Chimeric Investigations into the Diamide Binding Site on the Lepidopteran Ryanodine Receptor.

Alterations to amino acid residues G4946 and I4790, associated with resistance to diamide insecticides, suggests a location of diamide interaction within the pVSD voltage sensor-like domain of the insect ryanodine receptor (RyR). To further delineate the interaction site(s), targeted alterations were made within the same pVSD region on the diamondback moth (Plutella xylostella) RyR channel. The editing of five amino acid positions to match those found in the diamide insensitive skeletal RyR1 of humans (hRyR1) in order to generate a human-Plutella chimeric construct showed that these alterations strongly reduce diamide efficacy when introduced in combination but cause only minor reductions when introduced individually. It is concluded that the sites of diamide interaction on insect RyRs lie proximal to the voltage sensor-like domain of the RyR and that the main site of interaction is at residues K4700, Y4701, I4790 and S4919 in the S1 to S4 transmembrane domains.

Evolutionary trade-offs of insecticide resistance - The fitness costs associated with target-site mutations in the nAChR of Drosophila melanogaster.

The evolution of resistance to drugs and pesticides poses a major threat to human health and food security. Neonicotinoids are highly effective insecticides used to control agricultural pests. They target the insect nicotinic acetylcholine receptor and mutations of the receptor that confer resistance have been slow to develop, with only one field-evolved mutation being reported to date. This is an arginine-to-threonine substitution at position 81 of the nAChR_ß1 subunit in neonicotinoid-resistant aphids. To validate the role of R81T in neonicotinoid resistance and to test whether it may confer any significant fitness costs to insects, CRISPR/Cas9 was used to introduce an analogous mutation in the genome of Drosophila melanogaster. Flies carrying R81T showed an increased tolerance (resistance) to neonicotinoid insecticides, accompanied by a significant reduction in fitness. In comparison, flies carrying a deletion of the whole nAChR_α6 subunit, the target site of spinosyns, showed an increased tolerance to this class of insecticides but presented almost no fitness deficits.

Diamide insecticide resistance in transgenic Drosophila and Sf9-cells expressing a full-length diamondback moth ryanodine receptor carrying an I4790M mutation.

BACKGROUND: Resistance to diamide insecticides in Lepidoptera is known to be caused primarily by amino acid changes on the ryanodine receptor (RyR). Recently, two new target site mutations, G4946V and I4790M, have emerged in populations of diamondback moth, Plutella xylostella, as well as in other lepidopteran species, and both mutations have been shown empirically to decrease diamide efficacy. Here, we quantify the impact of the I4790M mutation on diamide activation of the receptor, as compared to alterations at the G4946 locus. RESULTS: I4790M when introduced into P. xylostella RyR expressed in an insect-derived Sf9 cell line was found to mediate just a ten-fold reduction in chlorantraniliprole efficacy (compared to 104- and 146-fold reductions for the G4946E and G4946V variants, respectively), whilst in the field its presence is associated with a ≥150-fold reduction. I4790M-mediated resistance to flubendiamide was estimated to be >24-fold. When the entire coding sequence of P. xylostella RyR was integrated into Drosophila melanogaster, the I4790M variant conferred ~4.4-fold resistance to chlorantraniliprole and 22-fold resistance to flubendiamide in the 3rd instar larvae, confirming that it imparts only a moderate level of resistance to diamide insecticides. Although the I4790M substitution appears to bear no fitness costs in terms of the flies' reproductive capacity, when assessed in a noncompetitive environment, it does, however, have potentially major impacts on mobility at both the larval and adult stages. CONCLUSIONS: I4790M imparts only a moderate level of resistance to diamide insecticides and potentially confers significant fitness costs to the insect.

An analysis of variability in genome organisation of intracellular calcium release channels across insect orders.

Using publicly available genomic data, combined with RT-PCR validation, we explore structural genomic variation for two major ion channels across insect classes. We have manually curated ryanodine receptor (RyR) and inositol 1,4,5-trisphosphate receptor (IP3R) ORFs and their corresponding genomic structures from 26 different insects covering major insect orders. We found that, despite high protein identity for both RyRs (>75%) and IP3Rs (~67%), the overall complexity of the gene structure varies greatly between different insect orders with the simplest genes (fewest introns) found in Diptera and the most complex in Lepidoptera. Analysis of intron conservation patterns indicated that the majority of conserved introns are found close to the 5' end of the channels and in RyR around the highly conserved mutually exclusive splice site. Of the two channels the IP3Rs appear to have a less well conserved organisation with a greater overall number of unique introns seen between insect orders. We experimentally validated two of the manually curated ORFs for IP3Rs and confirmed an atypical (3799aa) IP3R receptor in Myzus persicae, which is approximately 1000 amino acids larger than previously reported for IP3Rs.