The development of an egg-soaking method for delivering dsRNAs into spider mites.

Recently, the first sprayable RNAi biopesticide, Ledprona, against the Colorado potato beetle, Leptinotarsa decemlineata, has been registered at the United States Environmental Protection Agency. Spider mites (Acari: Tetranychidae), a group of destructive agricultural and horticultural pests, are notorious for rapid development of insecticide/acaricide resistance. The management options, on the other hand, are extremely limited. RNAi-based biopesticides offer a promising control alternative to address this emerging issue. In this study, we i) developed an egg-soaking dsRNA delivery method; ii) evaluated the factors influencing RNAi efficiency, and finally iii) investigated the potential mode of entry of this newly developed egg-soaking RNAi method. In comparison to other dsRNA delivery methods, egg-soaking method was the most efficient, convenient/practical, and cost-effective method for delivering dsRNAs into spider mites. RNAi efficiency of this RNAi method was affected by target genes, dsRNA concentration, developmental stages, and mite species. In general, the hawthorn spider mite, Amphitetranychus viennensis, is more sensitive to RNAi than the two-spotted spider mite, Tetranychus urticae, and both of them have dose-dependent RNAi effect. For different life stages, egg and larvae are the most sensitive life stages to dsRNAs. For different target genes, there is no apparent association between the suppression level and the resultant phenotype. Finally, we demonstrated that this egg-soaking RNAi method acts as both stomach and contact toxicity. Our combined results demonstrate the effectiveness of a topically applied dsRNA delivery method, and the potential of a spray induced gene silencing (SIGS) method as a control alternative for spider mites.

Unveiling fenpropathrin resistance levels in field populations of Tetranychus cinnabarinus (Boisduval): Insights, risks, and RNAi strategy.

Indoor cases of Tetranychus cinnabarinus displaying resistance have been documented, but the resistance level in field populations remains unexplored in China. This study delves into the resistance dynamics of T. cinnabarinus to fenpropathrin in various field populations across China, a pressing concern in contemporary agricultural pest control. The conventional bioassay and amplicon sequencing reveal a notable absence of significant fenpropathrin resistance in field populations, contrasting with known resistance in indoor cases. Current study highlights the limitations of traditional bioassays in detecting early-stage resistance and underscores the nuanced capabilities and constraints of amplicon sequencing in resistance gene frequency analysis. By employing an integrated approach, we combined dose-response bioassays, amplicon sequencing, and statistical modeling to assess resistance levels and investigate underlying genetic factors. The model with empirical data indicates that a 5% mutation frequency represents the threshold before resistance emerges. However, the detection of the kdr mutation in certain populations ranging from 0 to 1.2%, signals an early looming threat of future resistance emergence. Additionally, we further assessed a specific dsRNA targeting VGSC genes at two concentrations (10 ng/µL and 100 ng/µL), both inducing substantial mortality by silencing target genes effectively. The exploration of RNA interference (RNAi) as a novel, more environmentally friendly pest control measure opens new avenues, despite the ongoing challenge of resistance evolution. Overall, this study underscores the necessity for evolving pest management strategies, integrating advanced biotechnological approaches with traditional methods, to effectively counter pesticide resistance and ensure sustainable agricultural productivity.

Effects of the novel acaricide acynonapyr on the calcium-activated potassium channel.

Resistance to insecticides and acaricides is a major impediment to effectively controlling insect pests worldwide. These pests include the two-spotted spider mite Tetranychus urticae (T. urticae), which exists globally. This polyphagous herbivore causes major agricultural problems and can develop resistance to the agents above. Therefore, the continuous development of acaricides with new modes of action is important to circumvent the resistance of insects to pesticides. Acynonapyr is a novel class of acaricides containing an azabicyclo ring. In this study, we determined the activity of acynonapyr and its analogs on calcium-activated potassium (KCa2) channels in two-spotted spider mites using electrophysiological techniques (patch-clamp). We also examined their acaricidal efficacy against mites in the laboratory. The acynonapyr and analogs blocked T. urticae KCa2 (TurKCa2) channels in a concentration-dependent manner. A comparison of acaricidal activity against T. urticae with inhibitory activity against TurKCa2 revealed that TurKCa2 channels are the primary toxicological targets. Finally, we examined the effect of acynonapyr on Homo sapiens KCa2 (HsaKCa2.2) channels and demonstrated that the compound at 10 µM had a limited effect on the activity of this channel.

The cytochrome P450 subfamilies CYP392A and CYP392D are key players in acaricide metabolism in Tetranychusurticae.

The spider mite Tetranychus urticae is a major agricultural pest with a global distribution, extremely diverse host range and a remarkable ability to develop resistance to a wide variety of acaricides. P450 mono-oxygenases have been frequently associated with resistance development in this species. In particular enzymes of the CYP392A-subfamily were shown to metabolize a number of key acaricides, including abamectin, amitraz, fenpyroximate and the active metabolite of pyflubumide. However, transcriptomic studies comparing highly resistant and susceptible populations have often revealed high expression of members of the CYP392D-subfamily, but these have been only poorly studied. Here, we conducted a meta-analysis of gene expression data of 20 populations and identified two key enzymes of this family, CYP392D2 and CYP392D8, whose expression is associated with resistance. We subsequently functionally expressed these enzymes, together with CYP392A11 and CYP392A16 as known metabolizers, and compared their potential to accept a wide diversity of acaricides as substrate. This study overall confirms previous discovered substrates for CYP392A11 and CYP392A16, but also reveals unreported metabolic activity towards new acaricides. These include carbaryl, chlorpyrifos and etoxazole for CYP392A16 and carbaryl, chlorpyrifos and NNI-0711-NH pyflubumide for CYP392A11. For the newly studied CYP392D-family, we show that CYP392D2 metabolizes pyridaben, fenpyroximate, etoxazole and chlorpyrifos, while CYP392D8 metabolizes carbaryl, fenazaquin and tebufenpyrad. Last, we observed that both CYP392A- and CYP392D-subfamily enzymes activate chlorpyrifos to its corresponding oxon. Our study indicates that there is both overlap and specificity in the activity of A- and D-subfamily enzymes against acaricides and model substrates. With the recent advent of highly efficient CRISPR/Cas9 gene editing protocols in T. urticae, the way is now paved to conduct further genetic experiments revealing and quantifying the role of these enzymes in the resistance phenotype in field populations.

Overexpression of a nuclear receptor HR96 contributes to spirodiclofen susceptibility in Panonychus citri (McGregor).

The citrus red mite, Panonychus citri, is one of the most notorious and devastating citrus pests around the world that has developed resistance to multiple chemical acaricides. In previous research, we found that spirodiclofen-resistant is related to overexpression of P450, CCE, and ABC transporter genes in P. citri. However, the regulatory mechanisms of these detoxification genes are still elusive. This study identified all hormone receptor 96 genes of P. citri. 8 PcHR96 genes contained highly conserved domains. The expression profiles showed that PcHR96h was significantly upregulated in spirodiclofen resistant strain and after exposure to spirodiclofen. RNA interference of PcHR96h decreased expression of detoxification genes and increased spirodiclofen susceptibility in P. citri. Furthermore, molecular docking, heterologous expression, and drug affinity responsive target stability demonstrated that PcHR96h can interact with spirodiclofen in vitro. Our research results indicate that PcHR96h plays an important role in regulating spirodiclofen susceptibility and provides theoretical support for the resistance management of P. citri.

A novel spider venom peptide from the predatory mite Neoseiulus barkeri shows lethal effect on phytophagous pests.

The long-term use of pesticides in the field, and the high fertility and adaptability of phytophagous mites have led to resistance problems; consequently, novel safe and efficient active substances are necessary to broaden the tools of pest mite control. Natural enemies of arthropods typically secrete substances with paralytic or lethal effects on their prey, and those substances are a resource for future biopesticides. In this study, two putative venom peptide genes were identified in a parasitic mite Neoseiulus barkeri transcriptome. Recombinant venom NbSP2 peptide injected into Tetranychus cinnabarinus mites was significantly more lethal than recombinant NBSP1. NbSP2 was also lethal to Spodoptera litura when injected but not when fed to third instar larvae. The interaction proteins of NbSP2 in T. cinnabarinus and S. litura were identified by affinity chromatography. Among these proteins, ATP synthase subunit ß (ATP SSß) was deduced as a potential target. Four binding sites were predicted between NBSP2 and ATP SSß of T. cinnabarinus and S. litura. In conclusion, we identified a venom peptide with activity against T. cinnabarinus and S. litura. This study provides a novel component for development of a new biological pesticide.

Design, Synthesis and Insecticidal Activity of Novel meta-Diamide Compounds Containing Pyridine Rings.

In order to discover new meta-diamide compounds with good activity and novel structure, 15 related compounds were designed and synthesized by the bioisosterism principle with cyproflanilide as the lead compound. The insecticidal activities of these compounds against Plutella xylostella and Tetranychus cinnabarinus were tested, and the results of biological activity test showed that some compounds had more than 90 % insecticidal activity against Plutella xylostella at 1 mg/L and Tetranychus cinnabarinus at 100 mg/L. Especially, N-(2-bromo-6-(difluoromethoxy)-4-(perfluoro propan-2-yl)phenyl)-6-(isonicotinamido)picolinamide against Tetranychus cinnabarinus at 10 mg/L was 100 %, which was better than that of cyproflanilide. Molecular docking studies suggested that N-(2-bromo-6-(difluoromethoxy)-4-(perfluoropropan-2-yl)phenyl)-6-(4-cyano-2-methylbenzamido)picolinamide had a closely combined with the Plutella xylostella 3RHW (a glutamate-gated chloride channel). This study provides an avenue for designing and synthesizing a new generation of more effective pesticides.

Two Novel Diterpenoid Alkaloids from Aconitum Pendulum.

Two previously uncharacterized compounds, an aconitine-type C19-diterpenoid alkaloid (1) and a napelline-type diterpenoid alkaloid C20-diterpenoid alkaloid (2), as well as ten known compounds (3-12), were isolated from Aconitum pendulum. Their structures were elucidated based on spectroscopic data, including 1D and 2D NMR, IR, HR-ESI-MS, and single-crystal X-ray diffraction analysis. The anti-insecticidal activities of these compounds were evaluated by contact toxicity tests against two-spotted spider mites, and compounds 1, 2, and 9 showed moderate contact toxicity, with LC50 values of 0.86±0.09, 0.95±0.23, and 0.89±0.19 mg/mL, respectively. This study highlights the potential use of diterpenoid alkaloids as natural plant-derived pesticides for the management of plant pests.

Monitoring detoxification enzyme levels and resistance of Tetranychus urticae against some METI-group chemicals in Türkiye cotton fields.

Acaricides used against Tetranychus urticae Koch, 1836 (Acari: Tetranychidae) in cotton fields cause control failure over time. To determine the resistance status of T. urticae populations to tebufenpyrad and bifenazate, different populations collected from Aydin (AYD), Adana (ADA), Sanliurfa (SAN), and Diyarbakir (DIY) provinces of Türkiye, between 2019 and 2020, were subjected to diagnostic dose bioassays. Firstly, the spider mites were eliminated with a discriminating dose. Afterwards, LC50 and LC90 of the remaining populations were determined and the ten highest resistant populations were selected. The highest phenotypic resistance to bifenazate was observed in AYD4 and DIY2 (LC50 57.14 mg L- 1 with 85.01-fold and LC50 30.15 mg L- 1with 44.86-fold, respectively), while the lowest phenotypic resistance was found in SAN6 (LC50 1.5 mg L- 1; 2.28-fold). Considering the phenotypic resistance to tebufenpyrad, the highest resistance was found in AYD4 population (LC50 96.81 mg L- 1; 12.92-fold), while the lowest - in DIY28 population (LC50 21.23 mg L- 1; 2.83-fold). In pharmacokinetic studies, the ADA16 population was compared with the sensitive German Susceptible Strain population and it was determined that carboxylesterase activity was statistically higher (1.46 ± 0.04 nmol/min/mg protein enzyme activation 2.70-fold). The highest activation of glutathione S-transferase was detected in ADA16 (1.49 ± 0.01 nmol/min/mg protein; 2.32-fold). No mutations were found in PSST (METI 1), the point mutation site for tebufenpyrad, and Cytb (METI 3), the point mutation site for bifenazate. In terms of phenotypic resistance, bifenazate was found to be moderately resistant in two populations (85.01 and 44.86-fold), while tebufenpyrad was moderately resistant in one population (12.92-fold). This study showed that both acaricides are still effective against T. urticae populations.

Compatibility of synthetic and biological pesticides with a biocontrol agent Phytoseiulus longipes (Acari: Phytoseiidae).

Phytoseiulus longipes is a predatory mite of Tetranychus evansi, which is an invasive pest in Africa and elsewhere. The introduction of this predator in Africa has considerable potential, but little is known about the compatibility of P. longipes with commonly used pesticides. Here, we examined lethal and sublethal effects of two pyrethroids (cypermethrin and deltamethrin), two organophosphates (dimethoate and chlorpyrifos), one nicotinoid (imidacloprid), two acaricides (propargite and abamectin), two naturally derived pesticides (oxymatrine and azadirachtin), and one entomopathogenic fungal-based formulation (Hirsutella thompsonii) on P. longipes eggs and adults. The pesticides were sprayed at their maximum recommended concentrations. Topical exposures to azadirachtin, imidacloprid, propargite, abamectin, oxymatrine, and H. thompsonii significantly reduced the net reproductive rate (R0), intrinsic rate of increase (r) and finite rate of increase (λ)of P. longipes. Pesticide lethal and sublethal effects on the predator were summarized in a reduction coefficient (Ex) for the classification based on IOBC toxicity categories. Results revealed that Azadirachtin and H. thompsonii were slightly harmful effects to adults. Imidacloprid, propargite, abamectin, and oxymatrine were moderately harmful to both eggs and adults. Residual persistence bioassays revealed that 4-day-old residue of azadirachtin had no harmful effect on the predator. Abamectin, oxymatrine, and H. thompsonii became harmless to it 10 days post-spraying, and propargite and imidacloprid were considered harmless after 20 days. Cypermethrin, deltamethrin, dimethoate, and chlorpyrifos were highly harmful to both eggs and adults, persistence remaining high even after 31 days of application. These findings provide valuable insights into decision-making when considering P. longipes for use in IPM programs.

Effect of Eucalyptus globulus and Ferula assafoetida essential oils and their nanoformulations on the life table parameters of Tetranychus urticae (Acari: Tetranychidae).

One of the most damaging pests of agricultural crops across the globe is the two-spotted spider mite, Tetranychus urticae Koch. A wide variety of arthropods and plant pathogens can be controlled by essential oils, which are secondary metabolites produced by plants. It is possible to enhance the stability as well as the anti-pest efficiency of plant essential oils by encapsulation. Water distillation was used to extract the essential oils from Eucalyptus globulus and Ferula assafoetida. The chitosan nanoparticles were used to load both essential oils into nanoformulations. Studies were conducted on T. urticae life table characteristics under experimental circumstances to determine the sublethal impacts of essential oils and their nanoformulations. Intrinsic growth rate (r) for population exposed to E. globulus, F. assafoetida essential oils, their nanoformulations and the control were 0.1, 0.069, 0.051, 0.018 and 0.21 per day, respectively. F. assafoetida and E. globulus nanoformulations resulted the lowest fecundity compared to the other treatments. According the result of the lethal and sublethal effects of purified essential oils and nanoformulations of F. assafoetida and E. globulus, they would be recommended for controlling the two-spotted spider mites, T. urticae.

Sublethal effects of a spiromesifen and abamectin combination on Tetranychus urticae (Acari: Tetranychidae) and its predators Phytoseiulus persimilis and Amblyseius swirskii (Acari: Phytoseiiidae).

The two-spotted spider mite, Tetranychus urticae Koch (TSSM), is an important cosmopolitan pest of agricultural crops that is often managed in greenhouses by augmentation of predatory mites in combination with acaricides. Here we examined the transgenerational effects of low lethal concentrations of a widely-used acaricide, Oberon Speed® (a combination of spiromesifen and abamectin), on the life history traits and population growth of T. urticae and two of its predators, Phytoseiulus persimilis Athias-Henriot and Amblyseius swirskii Athias-Henriot (Acari: Phytoseiidae). The concentrations employed corresponded to the LC10, LC20 and LC30 values estimated for TSSM protonymphs 48 h post-exposure in a topical bioassay, which yielded an LC50 value of 207.2 ppm. Parental exposure of TSSM to all three low concentrations increased the total developmental time of progeny; both the LC20 and LC30 treatments reduced adult longevity and number of oviposition days, but only the LC30 treatment increased the preoviposition period. Similarly, both the LC20 and LC30 treatments significantly reduced life table parameters (r, R0, λ, and GRR), and increased generation time (T) and population doubling time (DT). Although maternal exposure to the acaricide had various impacts on progeny life history, A. swirskii was less affected than P. persimilis, suggesting the former species would be more compatible for integration with Oberon Speed® for control of T. urticae in greenhouse vegetable production.

Transovarial toxicity matters: lethal and sublethal effects of hexythiazox on the two-spotted spider mite (Acari: Tetranychidae).

The effects of hexythiazox on life-history traits and demographic parameters of Tetranychus urticae Koch (Acari: Tetranychidae) were evaluated using the age-stage two-sex life table (in fecundity-based and fertility-based variants), with emphasis on its transovarial toxicity. Hexythiazox was applied when T. urticae females were either in the preovipositional period or in the first day of oviposition. In the F0 generation bioassay, treatments with concentrations of 50, 12.5 and 3.125 mg/l significantly reduced the longevity of females and their fecundity. These effects were mostly the result of mortality of treated females (18-23%) over the 24-h exposure period. Even though the net reproductive rate (R0) decreased significantly, the intrinsic rate of increase (r), finite rate of increase (λ) and doubling time (D) were not significantly different from the control. The strongest transovarial toxic effect occurred within the first 4 days following treatment, when 52-89% of the eggs laid by treated females (96% in control) hatched. Fertility was significantly reduced by concentrations of 50, 12.5, 3.125, 0.781 and 0.195 mg/l. These concentrations caused significant reductions in R0 (34-54%), r (12-24%) and λ (3-5%), whereas D was extended for 0.4-0.7 days. In the F1 generation bioassay, 50, 12.5, 3.125, 0.781, 0.049 and 0.012 mg/l caused significant reductions in R0 (34-92%), r (10-68%) and λ (3-17%), whereas extending D for 0.3-5.6 days. These effects were mostly the consequence of transovarial toxicity. Application of the fecundity-based life table underestimated population-level effects of hexythiazox on T. urticae.

Agrochemical properties evaluation of some imines alkaloids of matrine/oxymatrine.

To discover non-food renewable forest bioactive products as new potential pesticidal alternatives for crop protection, a series of C15-imines alkaloids were obtained by structural modification of matrine and oxymatrine. Compounds Id, Ih, Ii and IIg (>2-3 folds of their precursors) showed the most potent antifeedant activity against armyworm. Against red spider mite, compounds Ie, Il, IIb, IIc and IIg displayed 1.8-3.1 folds acaricidal activity of their precursors. Notably, compound IIg exhibiting the most pronounced pesticidal activities, can be used as a promising bio-sourced agrochemical agent. The study of stress responses showed that the nAChR subunit α5 and VGSC might be the targets of action of matrine, oxymatrine and IIg against red spider mite.

A unique primary structure of RDL (resistant to dieldrin) confers resistance to GABA-gated chloride channel blockers in the two-spotted spider mite Tetranychus urticae Koch.

The primary structure of the second transmembrane (M2) segment of resistant to dieldrin (RDL), an ionotropic γ-aminobutyric acid receptor (GABAR) subunit, and the structure-function relationships in RDL are well conserved among insect species. An amino acid substitution at the 2' position in the M2 segment (Ala to Ser or Gly) confers resistance to non-competitive antagonists (NCAs) of GABARs. Here, a cDNA encoding RDL was cloned from the two-spotted spider mite Tetranychus urticae Koch. Unlike insect homologs, native TuRDL has His at the 2' position (H305) and Ile at 6' (I309) in the M2 segment and is insensitive to NCAs. Single and multiple mutations were introduced in the M2 segment of TuRDL, and the mutant proteins were expressed in Xenopus oocytes and examined for the restoration of sensitivity to NCAs. The sensitivity of a double mutant (H305A and I309T in the M2 segment) was greatly increased but was still considerably lower than that of insect RDLs. We therefore constructed chimeric RDLs consisting of TuRDL and Drosophila melanogaster RDL and examined their sensitivities to NCAs. The results show that the N-terminal region containing the Cys-loop as well as the M2 segment confers functional specificity; thus, our current understanding of the mechanism underlying NCA binding to GABARs requires reappraisal.

Non-food bioactive products for insecticides (II): Investigation on stress responses of Tetranychus cinnabarinus Boisduval against a derivative of the alkaloid matrine.

Besides structural modification of natural bioactive products to afford promising agrochemical candidates, investigation of their mechanisms of action against pests is also an important strategy to obtain novel potentially botanical pesticides. N-(p-Ethyl)phenylsulfonylmatrinic acid (2), derived from an natural alkaloid matrine (1), exhibited about 5.9-fold more pronounced acaricidal activity than 1 against the adult females of Tetranychus cinnabarinus Boisduval, and good control efficiency in the greenhouse. By comparison of nAChR, AChE and VGSC of treated and untreated T. cinnabarinus via RT-PCR and qRT-PCR analysis, it was found that compound 2 could activate nAChR and VGSC via up-regulation of nAChR α1, α4 and α5 subunits and VGSC expressions; compound 2 may be the AChE and AChE enzyme inhibitor. Importantly, a scheme of compound 2 interaction with nAChR, AChE and VGSC of T. cinnabarinus was proposed. It will lay the foundation for future optimization and application of matrine derivatives as agrochemicals.

Resistance to pyridaben in Canadian greenhouse populations of two-spotted spider mites, Tetranychus urticae (Koch).

Two-spotted spider mite (TSSM) Tetranychus urticae (Koch) is an important agricultural pest that causes considerable yield losses to over 150 field and greenhouse crops. Mitochondrial electron transport inhibitors (METI) acaricides are commonly used to control mite species in commercial Canadian greenhouses. Development of resistance to METIs in TSSM populations have been reported worldwide, but not until recently in Canada. The objectives of this study were to: 1) monitor the acaricide-susceptibility in greenhouse TSSM populations, and 2) investigate the resistance to pyridaben, a METI acaricide, in greenhouse resistant and pyridaben-selected (SRS) mite strains. The increased mortality to the pyridaben sub-lethal concentration (LC30) when SRS mites were exposed to piperonyl butoxide (PBO), a general cytochrome P450 monooxygenase inhibitor, and higher P450 activity compared to the greenhouse strain (RS) mites, indicated that P450s may be at least partially responsible for the resistance. The molecular mechanisms of target site insensitivity-mediated resistance in the pyridaben resistant strain of TSSM were investigated by comparing the DNA sequence of NADH dehydrogenase subunits TYKY and PSST, NADH-ubiquinone oxidoreductase chain 1 and 5 (ND1, ND5) and the NADH-ubiquinone oxidoreductase subunit 49 kDa from SRS to the reference strain (SS) and RS. Despite a number of nucleotide substitutions, none correlated with the pyridaben resistance. Understanding the underlying mechanisms of TSSM adaptation to acaricides is an essential part of resistance management strategy in any IPM program. The findings of this study will encourage growers to apply acaricides with different modes of action to reduce the rate at which acaricide resistance will occur in greenhouse TSSM populations.

Design, Synthesis, and Acaricidal Activity of Phenyl Methoxyacrylates Containing 2-Alkenylthiopyrimidine.

A series of novel phenyl methoxyacrylate derivatives containing a 2-alkenylthiopyrimidine substructure were designed, synthesized, and evaluated in terms of acaricidal activity. The structures of the title compounds were identified by 1H NMR, 13C NMR and high-resolution mass spectra (HRMS). Compound (E)-methyl 2-(2-((2-(3,3-dichloroallylthio)-6-(trifluoromethyl)pyrimidin-4-yloxy)methyl)phenyl)-3-methoxyacr-ylate (4j) exhibited significant acaricidal activity against Tetranychus cinnabarinus (T. cinnabarinus) in greenhouse tests possessing nearly twice the larvicidal and ovicidal activity compared to fluacrypyrim. Furthermore, the results of the field trials demonstrated that compound 4j could effectively control Panonychuscitri with long-lasting persistence and rapid action. The toxicology data in terms of LD50 value confirmed that compound 4j has a relatively low acute toxicity to mammals, birds, and honeybees.

Evaluation of andrographolide-based analogs derived from Andrographis paniculata against Mythimna separata Walker and Tetranychus cinnabarinus Boisduval.

To discover new natural-product-based pesticides, we structurally modified andrographolide, a labdane diterpenoid isolated from Andrographis paniculata, and stereoselectively prepared a series of 12α-(substituted)benzylamino-14-deoxyandrographolide derivatives (I-V). Three-dimensional structures of compounds 3c, 3d, IIIa and IIIb were further determined by single-crystal X-ray diffraction. Compounds IIa (R1 = n-C3H7, R2 = PhCH2) exhibited more promising insecticidal activity against Mythimna separata than toosendanin. Compounds 3a (R1 = H), Ib (R1 = H, R2 = 4-ClPhCH2), and IVa (R1 = 4-ClPh, R2 = PhCH2) showed potent acaricidal activity against Tetranychus cinnabarinus.

Acaricidal effect of cell-free supernatants from Xenorhabdus and Photorhabdus bacteria against Tetranychus urticae (Acari: Tetranychidae).

The effects of secondary metabolites produced by the following symbiotic bacteria, Xenorhabdus szentirmaii, X. nematophila, X. bovienii, X. cabanillasii, Photorhabdus luminescens and P. temperata, associated with entomopathogenic nematodes, were investigated against various developmental stages of Tetranychus urticae (Acari: Tetranychidae) using cell-free bacterial supernatants in Petri dishes. In addition, the effects of the most active bacterial supernatant(s) found in Petri dish experiments were tested on T. urticae in pot experiments. All studies were conducted at 25 ±â€¯1 °C temperature, 70 ±â€¯5% relative humidity and a light cycle of 16 h in a climate room. The result of the Petri dish experiments showed that the supernatants had little or no effect on the egg stage, as less than 4% mortality was recorded. Depending on the bacterial supernatant, mortality in the other stages was 46-97% for larvae, 30-96% for protonymphs, 41-92% for deutonymphs, 92-100% for adult males and 46-93% for adult females. Control mortalities ranged from 1-7% for larvae, 2-9% for protonymphs, 4-10% for deutonymphs, 6-10% for adult males and 4-8% for adult females. Among supernatants tested, X. szentirmaii and X. nematophila were the most efficacious with mortality greater than 90% on the mobile stages of T. urticae. According to the results from pot experiments, the supernatants of X. szentirmaii and X. nematophila, singularly and in combination, significantly reduced the T. urticae population. However, the mixture of X. szentirmaii and X. nematophila supernatants did not increase efficiency to reduce T. urticae population compared to each supernatant alone. Further studies are warranted to find the active compound(s) in the supernatants of X. szentirmaii or and X. nematophila and assess whether the supernatant(s) has the potential of being a practical and economical control agent for T. urticae.