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

Life stage and resistance effects in modelling phosphine fumigation of Rhyzopertha dominica (F.)

Resistance to phosphine in insect pests of stored grain is a serious problem and there is a world-wide need for the development of sustainable resistance management strategies. Here we introduce results from a new mathematical model of resistance development that includes all life stages, rates of oviposition, natural mortality and mortality under fumigation in relation to resistant genotype. The example we discuss is phosphine resistance in the lesser grain borer, Rhyzopertha dominica where resistance is known to be controlled by two major genes that are close to recessive in expression, so that resistance is not fully expressed unless both resistant genes are present and homozygous. An example of a scenario where this model could be used concerns the repeat application of phosphine in a situation where control of all life stages has not been achieved. We determined a critical interval within which a second fumigation must occur to stop a rapidly recovering population of resistant genotypes. Such scenarios can be readily investigated using this approach to provide the grain industry with resistance management options and strategies. Keywords: Rhyzopertha dominica, Population dynamics, Stored wheat, Phosphine fumigant, Low concentratio

Diagnostic Molecular Markers for Phosphine Resistance in US Populations of Tribolium castaneum and Rhyzopertha dominica

Citation: Chen, Z., Schlipalius, D., Opit, G., Subramanyam, B., & Phillips, T. W. (2015). Diagnostic Molecular Markers for Phosphine Resistance in US Populations of Tribolium castaneum and Rhyzopertha dominica. Plos One, 10(3), 14. doi:10.1371/journal.pone.0121343Stored product beetles that are resistant to the fumigant pesticide phosphine (hydrogen phosphide) gas have been reported for more than 40 years in many places worldwide. Traditionally, determination of phosphine resistance in stored product beetles is based on a discriminating dose bioassay that can take up to two weeks to evaluate. We developed a diagnostic cleaved amplified polymorphic sequence method, CAPS, to detect individuals with alleles for strong resistance to phosphine in populations of the red flour beetle, Tribolium castaneum, and the lesser grain borer, Rhyzopertha dominica, according to a single nucleotide mutation in the dihydrolipoamide dehydrogenase (DLD) gene. We initially isolated and sequenced the DLD genes from susceptible and strongly resistant populations of both species. The corresponding amino acid sequences were then deduced. A single amino acid mutation in DLD in populations of T. castaneum and R. dominica with strong resistance was identified as P45S in T. castaneum and P49S in R. dominica, both collected from northern Oklahoma, USA. PCR products containing these mutations were digested by the restriction enzymes MboI and BstNI, which revealed presence or absence, respectively of the resistant (R) allele and allowed inference of genotypes with that allele. Seven populations of T. castaneum from Kansas were subjected to discriminating dose bioassays for the weak and strong resistance phenotypes. Application of CAPS to these seven populations confirmed the R allele was in high frequency in the strongly resistant populations, and was absent or at a lower frequency in populations with weak resistance, which suggests that these populations with a low frequency of the R allele have the potential for selection of the strong resistance phenotype. CAPS markers for strong phosphine resistance will help to detect and confirm resistant beetles and can facilitate resistance management actions against a given pest population

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 disinfectant 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

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 disinfectant 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

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

Genes related to mitochondrial functions are differentially expressed in phosphine-resistant and -susceptible Tribolium castaneum

Background: Phosphine is a valuable fumigant to control pest populations in stored grains and grain products. However, recent studies indicate a substantial increase in phosphine resistance in stored product pests worldwide.Results: To understand the molecular bases of phosphine resistance in insects, we used RNA-Seq to compare gene expression in phosphine-resistant and susceptible laboratory populations of the red flour beetle, Tribolium castaneum. Each population was evaluated as either phosphine-exposed or no phosphine (untreated controls) in triplicate biological replicates (12 samples total). Pairwise analysis indicated there were eight genes differentially expressed between susceptible and resistant insects not exposed to phosphine (i.e., basal expression) or those exposed to phopshine (>8-fold expression and 90 % C.I.). However, 214 genes were differentially expressed among all four treatment groups at a statistically significant level (ANOVA, p < 0.05). Increased expression of 44 cytochrome P450 genes was found in resistant vs. susceptible insects, and phosphine exposure resulted in additional increases of 21 of these genes, five of which were significant among all treatment groups (p < 0.05). Expression of two genes encoding anti-diruetic peptide was 2- to 8-fold reduced in phosphine-resistant insects, and when exposed to phosphine, expression was further reduced 36- to 500-fold compared to susceptible. Phosphine-resistant insects also displayed differential expression of cuticle, carbohydrate, protease, transporter, and many mitochondrial genes, among others. Gene ontology terms associated with mitochondrial functions (oxidation biological processes, monooxygenase and catalytic molecular functions, and iron, heme, and tetrapyyrole binding) were enriched in the significantly differentially expressed dataset. Sequence polymorphism was found in transcripts encoding a known phosphine resistance gene, dihydrolipoamide dehydrogenase, in both susceptible and resistant insects. Phosphine-resistant adults also were resistant to knockdown by the pyrethroid deltamethrin, likely due to the increased cytochrome P450 expression.Conclusions: Overall, genes associated with the mitochondria were differentially expressed in resistant insects, and these differences may contribute to a reduction in overall metabolism and energy production and/or compensation in resistant insects. These data provide the first gene expression data on the response of phosphine-resistant and -susceptible insects to phosphine exposure, and demonstrate that RNA-Seq is a valuable tool to examine differences in insects that respond differentially to environmental stimuli.Peer reviewedEntomology and Plant Patholog

New tools for management of phosphine resistance

Phosphine is the primary fumigant used to protect the majority of the world' s grain and a variety of other stored commodities from insect pests. Phosphine is playing an increasingly important role in the protection of commodities for two primary reasons. Firstly, use of the alternative fumigant, methyl bromide, has been sharply curtailed and is tightly regulated due to its role in ozone depletion, and secondly, consumers are becoming increasingly intolerant of contact pesticides. Niche alternatives to phosphine exist, but they suffer from a range of factors that limit their use, including: 1) Limited commercial adoption due to expense or slow mode of action; 2) Poor efficacy due to low toxicity, rapid sorption, limited volatility or high density; 3) Public health concerns due to toxicity to handlers or nearby residents, as well as risk of explosion; 4) Poor consumer acceptance due to toxic residues or smell. These same factors limit the prospects of quickly identifying and deploying a new fumigant. Given that resistance toward phosphine is increasing among insect pests, improved monitoring and management of resistance is a priority. Knowledge of the mode of action of phosphine as well as the mechanisms of resistance may also greatly reduce the effort and expense of identifying synergists or novel replacement compounds