Project description:Sitophilus oryzae (Linnaeus) is a major pest of stored grain across Southeast Asia and is of increasing concern in other regions due to the advent of strong resistance to phosphine, the fumigant used to protect stored grain from pest insects. We investigated the inheritance of genes controlling resistance to phosphine in a strongly resistant S. oryzae strain (NNSO7525) collected in Australia and find that the trait is autosomally inherited and incompletely recessive with a degree of dominance of -0.66. The strongly resistant strain has an LC50 52 times greater than a susceptible reference strain (LS2) and 9 times greater than a weakly resistant strain (QSO335). Analysis of F2 and backcross progeny indicates that two or more genes are responsible for strong resistance, and that one of these genes, designated So_rph1, not only contributes to strong resistance, but is also responsible for the weak resistance phenotype of strain QSO335. These results demonstrate that the genetic mechanism of phosphine resistance in S. oryzae is similar to that of other stored product insect pests. A unique observation is that a subset of the progeny of an F1 backcross generation are more strongly resistant to phosphine than the parental strongly resistant strain, which may be caused by multiple alleles of one of the resistance genes.
Project description:High levels of resistance to phosphine in the rice weevil Sitophilus oryzae have been detected in Asian countries including China and Vietnam, however there is limited knowledge of the genetic mechanism of resistance in these strains. We find that the genetic basis of strong phosphine resistance is conserved between strains of S. oryzae from China, Vietnam, and Australia. Each of 4 strongly resistant strains has an identical amino acid variant in the encoded dihydrolipoamide dehydrogenase (DLD) enzyme that was previously identified as a resistance factor in Rhyzopertha dominica and Tribolium castaneum. The unique amino acid substitution, Asparagine > Threonine (N505T) of all strongly resistant S. oryzae corresponds to the position of an Asparagine > Histidine variant (N506H) that was previously reported in strongly resistant R. dominica. Progeny (F16 and F18) from 2 independent crosses showed absolute linkage of N505T to the strong resistance phenotype, indicating that if N505T was not itself the resistance variant that it resided within 1 or 2 genes of the resistance factor. Non-complementation between the strains confirmed the shared genetic basis of strong resistance, which was supported by the very similar level of resistance between the strains, with LC50 values ranging from 0.20 to 0.36 mg L(-1) for a 48-h exposure at 25 °C. Thus, the mechanism of high-level resistance to phosphine is strongly conserved between R. dominica, T. castaneum and S. oryzae. A fitness cost associated with strongly resistant allele was observed in segregating populations in the absence of selection.
Project description:A cDNA clone encoding pectinmethylesterase of the rice weevil, Sitophilus oryzae (L.) has been isolated and sequenced. The cDNA clone was expressed in cultured insect cells and active pectinmethylesterase was purified from the culture medium, thus confirming that the cDNA encodes pectinmethylesterase. In situ hybridization indicated that the enzyme's transcript was present in the midgut. Weevils treated with tetracycline so that they lack genes of known symbiotic organisms still contained the pectinmethylesterase gene, indicating that the gene is encoded by the rice weevil genome. The rice weevil enzyme is most similar in sequence to bacterial pectinmethylesterases. Given this and the enzyme's apparently rather general absence from animal species, we suggest the possibility that this gene was transferred horizontally to an ancient weevil, possibly from a bacterial symbiont, and exists in Sitophilus species now as a result of that ancestral horizontal transfer.
Project description:Tyramine receptor (TyrR) is a biogenic amine G protein-coupled receptor (GPCR) associated with many important physiological functions in insect locomotion, reproduction, and pheromone response. Binding of specific ligands to the TyrR triggers conformational changes, relays the signal to G proteins, and initiates an appropriate cellular response. Here, we monitor the binding effect of agonist compounds, tyramine and amitraz, to a Sitophilus oryzae tyramine receptor (SoTyrR) homology model and their elicited conformational changes. All-atom molecular dynamics (MD) simulations of SoTyrR-ligand complexes have shown varying dynamic behavior, especially at the intracellular loop 3 (IL3) region. Moreover, in contrast to SoTyrR-tyramine, SoTyrR-amitraz and non-liganded SoTyrR shows greater flexibility at IL3 residues and were found to be coupled to the most dominant motion in the receptor. Our results suggest that the conformational changes induced by amitraz are different from the natural ligand tyramine, albeit being both agonists of SoTyrR. This is the first attempt to understand the biophysical implication of amitraz and tyramine binding to the intracellular domains of TyrR. Our data may provide insights into the early effects of ligand binding to the activation process of SoTyrR.
Project description:Infestation of phosphine (PH3) resistant insects threatens global grain reserves. PH3 fumigation controls rice weevil (Sitophilus oryzae) but not highly resistant insect pests. Here, we investigated naturally occurring strains of S. oryzae that were moderately resistant (MR), strongly resistant (SR), or susceptible (wild-type; WT) to PH3 using global proteome analysis and mitochondrial DNA sequencing. Both PH3 resistant (PH3-R) strains exhibited higher susceptibility to ethyl formate-mediated inhibition of cytochrome c oxidase than the WT strain, whereas the disinfectant PH3 concentration time of the SR strain was much longer than that of the MR strain. Unlike the MR strain, which showed altered expression levels of genes encoding metabolic enzymes involved in catabolic pathways that minimize metabolic burden, the SR strain showed changes in the mitochondrial respiratory chain. Our results suggest that the acquisition of strong PH3 resistance necessitates the avoidance of oxidative phosphorylation through the accumulation of a few non-synonymous mutations in mitochondrial genes encoding complex I subunits as well as nuclear genes encoding dihydrolipoamide dehydrogenase, concomitant with metabolic reprogramming, a recognized hallmark of cancer metabolism. Taken together, our data suggest that reprogrammed metabolism represents a survival strategy of SR insect pests for the compensation of minimized energy transduction under anoxic conditions. Therefore, understanding the resistance mechanism of PH3-R strains will support the development of new strategies to control insect pests.
Project description:BACKGROUND: Insects thriving on nutritionally poor habitats have integrated mutualistic intracellular symbiotic bacteria (endosymbionts) in a bacteria-bearing tissue (the bacteriome) that isolates the endosymbionts and protects them against a host systemic immune response. Whilst the metabolic and physiological features of long-term insect associations have been investigated in detail over the past decades, cellular and immune regulations that determine the host response to endosymbionts and pathogens have attracted interest more recently. RESULTS: To investigate bacteriome cellular specificities and weevil immune responses to bacteria, we have constructed and sequenced 7 cDNA libraries from Sitophilus oryzae whole larvae and bacteriomes. Bioinformatic analysis of 26,886 ESTs led to the generation of 8,941 weevil unigenes. Based on in silico analysis and on the examination of genes involved in the cellular pathways of potential interest to intracellular symbiosis (i.e. cell growth and apoptosis, autophagy, immunity), we have selected and analyzed 29 genes using qRT-PCR, taking into consideration bacteriome specificity and symbiosis impact on the host response to pathogens. We show that the bacteriome tissue accumulates transcripts from genes involved in cellular development and survival, such as the apoptotic inhibitors iap2 and iap3, and endosomal fusion and trafficking, such as Rab7, Hrs, and SNARE. As regards our investigation into immunity, we first strengthen the bacteriome immunomodulation previously reported in S. zeamais. We show that the sarcotoxin, the c-type lysozyme, and the wpgrp2 genes are downregulated in the S. oryzae bacteriome, when compared to aposymbiotic insects and insects challenged with E. coli. Secondly, transcript level comparison between symbiotic and aposymbiotic larvae provides evidence that the immune systemic response to pathogens is decreased in symbiotic insects, as shown by the relatively high expression of wpgrp2, wpgrp3, coleoptericin-B, diptericin, and sarcotoxin genes in aposymbiotic insects. CONCLUSIONS: Library sequencing significantly increased the number of unigenes, allowing for improved functional and genetic investigations in the cereal weevil S. oryzae. Transcriptomic analyses support selective and local immune gene expression in the bacteriome tissue and uncover cellular pathways that are of potential interest to bacteriocyte survival and homeostasis. Bacterial challenge experiments have revealed that the systemic immune response would be less induced in a symbiotic insect, thus highlighting new perspectives on host immunity in long-term invertebrate co-evolutionary associations.