Synthesis and Structure Elucidation of Celangulin Derivatives

Celangulins are sesquiterpene polyol esters based on a core structure known as β-dihydroagarofuran from Celastrus angulatus Max. Some of them are environment acceptable-pesticides. In order to find new active compounds and study their SAR, sesquiterpene polyol esters libraries were synthesized based on the basic hydrolysis of the extract of the root bark of Celastrus angulatus which could offer the β-dihydroagarofuran polyol by combinatorial chemical method and the compounds with insecticidal activity were isolated by bioassay-guided fractionation. Two new compounds with insecticidal activity against Mythimna separata, were isolated and elucidated as 2β,6α,8β, 13-tetraisobutanoyloxy-1β,4,9α-trihydroxy-β-dihydroagarofuran (A) and 1β,2β,6α,8β, 13-pentaisobutanoyloxy-4α,9α-dihydroxy-β-dihydroagarofuran (B) mainly by 1H NMR, 13C NMRH-HCOSY, HMQC, HMBC and HRMS spectral data. A: [α]20D -7.3 (c 0.12, CHCl3), B: [α]20D -34.6 (c 0.8,CHCl3).……

Role of DSC1 in Drosophila melanogaster synaptic activities in response to haedoxan A

Drosophila sodium channel 1 (DSC1) encodes a voltage-gated divalent cation channel that mediates neuronal excitability in insects. Previous research revealed that DSC1 knockout Drosophila melanogaster conferred different susceptibility to insecticides, which indicated the vital regulation role of DSC1 under insecticide stress. Haedoxan A (HA) is a lignan compound isolated from Phryma leptostachya, and we found that HA has excellent insecticidal activity and is worthy of further study as a botanical insecticide. Herein, we performed bioassay and electrophysiological experiments to test the biological and neural changes in the larval Drosophila with/without DSC1 knockout in response to HA. Bioassay results showed that knockout of DSC1 reduced the sensitivity to HA in both w1118 (a common wild-type strain in the laboratory) and parats1 (a pyrethroid-resistant strain) larvae. Except for parats1/DSC1−/−, electrophysiology results implicated that HA delayed the decay rate and increased the frequency of miniature excitatory junctional potentials of Drosophila from w1118, parats1, and DSC1−/− strains. Moreover, the neuromuscular synapse excitatory activities of parats1/DSC1−/− larvae were more sensitive to HA than DSC1−/− larvae, which further confirmed the functional contribution of DSC1 to neuronal excitability. Collectively, these results indicated that the DSC1 channel not only regulated the insecticidal activity of HA, but also maintained the stability of neural circuits through functional interaction with voltage-gated sodium channels. Therefore, our study provides useful information for elucidating the regulatory mechanism of DSC1 in the neural system of insects involving the action of HA derived from P. leptostachya.

Negative cross-resistance of a pyrethroid-resistant Drosophila mutant to Phryma leptostachya-derived haedoxan A

Voltage-gated sodium channels are the primary target of pyrethroid insecticides.Mutations in sodium channel confer knockdown resistance(kdr)to pyrethroids in various arthropod pests.Haedoxan A(HA)is the major insecticidal component from Phryma leptostachya.It has been shown that HA alters electrical responses at the Drosophila neuromuscular junction and modifies the gating properties of cockroach sodium channels expressed in Xenopus oocytes.However,whether sodium channel mutations that confer pyrethroid resistance also affect the action of HA is unknown.In this study,we conducted bioassays using HA and permethrin in two Drosophila melanogaster strains:w^(1118),an insecticide-susceptible strain,and para^(tsl),a pyrethroid-resistant strain due to a I265N mutation in the sodium channel,and identified a new case of negative cross-resistance(NCR)between permethrin and HA.Both para^(tsl) larvae and adults were more resistant to permethrin,as expected.However,both para^(tsl) larvae and adults were more sensitive to HA compared to w^(1118).We confirmed that the I265N mutation reduced the sensitivity to permethrin of a Drosophila sodium channel variant,DmNa_(v)22,expressed in Xenopus oocytes.Interestingly,the I265N mutation also abolished the effect of HA on sodium channels.Further characterization showed that I265 on the sodium channels is critical for the action of both pyrethroids and HA on sodium channels,pointing to an overlapping mode of action between pyrethroids and HA on the sodium channel.Overall,our results suggest an I265N-independnt mechanism(s)in para^(tsl) flies that is responsible for the NCR between permethrin and HA at the whole insect level.

Knockin of the G275E mutation of the nicotinic acetylcholine receptor (nAChR) α6 confers high levels of resistance to spinosyns in Spodoptera exigua

Spinosyns,including spinosad and spinetoram,act on the insect central nervous system,gradually paralyzing or destroying the target insect.Spinosad resistance is associated with loss-of-function mutations in the nicotinic acetylcholine receptor(nAChR)α6 subunit in a number of agricultural pests.Using gene editing,nAChRα6 has been verified as a target for spinosyns in five insect species.Recently,a point mutation(G275E)in exon 9 of nAChRα6 was identified in spinosad-resistant strains of Thrips palmi and Tuta absoluta.To date,no in vivo functional evidence has been obtained to support that this mutation is involved in spinosyn resistance in lepidopteran pests.In this study,the G275E mutation was introduced into the nAChR of Spodoptera exigua using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)gene-editing technology.Reverse transcriptase-polymerase chain reaction and sequencing confirmed that this mutation was present in exon 9 of the nAChR transcripts in the edited 275E strain.The results of bioassays showed that the 275E strain was highly resistant to spinosad(230-fold)and spinetoram(792-fold)compared to the unedited background strain,directly confirming that the G275E mutation of the nAChRα6 subunit confers high levels of spinosyn resistance in S.exigua.Inheritance analysis showed that the resistance trait is autosomal and incompletely recessive.This study employs a reverse genetics approach to validate the functional role played by the G275E mutation in nAChRα6 of S.exigua in spinosyns resistance and provides another example of the use of CRISPR/Cas9 gene-editing technology to confirm the role played by candidate target site mutations in insecticide resistance.