Identification and detection of the V1848I indoxacarb resistance mutation in the beet armyworm, Spodoptera exigua.

Indoxacarb is a pivotal insecticide used worldwide to manage Spodoptera exigua, a devastating agricultural pest. This active compound plays a crucial role in resistance management strategies due to its distinctive mode of action. A field population of S. exigua (SH23) from Shanghai, China, exhibited significantly reduced susceptibility to indoxacarb, with a resistance ratio of 113.84-fold in biological assays. Following two rounds of laboratory screening with indoxacarb, the resistance of the new strain (SH23-S2) escalated steeply to 876.15-fold. Genetic analyses of both the SH23 and SH23-S2 strains demonstrated autosomal inheritance and incompletely dominant resistance patterns. Synergist assays indicated a minor role of detoxification enzymes (glutathione s-transferases and cytochrome P450) of SH23-S2 strain in this resistance, implicating target-site resistance as the primary mechanism. To explore the impact of target-site resistance, segment 1-6 of domain IV (IVS1-6) of the sodium channel in S. exigua was cloned, and the sequences from susceptible and indoxacarb-resistant S. exigua were compared. The V1848I mutation, linked to indoxacarb resistance in Plutella xylostella, Tuta absoluta and Liriomyza trifolii, was identified and strongly associated with the indoxacarb-resistant phenotype in the S. exigua SH23-S2 strain, whereas the F1845Y mutation was not detected. Furthermore, a molecular test for the V1848I mutation in field populations was created using an allele-specific PCR (AS-PCR). The discovery of indoxacarb resistance mutation and the creation of diagnostic tool will enable the early detection of indoxacarb resistance, which will facilitate the implementation of targeted resistance management strategies, ultimately delaying the proliferation of resistance.

Identification and functional characterization of the dirigent gene family in Phryma leptostachya and the contribution of PlDIR1 in lignan biosynthesis.

BACKGROUND: Furofuran lignans, the main insecticidal ingredient in Phryma leptostachya, exhibit excellent controlling efficacy against a variety of pests. During the biosynthesis of furofuran lignans, Dirigent proteins (DIRs) are thought to be dominant in the stereoselective coupling of coniferyl alcohol to form ( ±)-pinoresinol. There are DIR family members in almost every vascular plant, but members of DIRs in P. leptostachya are unknown. To identify the PlDIR genes and elucidate their functions in lignan biosynthesis, this study performed transcriptome-wide analysis and characterized the catalytic activity of the PlDIR1 protein. RESULTS: Fifteen full-length unique PlDIR genes were identified in P. leptostachya. A phylogenetic analysis of the PlDIRs classified them into four subfamilies (DIR-a, DIR-b/d, DIR-e, and DIR-g), and 12 conserved motifs were found among them. In tissue-specific expression analysis, except for PlDIR7, which displayed the highest transcript abundance in seeds, the other PlDIRs showed preferential expression in roots, leaves, and stems. Furthermore, the treatments with signaling molecules demonstrated that PlDIRs could be significantly induced by methyl jasmonate (MeJA), salicylic acid (SA), and ethylene (ETH), both in the roots and leaves of P. leptostachya. In examining the tertiary structure of the protein and the critical amino acids, it was found that PlDIR1, one of the DIR-a subfamily members, might be involved in the region- and stereo-selectivity of the phenoxy radical. Accordingly, LC-MS/MS analysis demonstrated the catalytic activity of recombinant PlDIR1 protein from Escherichia coli to direct coniferyl alcohol coupling into ( +)-pinoresinol. The active sites and hydrogen bonds of the interaction between PlDIR1 and bis-quinone methide (bisQM), the intermediate in ( +)-pinoresinol formation, were analyzed by molecular docking. As a result, 18 active sites and 4 hydrogen bonds (Asp-42, Ala-113, Leu-138, Arg-143) were discovered in the PlDIR1-bisQM complex. Moreover, correlation analysis indicated that the expression profile of PlDIR1 was closely connected with lignan accumulations after SA treatment. CONCLUSIONS: The results of this study will provide useful clues for uncovering P. leptostachya's lignan biosynthesis pathway as well as facilitate further studies on the DIR family.

The knockout of the nicotinic acetylcholine receptor subunit gene α1 (nAChR α1) through CRISPR/Cas9 technology exposes its involvement in the resistance of Spodoptera exigua to insecticides.

Insect nicotinic acetylcholine receptors (nAChRs) are the directed targets of many insecticides. However, there have been no reports on the molecular characterization of the nAChR gene family or the causal association between nAChR α1 and resistance to insecticides in S. exigua, which is a significant agricultural pest. In this study, we identified a total of 9 candidate nAChR subunits in S. exigua, namely nAChR α1-α7 and nAChR ß1-ß2. For functional validation roles of Seα1 in insecticide resistance of S. exigua, we introduced a âˆ¼ 1041-bp deletion of the Seα1 gene in a homozygous mutant strain (Seα1-KO) by CRISPR/Cas9 genome editing system, resulting in a premature truncation of the Seα1 protein and the subsequent loss of functional transmembrane (TM) 3 and TM4 elements. Compared with WH-S strain (wild-type strain), the Seα1-KO strain exhibited 2.62-folds resistant to trifluoropyrimidine, 8.3-folds resistant to dimehypo, and 5.28-folds resistant to dinotefuran, but no significant change in susceptibility to emamectin benzoate, spinetoram, lambda-cyhalothrin, permethrin and chlorpyrifos. Thus, this study has laid a solid foundation for investigating the role of nAChRs in S. exigua, and provides evidence for the crucial involvement of the α1 subunit in the mechanism of trifluoropyrimidine, dimehypo, and dinotefuran in S. exigua. Moreover, it provides a reference for the value of Seα1 subunit and its homologues in other species as insecticide targets.

Effects of periplocoside T isolated from Periploca sepium on behavior and sensory-CNS-motor circuits in Drosophila melanogaster larvae.

Periplocoside T (PST) from Periploca sepium has insecticidal activity against some lepidopterans, which can significantly inhibit the activity of vacuolar-type H+-ATPases (V-ATPase). V-ATPase is involved in the release of neurotransmitters in vesicles during nerve signal transduction. However, there are actions of PST on behavior and sensory-central nervous system (CNS)-motor neural circuit which are commonly overlooked. After exposure to 500 mg/L PST for 48 h, the difference of the proportion of larvae responding to stimuli in the four Drosophila strains was not significant as compared to controls, but larval mouth hook movement and body wall motion were significantly decreased as compared to controls, and the decrease was more obvious in parats1; DSC1-/- and DSC1-/- strains, especially in parats1; DSC1-/- strain. Compared with control (DMSO), the excitatory junction potential (EJP) frequencies of sensory-CNS-motor circuits in the four Drosophila strains after PST or bafiloymcin A1 (BA1, a V-ATPase specific inhibitor) treatment gradually decreased with time, and the decreasing amplitude of BA1 treatment was greater than that of PST treatment, but both were higher than that of the control. The decay amplitude of EJP frequency in two strains with DSC1 channel knockout was lower than that of w1118 and parats1 strains without DSC1 channel knockout. Thus, the results indicated that PST, similar to BA1, could inhibit the transmission of sensory-CNS-motor circuit excitability of Drosophila larvae by inhibiting the activity of V-ATPase, and DSC1 channel play a role of in regulating the stability of nervous system.

Design, synthesis and insecticidal activities of 4-propargyloxybenzene sulfonamide derivatives substituted with amino acids.

Sixty-nine 4-propargyloxybenzene sulfonamide derivatives with different amino acids as amino substituent were synthesized and evaluated for their insecticidal activity against third-instar Mythimna separate. The bioassay results revealed that some derivatives bearing amino acid ester group performed good insecticidal activity against third-instar M.separata, such as the LC50 values of D18 and D19 were 4.28 and 2.96 mg/ml after 48 h, in particular, the LC50 of D16 was 2.38 mg/ml and the activity was improved by 14 times compared to celangulin V (34.48 mg/ml). The above results provided theoretical and experimental basis for the discovery of novel insecticidal active compounds.

De novo leaf and root transcriptome analysis to explore biosynthetic pathway of Celangulin V in Celastrus angulatus maxim.

BACKGROUND: Celastrus angulatus Maxim is a kind of crucial and traditional insecticidal plant widely distributed in the mountains of southwest China. Celangulin V is the efficient insecticidal sesquiterpenoid of C. angulatus and widely used in pest control in China, but the low yield and discontinuous supply impeded its further popularization and application. Fortunately, the development of synthetic biology provided an opportunity for sustainable supply of Celangulin V, for which understanding its biosynthetic pathway is indispensable. RESULTS: In this study, six cDNA libraries were prepared from leaf and root of C. angulatus before global transcriptome analyses using the BGISEQ-500 platform. A total of 104,950 unigenes were finally obtained with an average length of 1200 bp in six transcriptome databases of C. angulatus, in which 51,817 unigenes classified into 25 KOG classifications, 39,866 unigenes categorized into 55 GO functional groups, and 48,810 unigenes assigned to 135 KEGG pathways, 145 of which were putative biosynthetic genes of sesquiterpenoid and triterpenoid. 16 unigenes were speculated to be related to Celangulin V biosynthesis. De novo assembled sequences were verified by Quantitative Real-Time PCR (qRT-PCR) analysis. CONCLUSIONS: This study is the first report on transcriptome analysis of C. angulatus, and 16 unigenes probably involved in the biosynthesis of Celangulin V were finally collected. The transcriptome data will make great contributions to research for this specific insecticidal plant and the further gene mining for biosynthesis of Celangulin V and other sesquiterpene polyol esters.

Insecticidal Activity of Four Lignans Isolated from Phryma leptostachya.

A new lignan (T4) and three known lignans (T1, T2, and T3) were isolated from the methanol extract of the roots of Phryma leptostachya using bioassay-guided method, and their structures were identified as phrymarolin I (T1), II (T2), haedoxan A (T3), and methyl 4-((6a-acetoxy-4-(6-methoxybenzo[d][1,3]dioxol-5-yl)tetrahydro-1H,3H-furo[3,4-c]furan-1-yl)oxy)-1-hydroxy-2,2-dimethoxy-5-oxocyclopent-3-ene-1-carboxylate (T4) byNMR and ESI-MS spectral data. Bioassay results revealed that haedoxan A exhibited remarkably high insecticidal activity against Mythimna separata with a stomach toxicity LC50 value of 17.06 mg/L and a topical toxicity LC50 value of 1123.14 mg/L at 24 h, respectively. Phrymarolin I and compound T4 also showed some stomach toxicity against M. separata with KD50 values of 3450.21 mg/L at 4 h and 2807.10 mg/L at 8 h, respectively. In addition, phrymarolin I and haedoxan A exhibited some stomach toxicity against Plutella xylostella with an LC50 value of 1432.05 and 857.28 mg/L at 48 h, respectively. In conclusion, this study demonstrated that lignans from P. leptostachya are promising as a novel class of insecticides or insecticide lead compounds for developing botanical pesticides.

DSC1 channel-dependent developmental regulation of pyrethroid susceptibility in Drosophila melanogaster.

Pyrethroid insecticides modify the gating of voltage-gated sodium channels, thus disrupting the function of the nervous system. In Drosophila melanogaster, para encodes a functional sodium channel. Drosophila Sodium Channel 1 (DSC1), although considered as a putative sodium channel gene for decades due to its high sequence similarity with sodium channels, encodes a voltage-gated cation channel with high permeability to Ca2+. Previous study showed that knockout of the DSC1 gene (DSC1-/-) caused Drosophila adults to be more susceptible to pyrethroids and the adult giant fiber (GF) neural circuit were more susceptible to pyrethroids. Considering distinct expression of DSC1 transcripts in adults and larvae, we examined the role of DSC1 channels in regulating pyrethroid susceptibility in Drosophila larvae. We conducted insecticide bioassays and examined the susceptibility of the larval neuromuscular junction (NMJ) to pyrethroids using w1118, an insecticide-susceptible line, DSC1-/-, parats1 (a pyrethroid-resistant line carrying a mutation in para) and a double mutation line parats1; DSC1-/-. We found that, like the adult GF system, the NMJ of DSC1-/- flies is more susceptible to pyrethroids than that of w1118 with the pyrethroid susceptibility ranked as DSC1-/- > w1118 > parats1; DSC1-/- > parats1. However, DSC1-/- larvae were about two-fold more resistant to pyrethroids than w1118 larvae, and the pyrethroid susceptibility of larvae ranked as w1118 > DSC1-/- > parats1; DSC1-/- > parats1. These results reveal common and distinct roles of DSC1 channels in regulating the action of pyrethroids in adults and larvae of D. melanogaster.

Molecular evidence for dual pyrethroid-receptor sites on a mosquito sodium channel.

Pyrethroid insecticides are widely used as one of the most effective control measures in the global fight against agricultural arthropod pests and mosquito-borne diseases, including malaria and dengue. They exert toxic effects by altering the function of voltage-gated sodium channels, which are essential for proper electrical signaling in the nervous system. A major threat to the sustained use of pyrethroids for vector control is the emergence of mosquito resistance to pyrethroids worldwide. Here, we report the successful expression of a sodium channel, AaNav1-1, from Aedes aegypti in Xenopus oocytes, and the functional examination of nine sodium channel mutations that are associated with pyrethroid resistance in various Ae. aegypti and Anopheles gambiae populations around the world. Our analysis shows that five of the nine mutations reduce AaNav1-1 sensitivity to pyrethroids. Computer modeling and further mutational analysis revealed a surprising finding: Although two of the five confirmed mutations map to a previously proposed pyrethroid-receptor site in the house fly sodium channel, the other three mutations are mapped to a second receptor site. Discovery of this second putative receptor site provides a dual-receptor paradigm that could explain much of the molecular mechanisms of pyrethroid action and resistance as well as the high selectivity of pyrethroids on insect vs. mammalian sodium channels. Results from this study could impact future prediction and monitoring of pyrethroid resistance in mosquitoes and other arthropod pests and disease vectors.

Effects of Celangulin IV and V From Celastrus angulatus Maxim on Na+/K+-ATPase Activities of the Oriental Armyworm (Lepidoptera: Noctuidae).

Na(+)/K(+)-ATPase (sodium pump) is an important target for the development of botanical pesticide as it is responsible for transforming chemical energy in ATP to osmotic work and maintaining electrochemical Na(+ )and K(+ )gradients across the cell membrane of most animal cells. Celangulin IV (C-IV) and V (C-V), which are isolated from the root bark of Celastrus angulatus, are the major active ingredients of this insecticidal plant. The activities of C-IV and C-V on Na(+)/K(+)-ATPase were investigated by ultramicro measuring method to evaluate the effects of C-IV and C-V on Na(+)/K(+)-ATPase activities of the brain from the fifth Mythimna separata larvae and to discuss the insecticidal mechanism of C-IV and C-V. Results indicate that inhibitory activities of Na(+)/K(+)-ATPase by C-IV and C-V possess an obvious concentration-dependent in vitro. Compared with C-IV, the inhibition of C-V on Na(+)/K(+)-ATPase was not striking. In vivo, at a concentration of 25 mg/liter, the inhibition ratio of C-IV on Na(+)/K(+)-ATPase activity from the brain in narcosis and recovery period was more remarkable than that of C-V. Furthermore, the insects were fed with different mixture ratios of C-IV and C-V. The inhibition extent of Na(+)/K(+)-ATPase activity was corresponded with the dose of C-IV. However, C-V had no notable effects. This finding may mean that the mechanism of action of C-IV and C-V on Na(+)/K(+)-ATPase were different. Na(+)/K -ATPase may be an action target of C-IV and C-V.

Histopathological effects and immunolocalization of periplocoside NW from Periploca sepium Bunge on the midgut epithelium of Mythimna separata Walker larvae.

Periplocoside NW (PSNW) with pregnane glycoside skeleton is a novel insecticidal compound isolated from the root bark of Periploca sepium Bunge. This compound has a potent stomach poisoning activity against several insect pests. In this study, we observed the intoxication symptoms, investigated the histopathological effects and carried out immuno-electron microscopic localization of PSNW on the midgut epithelium of oriental armyworm Mythimna separata Walker larvae for better understanding its action mechanism against insects. Ultrastructural observations showed that cell damages caused by PSNW in the midgut of M. separata larvae are related to the degeneration of brush border microvilli. The dissolution of cytoskeletal structures in the interior and on the surface of microvilli was responsible for the decrease in size and eventual disappearance of microvilli when bubbles of cytoplasmic substances protrude into the midgut lumen of M. separata, thus resulting in cell death. The immuno-electron microscopic localization research showed that gold particle appeared on the microvilli layer of the midgut of M. separate larvae firstly. The density of gold particle gradually added with the time, and finally microvilli layer was destructed severely. Meantime, the gold particles were also presented to the intracellular organelle membrane and the organelles also were destructed. Therefore, we proposed that this membrane system on insect midgut epithelium cells is the initial acting site of PSNW against insects.

Discovery of the Chlorinated and Ammoniated Derivatives of Vanillin as Potential Insecticidal Candidates Targeting V-ATPase: Structure-Based Virtual Screening, Synthesis, and Bioassay.

Vacuolar-type H+-ATPases (V-ATPases) play a crucial role in the life cycle of agricultural pests and represent a promising target for the development of novel insecticides. In this study, S18, a derivative of vanillin acquired from Specs database using a structure-based virtual screening methodology, was first identified as a V-ATPase inhibitor. It binds to subunit A of the enzyme with a Kd of 1 nM and exhibits insecticidal activity against M. separata. Subsequently, using S18 as the lead compound, a new series of vanillin derivatives were rationally designed and efficiently synthesized. and their biological activities were assessed. Among them, compound 3b-03 showed the strongest insecticidal activity against M. separata by effectively targeting the V-ATPase subunit A with Kd of 0.803 µM. Isothermal titration calorimetric measurements and docking results provided insights into its interaction with subunit A of V-ATPase, which could facilitate future research aimed at the development of novel chemical insecticides.

Single Electron Transfer Reductive Deuteration of Acyl Chlorides for the Synthesis of Deuterated Alcohols with a High Deuterium Atom Economy.

We present a highly deuterium atom economical approach for the synthesis of deuterated alcohols via the single electron transfer (SET) reductive deuteration of acyl chlorides. Cost-effective sodium dispersion and EtOD-d1 were used as the single electron donor and deuterium donor, respectively. Our approach achieved up to 49% deuterium atom economy, which represents the highest deuterium atom economy yet achieved in SET reductive deuteration reactions. With all 20 tested substrates, excellent regioselectivity and >92% deuterium incorporations were obtained. Furthermore, we demonstrated the potential of this methodology by synthesizing four deuterated analogues of pesticides.

Synthesis and larvicidal activity against Culex pipiens pallens of new triazole derivatives of phrymarolin from Phryma leptostachya L.

Twelve new triazole derivatives of Phrymarolin were prepared from Phrymarolin I and the structures of all the derivatives were fully characterized by (1)H-NMR, (13)C-NMR and MS spectral data analyses. Larvicidal activities against 4rd instar larvae of Culex pipiens pallens of these Phrymarolin analogues were assayed. Although the triazole derivatives of Phrymarolin showed certain larvicidal activity, they showed lower activity than Phrymarolin I. The typical non-natural groups triazole substituents reduced the larvicidal activity of Phrymarolin derivatives.

Periplocoside P affects synaptic transmission at the neuromuscular junction and reduces synaptic excitability in Drosophila melanogaster by inhibiting V-ATPase.

BACKGROUND: Periplocoside P (PSP) is a major component of Periploca sepium Bunge known for its potent insecticidal activity. V-Type adenosine triphosphatase (V-ATPase), which is widely distributed in the cytoplasmic membranes and organelles of eukaryotic cells, plays a crucial role in synaptic excitability conduction. Previous research has shown that PSP targets the apical membrane of goblet cells in the insect midgut. However, the effects of PSP on synaptic transmission at the neuromuscular junction are often overlooked. RESULTS: The bioassay revealed that Drosophila adults with different genetic backgrounds showed varying levels of susceptibility to PSP in the order: parats1 > parats1 ;DSC1-/- ≈ w1118 > DSC1-/- . Intracellular electrode recording demonstrated that PSP, similar to bafilomycin A1, had an impact on the amplitude of the excitatory junction potential (EJP) and accelerated excitability decay. Furthermore, the alteration in EJP amplitude is concentration-dependent. Another surprising discovery was that the knockout DSC1 channel showed insensitivity to PSP. CONCLUSION: Our findings confirm that PSP can influence synaptic transmission at the neuromuscular junction of Drosophila larvae by targeting V-ATPase. These results provide a basis for investigating the mechanism of action of PSP and its potential application in designing novel insecticides. © 2023 Society of Chemical Industry.

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.

Functional validation of CYP304A1 associated with haedoxan A detoxification in Aedes albopictus by RNAi and transgenic drosophila.

BACKGROUND: Insect cytochrome P450 monooxygenases play important roles in the detoxification metabolism of endogenous and exogenous compounds. Haedoxan A (HA) from Phryma leptostachya L. is a highly efficient natural pesticide used to control houseflies and mosquitos. CYP4C21 and CYP304A1 were previously demonstrated to be transcriptionally increased in Aedes albopictus in response to HA exposure, but their involvement in HA metabolism is unknown. RESULTS: Our data showed that CYP304A1 expression levels in A. albopictus were highest in third-instar larvae, and the expression level of CYP4C21 decreased significantly with the growth of instars, with the lowest occurring in the pupal stage. Compared with the control, the silencing of CYP304A1 and CYP4C21 genes by chitosan nanoparticle-mediated RNA interference could deplete 58.2% and 54.0% of the expression of corresponding genes, respectively. The bioassay data showed that knocking down the expression of CYP304A1 increased the mortality of A. albopictus when exposed to HA at LC30 and LC50 doses, but did not significantly increase mortality after silencing CYP4C21. Our data demonstrated that CYP304A1, but not CYP4C21, may be involved in HA detoxification. Moreover, the resistance ratio of CYP304A1 overexpressing flies was approximately 2-fold higher than that of the control line. The metabolized product of HA by CYP304A1 needs to be further confirmed by in vitro expression. CONCLUSION: This finding showed that inducibility was not always linked to detoxifying capabilities, and enhanced our understanding of the molecular basis of HA metabolic detoxification in A. albopictus. © 2022 Society of Chemical Industry.

Larvicidal activity of lignans from Phryma leptostachya L. against Culex pipiens pallens.

The larvicidal activity of crude petroleum ether, ethyl acetate, and methanol extracts of the whole plants of Phryma leptostachya L. was assayed for its toxicity against the early fourth instar larvae of Culex pipiens pallens. The larval mortality was observed after 24 h of exposure. Among three solvent extracts from Phyrma leptostachya L., the petroleum ether extract exhibited the best larvicidal activity. The corresponding LC50 values of petroleum ether, ethyl acetate, and methanol extracts were 3.23, 5.23, and 61.86 ppm against the early fourth instar larvae of Culex pipiens pallens. The petroleum ether extract was successively subjected to column chromatography and preparative high performance liquid chromatography, and yielded the three lignans, phrymarolin-I, haedoxane A, and haedoxane E, which were isolated and identified as new mosquito larvicidal compounds. Phrymarolin-I, haedoxane A, and haedoxane E showed high larvicidal activity, for which the lethal doses LC50 were estimated at 1.21, 0.025, and 0.15 ppm against the early fourth instar larvae of Culex pipiens pallens, respectively. The structures were elucidated by analyses of IR, UV, MS, and NMR spectral data. This is the first report on the mosquito larvicidal activity of the three compounds, phrymarolin-I, haedoxane A, and haedoxane E from Phyrma leptostachya L.

[Diversity and antimicrobial activities of actinomycetes from pesticide-contaminated spots in Shandong Peninsula].

OBJECTIVE: In order to study diversity and find antimicrobial activities of actinomycetes from pesticide-contaminated spots in Shandong Peninsula. METHODS: The phylogenetic analysis of 154 isolated strains was done based on 16SrDNA sequences. Antimicrobial activities of 10 non-Streptomyces strains were tested by using cylinder-plate method and hypha growth rate method. RESULTS: Among the strains, 154 strains belonged to 7 families, 8 genera: Streptomyces (87.01%), Kocuria, Microbacterium, Nocardiopsis, Knoellia, Pseudonocardia, Micromonospora, Actinoplanes. The fermentation broths of 10 non-Streptomyces strains had inhibitory actives against all tested phytopathogenic fungi (Botrytis cinerea, Fusarium oxysporum f. sp. niveum, Gibberella zeae, Sclerotinia sclerotiorum, Colletotrichum gloesporioides) and bacteria (Staphylococcus aureus, Bacillus subtills, Bacillus cereus, Escherichai coli, Pseudomonas aeruginos) , especially Microbacterium oxydans JN853773 and Kocuria rosea JN192402 had strong inhibitory effects. CONCLUSION: Abundant diversity of actinomycetes existed in pesticide contaminated spots in Shandong Peninsula. Microbacterium oxydans JN853773 and Kocuria rosea JN192402 showed high antimicrobial activities and could be further exploited.

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: w1118 , an insecticide-susceptible strain, and parats1 , 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 parats1 larvae and adults were more resistant to permethrin, as expected. However, both parats1 larvae and adults were more sensitive to HA compared to w1118 . We confirmed that the I265N mutation reduced the sensitivity to permethrin of a Drosophila sodium channel variant, DmNav 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 parats1 flies that is responsible for the NCR between permethrin and HA at the whole insect level.