral Intensity Dimensions in Libyan Management Accountants’ Ethical Decision Making

This study investigates the association of a broad set of variables with the ethical decision making of management accountants in Libya. Adopting a cross-sectional methodology, a questionnaire including four different ethical scenarios was used to gather data from 229 participants. For each scenario, ethical decision making was examined in terms of the recognition, judgment and intention stages of Rest’s model. A significant relationship was found between ethical recognition and ethical judgment and also between ethical judgment and ethical intention, but ethical recognition did not significantly predict ethical intention—thus providing support for Rest’s model. Organizational variables, age and educational level yielded few significant results. The lack of significance for codes of ethics might reflect their relative lack of development in Libya, in which case Libyan companies should pay attention to their content and how they are supported, especially in the light of the under-development of the accounting profession in Libya. Few significant results were also found for gender, but where they were found, males showed more ethical characteristics than females. This unusual result reinforces the dangers of gender stereotyping in business. Personal moral philosophy and moral intensity dimensions were generally found to be significant predictors of the three stages of ethical decision making studied. One implication of this is to give more attention to ethics in accounting education, making the connections between accounting practice and (in Libya) Islam. Overall, this study not only adds to the available empirical evidence on factors affecting ethical decision making, notably examining three stages of Rest’s model, but also offers rare insights into the ethical views of practising management accountants and provides a benchmark for future studies of ethical decision making in Muslim majority countries and other parts of the developing world

Influence of environmental parameters on movements and habitat utilization of humpback whales (Megaptera novaeangliae) in the Madagascar breeding ground

© The Author(s), 2016. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Royal Society Open Science 3 (2016): 160616, doi:10.1098/rsos.160616.Assessing the movement patterns and key habitat features of breeding humpback whales is a prerequisite for the conservation management of this philopatric species. To investigate the interactions between humpback whale movements and environmental conditions off Madagascar, we deployed 25 satellite tags in the northeast and southwest coast of Madagascar. For each recorded position, we collated estimates of environmental variables and computed two behavioural metrics: behavioural state of ‘transiting’ (consistent/directional) versus ‘localized’ (variable/non-directional), and active swimming speed (i.e. speed relative to the current). On coastal habitats (i.e. bathymetry < 200 m and in adjacent areas), females showed localized behaviour in deep waters (191 ± 20 m) and at large distances (14 ± 0.6 km) from shore, suggesting that their breeding habitat extends beyond the shallowest waters available close to the coastline. Males' active swimming speed decreased in shallow waters, but environmental parameters did not influence their likelihood to exhibit localized movements, which was probably dominated by social factors instead. In oceanic habitats, both males and females showed localized behaviours in shallow waters and favoured high chlorophyll-a concentrations. Active swimming speed accounts for a large proportion of observed movement speed; however, breeding humpback whales probably exploit prevailing ocean currents to maximize displacement. This study provides evidence that coastal areas, generally subject to strong human pressure, remain the core habitat of humpback whales off Madagascar. Our results expand the knowledge of humpback whale habitat use in oceanic habitat and response to variability of environmental factors such as oceanic current and chlorophyll level.Funding was provided by Total Foundation to NeuroPSI, and by individuals and foundations to the WCS Ocean Giants Program