CRC20080 Diagnostic technologies for phosphine resistance management

Development of molecular markers for rapid diagnosis of phosphine resistance in insects

DOSE-RESPONSE MODELING WITH MARGINAL INFORMATION ON A MISSING CATEGORICAL COVARIATE

When the relationship between a dosage-type variable and a binary outcome depends on a categorical variable, a common analysis would employ a dose-response model with the categorical variable as a covariate. When the level of the categorical variable is not known for all subjects, however, the standard dose-response model alone cannot provide useful inference. We present an EM-based approach to account for the missing covariate in a dose-response model setting when additional knowledge about the marginal distribution of the covariate is available. This approach is motivated by a study of the beetle Rhyzopertha dominica, a pest of stored grain in Australia. Certain genotypes of this beetle have developed inheritable resistance to a widely-used insecticidal fumigant. In this study, the effects of various dosage levels of the fumigant were considered, and it was feasible to genotype only the surviving beetles

Sulfurous Gases As Biological Messengers and Toxins: Comparative Genetics of Their Metabolism in Model Organisms

Gasotransmitters are biologically produced gaseous signalling molecules. As gases with potent biological activities, they are toxic as air pollutants, and the sulfurous compounds are used as fumigants. Most investigations focus on medical aspects of gasotransmitter biology rather than toxicity toward invertebrate pests of agriculture. In fact, the pathways for the metabolism of sulfur containing gases in lower organisms have not yet been described. To address this deficit, we use protein sequences from Homo sapiens to query Genbank for homologous proteins in Caenorhabditis elegans, Drosophila melanogaster, and Saccharomyces cerevisiae. In C. elegans, we find genes for all mammalian pathways for synthesis and catabolism of the three sulfur containing gasotransmitters, H2S, SO2 and COS. The genes for H2S synthesis have actually increased in number in C. elegans. Interestingly, D. melanogaster and Arthropoda in general, lack a gene for 3-mercaptopyruvate sulfurtransferase, an enzym for H2S synthesis under reducing conditions

Mechanisms of Phosphine Toxicity

Fumigation with phosphine gas is by far the most widely used treatment for the protection of stored grain against insect pests. The development of high-level resistance in insects now threatens its continued use. As there is no suitable chemical to replace phosphine, it is essential to understand the mechanisms of phosphine toxicity to increase the effectiveness of resistance management. Because phosphine is such a simple molecule (PH3), the chemistry of phosphorus is central to its toxicity. The elements above and below phosphorus in the periodic table are nitrogen (N) and arsenic (As), which also produce toxic hydrides, namely, NH3 and AsH3. The three hydrides cause related symptoms and similar changes to cellular and organismal physiology, including disruption of the sympathetic nervous system, suppressed energy metabolism and toxic changes to the redox state of the cell. We propose that these three effects are interdependent contributors to phosphine toxicity

Potential for using pheromone trapping and molecular screening in phosphine resistance research: Presentation

Phosphine resistance monitoring typically involves bioassays of beetles from population samples collected from grain storage facilities. Insects are classified into susceptible or resistant phenotypes based on mortality or survival at one or more discriminating doses. Although valuable, phenotype testing has several drawbacks. First, phenotype testing needs live insects, and considerable effort is required to collect and maintain them before testing. Second, population samples may contain multiple genotypes expressing different levels of resistance that may not be distinguishable using discriminating dose bioassays. Third, collections are likely to be focussed around grain storages to maximise sampling success. Recent research shows that several key pests are actively dispersing through flight. The availability of commercial pheromone lures and recent advances in molecular screening provide an opportunity to provide information on resistance gene frequencies more broadly across the landscape. This approach is proving to be a valuable adjunct to traditional resistance testing in Australia.Phosphine resistance monitoring typically involves bioassays of beetles from population samples collected from grain storage facilities. Insects are classified into susceptible or resistant phenotypes based on mortality or survival at one or more discriminating doses. Although valuable, phenotype testing has several drawbacks. First, phenotype testing needs live insects, and considerable effort is required to collect and maintain them before testing. Second, population samples may contain multiple genotypes expressing different levels of resistance that may not be distinguishable using discriminating dose bioassays. Third, collections are likely to be focussed around grain storages to maximise sampling success. Recent research shows that several key pests are actively dispersing through flight. The availability of commercial pheromone lures and recent advances in molecular screening provide an opportunity to provide information on resistance gene frequencies more broadly across the landscape. This approach is proving to be a valuable adjunct to traditional resistance testing in Australia

Utility of biotechnology based decision making tools in postharvest grain pest management: An Australian case study

A major concern for the Australian grain industry in recent years is the constant threat of resistance to the key disinfestant phosphine in a range of stored grain pests. The need to maintain the usefulness of phosphine and to contain the development of resistance are critical to international market access for Australian grain. Strong levels of resistance have already been established in major pests including the lesser grain borer, Rhyzopertha dominica (F.), the red flour beetle, Tribolium castaneum (Herbst), and most recently in the rusty grain beetle Cryptolestes ferrugineus (Stephens). As a proactive integrated resistance management strategy, new fumigation protocols are being developed in the laboratory and verified in large-scale field trials in collaboration with industry partners. To aid this development, we have deployed advanced molecular diagnostic tools to accurately determine the strength and frequency of key phosphine resistant insect pests and their movement within a typical Australian grain value chain. For example, two major bulk storage facilities based at Brookstead and Millmerran in southeast Queensland, Australia, were selected as main nodes and several farms and feed mills located in and around these two sites at a scale of 25 to 100 km radius were selected and surveyed. We determined the type, pattern, frequency as well as the distribution of resistance alleles accurately for two major pests, R. dominica and T. castaneum. Overall, this information along with the phenotypic data, provide a basis for designing key intervention strategies in managing resistance problems in the study area.A major concern for the Australian grain industry in recent years is the constant threat of resistance to the key disinfestant phosphine in a range of stored grain pests. The need to maintain the usefulness of phosphine and to contain the development of resistance are critical to international market access for Australian grain. Strong levels of resistance have already been established in major pests including the lesser grain borer, Rhyzopertha dominica (F.), the red flour beetle, Tribolium castaneum (Herbst), and most recently in the rusty grain beetle Cryptolestes ferrugineus (Stephens). As a proactive integrated resistance management strategy, new fumigation protocols are being developed in the laboratory and verified in large-scale field trials in collaboration with industry partners. To aid this development, we have deployed advanced molecular diagnostic tools to accurately determine the strength and frequency of key phosphine resistant insect pests and their movement within a typical Australian grain value chain. For example, two major bulk storage facilities based at Brookstead and Millmerran in southeast Queensland, Australia, were selected as main nodes and several farms and feed mills located in and around these two sites at a scale of 25 to 100 km radius were selected and surveyed. We determined the type, pattern, frequency as well as the distribution of resistance alleles accurately for two major pests, R. dominica and T. castaneum. Overall, this information along with the phenotypic data, provide a basis for designing key intervention strategies in managing resistance problems in the study area

Phosphine Resistance in the Rust Red Flour Beetle, Tribolium castaneum (Coleoptera: Tenebrionidae): Inheritance, Gene Interactions and Fitness Costs

The recent emergence of heritable high level resistance to phosphine in stored grain pests is a serious concern among major grain growing countries around the world. Here we describe the genetics of phosphine resistance in the rust red flour beetle Tribolium castaneum (Herbst), a pest of stored grain as well as a genetic model organism. We investigated three field collected strains of T. castaneum viz., susceptible (QTC4), weakly resistant (QTC1012) and strongly resistant (QTC931) to phosphine. The dose-mortality responses of their test- and inter-cross progeny revealed that most resistance was conferred by a single major resistance gene in the weakly (3.2×) resistant strain. This gene was also found in the strongly resistant (431×) strain, together with a second major resistance gene and additional minor factors. The second major gene by itself confers only 12–20× resistance, suggesting that a strong synergistic epistatic interaction between the genes is responsible for the high level of resistance (431×) observed in the strongly resistant strain. Phosphine resistance is not sex linked and is inherited as an incompletely recessive, autosomal trait. The analysis of the phenotypic fitness response of a population derived from a single pair inter-strain cross between the susceptible and strongly resistant strains indicated the changes in the level of response in the strong resistance phenotype; however this effect was not consistent and apparently masked by the genetic background of the weakly resistant strain. The results from this work will inform phosphine resistance management strategies and provide a basis for the identification of the resistance genes

Deep sequencing-based transcriptome analysis of Plutella xylostella larvae parasitized by Diadegma semiclausum

Background: Parasitoid insects manipulate their hosts' physiology by injecting various factors into their host upon parasitization. Transcriptomic approaches provide a powerful approach to study insect host-parasitoid interactions at the molecular level. In order to investigate the effects of parasitization by an ichneumonid wasp (Diadegma semiclausum) on the host (Plutella xylostella), the larval transcriptome profile was analyzed using a short-read deep sequencing method (Illumina). Symbiotic polydnaviruses (PDVs) associated with ichneumonid parasitoids, known as ichnoviruses, play significant roles in host immune suppression and developmental regulation. In the current study, D. semiclausum ichnovirus (DsIV) genes expressed in P. xylostella were identified and their sequences compared with other reported PDVs. Five of these genes encode proteins of unknown identity, that have not previously been reported

Behavioral genomics of honeybee foraging and nest defense

The honeybee has been the most important insect species for study of social behavior. The recently released draft genomic sequence for the bee will accelerate honeybee behavioral genetics. Although we lack sufficient tools to manipulate this genome easily, quantitative trait loci (QTLs) that influence natural variation in behavior have been identified and tested for their effects on correlated behavioral traits. We review what is known about the genetics and physiology of two behavioral traits in honeybees, foraging specialization (pollen versus nectar), and defensive behavior, and present evidence that map-based cloning of genes is more feasible in the bee than in other metazoans. We also present bioinformatic analyses of candidate genes within QTL confidence intervals (CIs). The high recombination rate of the bee made it possible to narrow the search to regions containing only 17–61 predicted peptides for each QTL, although CIs covered large genetic distances. Knowledge of correlated behavioral traits, comparative bioinformatics, and expression assays facilitated evaluation of candidate genes. An overrepresentation of genes involved in ovarian development and insulin-like signaling components within pollen foraging QTL regions suggests that an ancestral reproductive gene network was co-opted during the evolution of foraging specialization. The major QTL influencing defensive/aggressive behavior contains orthologs of genes involved in central nervous system activity and neurogenesis. Candidates at the other two defensive-behavior QTLs include modulators of sensory signaling (Am5HT(7) serotonin receptor, AmArr4 arrestin, and GABA-B-R1 receptor). These studies are the first step in linking natural variation in honeybee social behavior to the identification of underlying genes

The Genome of Rhyzopertha dominica (Fab.) (Coleoptera: Bostrichidae): Adaptation for Success

The lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), is a major global pest of cereal grains. Infestations are difficult to control as larvae feed inside grain kernels, and many populations are resistant to both contact insecticides and fumigants. We sequenced the genome of R. dominica to identify genes responsible for important biological functions and develop more targeted and efficacious management strategies. The genome was assembled from long read sequencing and long-range scaffolding technologies. The genome assembly is 479.1 Mb, close to the predicted genome size of 480.4 Mb by flow cytometry. This assembly is among the most contiguous beetle assemblies published to date, with 139 scaffolds, an N50 of 53.6 Mb, and L50 of 4, indicating chromosome-scale scaffolds. Predicted genes from biologically relevant groups were manually annotated using transcriptome data from adults and different larval tissues to guide annotation. The expansion of carbohydrase and serine peptidase genes suggest that they combine to enable efficient digestion of cereal proteins. A reduction in the copy number of several detoxification gene families relative to other coleopterans may reflect the low selective pressure on these genes in an insect that spends most of its life feeding internally. Chemoreceptor genes contain elevated numbers of pseudogenes for odorant receptors that also may be related to the recent ontogenetic shift of R. dominica to a diet consisting primarily of stored grains. Analysis of repetitive sequences will further define the evolution of bostrichid beetles compared to other species. The data overall contribute significantly to coleopteran genetic research