Acaricide resistance status and identification of resistance mutations in populations of the two-spotted spider mite Tetranychus urticae from Ethiopia.

Efficacy of ketoenols on insecticide resistant field populations of two-spotted spider mite Tetranychus urticae and sweet potato whitefly Bemisia tabaci from Greece

The mechanisms underlying insecticide and acaricide resistance in insects and mites are often complex, including additive effects of target-site insensitivity, increased metabolism and transport. The extent to which target-site resistance mutations contribute to the resistance phenotype is, however, not well studied. Here, we used marker-assisted backcrossing to create 30 congenic lines carrying nine mutations (alone, or in combination in a few cases) associated with resistance to avermectins, pyrethroids, mite growth inhibitors and mitochondrial complex III inhibitors (QoI) in a polyphagous arthropod pest, the spider mite Tetranychus urticae. Toxicity tests revealed that mutations in the voltage-gated sodium channel, chitin synthase 1 and cytochrome b confer high levels of resistance and, when fixed in a population, these mutations alone can result in field failure of acaricide treatment. In contrast, although we confirmed the implication of mutations in glutamate-gated chloride channels in abamectin and milbemectin insensitivity, these mutations do not lead to the high resistance levels that are often reported in abamectin resistant strains of T. urticae. Overall, this study functionally validates reported target-site resistance mutations in T. urticae, by uncoupling them from additional mechanisms, allowing to finally investigate the strength of the conferred phenotype in vivo.

In 2015, we studied the efficiency of the predatory mite Neoseiulus californicus (McGregor) for suppression of the two-spotted spider mite Tetranychus urticae in a field experiment at a hop plantation. We randomly arranged four treatments in a three-block experiment. Fungicides were used in all treatments; insecticides were used in all treatments except the predatory mite treatment and acaricides were used in only two treatments. A single inundative release of the mite N. californicus was carried out on 4 July. On four different dates (10 June, 17 July, 29 July and 9 August), we counted the eggs and the mobile stages (larvae, nymphs and adults) of the two-spotted spider mite in all four treatments. In the treatment with the predatory mite, we established the fewest eggs and mobile stages of T. urticae 14 days after the release of the predator. The selected acaricides in our research acted in a primarily ovicidal manner, but we did not detect satisfactory effects on the mobile stages of the two-spotted spider mite. This result suggests the emergence of resistance of two-spotted spider mites to the acaricides hexythiazox and abamectin. Our research established comparable effects of the predatory mite N. californicus and acaricides, and further improvement of the efficiency would require release of the natural enemy into a hop plantation in mid-June, followed by a second release three weeks later. The costs of acaricide use in our experiment were from 12.7-fold (two sprayings of hexythiazox, and a single spraying with abamectin) to 17.8-fold (single treatments of hexythiazox and abamectin) lower than those of a single release of the biological control agent in question. The results of our study represent a starting point for future research, which could achieve satisfactory results in suppressing two-spotted spider mites on a hop plantation by repeated use of the predatory mite N. californicus.

The frequency of insecticide/acaricide target‐site resistance is increasing in arthropod pest populations and is typically underpinned by single point mutations that affect the binding strength between the insecticide/acaricide and its target‐site. Theory predicts that although resistance mutations clearly have advantageous effects under the selection pressure of the insecticide/acaricide, they might convey negative pleiotropic effects on other aspects of fitness. If such fitness costs are in place, target‐site resistance is thus likely to disappear in the absence of insecticide/acaricide treatment, a process that would counteract the spread of resistance in agricultural crops. Hence, there is a great need to reliably quantify the various potential pleiotropic effects of target‐site resistance point mutations on arthropod fitness. Here, we used near‐isogenic lines of the spider mite pest Tetranychus urticae that carry well‐characterized acaricide target‐site resistance mutations to quantify potential fitness costs. Specifically, we analyzed P262T in the mitochondrial cytochrome b, the combined G314D and G326E substitutions in the glutamate‐gated chloride channels, L1024V in the voltage‐gated sodium channel, and I1017F in chitin synthase 1. Five fertility life table parameters and nine single‐generation life‐history traits were quantified and compared across a total of 15 mite lines. In addition, we monitored the temporal resistance level dynamics of populations with different starting frequency levels of the chitin synthase resistant allele to further support our findings. Three target‐site resistance mutations, I1017F and the co‐occurring G314D and G326E mutations, were shown to significantly and consistently alter certain fitness parameters in T. urticae. The other two mutations (P262T and L1024V) did not result in any consistent change in a fitness parameter analyzed in our study. Our findings are discussed in the context of the global spread of T. urticae pesticide resistance and integrated pest management.

The two-spotted spider mite (Tetranychus urticae) is one of the most well-known pesticide-resistant agricultural pests, with resistance often attributed to changes such as target-site mutations and detoxification activation. Recent studies show that pesticide resistance can also be influenced by symbionts, but their involvement in this process in spider mites remains uncertain. Here, we found that infection with Wolbachia, a well-known bacterial reproductive manipulator, significantly increased mite survival after exposure to the insecticides abamectin, cyflumetofen, and pyridaben. Wolbachia-infected (WI) mites showed higher expression of detoxification genes such as P450, glutathione-S-transferase (GST), ABC transporters, and carboxyl/cholinesterases. RNA interference experiments confirmed the role of the two above-mentioned detoxification genes, TuCYP392D2 and TuGSTd05, in pesticide resistance. Increased GST activities were also observed in abamectin-treated WI mites. In addition, when wild populations were treated with abamectin, WI mites generally showed better survival than uninfected mites. However, genetically homogeneous mites with different Wolbachia strains showed similar survival. Finally, abamectin treatment increased Wolbachia abundance without altering the mite's bacterial community. This finding highlights the role of Wolbachia in orchestrating pesticide resistance by modulating host detoxification. By unraveling the intricate interplay between symbionts and pesticide resistance, our study lays the groundwork for pioneering strategies to combat agricultural pests.

Growers of greenhouse roses in Cyprus have recently reported failures of several acaricides against two-spotted spider mite (Tetranychus urticae Koch). To verify the cause of these failures we evaluated the effectiveness of seven acaricides against two rose and two cucumber spider mite populations in the laboratory. The acaricides included in our study represented the most important mode of action groups registered for use in greenhouses on the island. Label rates of all seven acaricides (abamectin, clofentezine, dicofol, fenbutatin oxide, fenazaquin, propargite and milbemectin—a product under registration) caused close to 100% mortality in the cucumber populations, but significantly lower mortality in the two rose populations. Mortality never exceeded 65% in the first rose population and 20% in the second population for any acaricide, suggesting development of resistance. The higher frequency of acaricide applications in roses and the perennial nature of the crop, that sustains genetically similar populations for long periods of time, may have facilitated the development of resistance. Judicious use of newer acaricides with distinct modes of action as they become registered may help control resistant spider mite populations in roses and other ornamental crops. Additionally, adoption of a mode of action labeling scheme of pesticides and training of growers on alternation of products will facilitate long term sustainable spider mite management in Cypriot agriculture.

In spider mites, mutations in the mitochondrial cytochrome b Qo pocket have been reported to confer resistance to the Qo inhibitors bifenazate and acequinocyl. In this study, we surveyed populations of the two-spotted spider mite Tetranychus urticae for mutations in cytochrome b, linked newly discovered mutations with resistance and assessed potential pleiotropic fitness costs. We identified two novel mutations in the Qo site: G132A (equivalent to G143A in fungi resistant to strobilurins) and G126S + A133T (previously reported to cause bifenazate and acequinocyl resistance in Panonychus citri). Two T. urticae strains carrying G132A were highly resistant to bifenazate but not acequinocyl, whereas a strain with G126S + A133T displayed high levels of acequinocyl resistance, but only moderate levels of bifenazate resistance. Bifenazate and acequinocyl resistance were inherited maternally, providing strong evidence for the involvement of these mutations in the resistance phenotype. Near isogenic lines carrying G132A revealed several fitness penalties in T. urticae; a lower net reproductive rate (R0 ), intrinsic rate of increase (rm) and finite rate of increase (LM); a higher doubling time (DT); and a more male-biased sex ratio. Several lines of evidence were provided to support the causal role of newly discovered cytochrome b mutations in bifenazate and acequinocyl resistance. Because of the fitness costs associated with the G132A mutation, resistant T. urticae populations might be less competitive in a bifenazate-free environment, offering opportunities for resistance management. © 2019 Society of Chemical Industry.

Pesticide resistance represents a clear and trackable case of adaptive evolution with a strong societal impact. Understanding the factors associated with the evolution and spread of resistance is imperative to develop sustainable crop management strategies. The two-spotted spider mite Tetranychus urticae, a major crop pest with worldwide distribution and a polyphagous lifestyle, has evolved resistance to most classes of pesticides. Tetranychus urticae exists as either a green- or a red-coloured morph. However, the extent of genetic divergence and reproductive compatibility vary across populations of these colour morphs, complicating their taxonomic resolution at the species level. Here, we studied patterns of genetic differentiation and barriers to gene flow within and between morphs of T. urticae in order to understand the factors that influence the spread of resistance mutations across its populations. We derived multiple iso-female lines from Tetranychus populations collected from agricultural crops. We generated genomic and morphological data, characterized their bacterial communities and performed controlled crosses. Despite morphological similarities, we found large genomic differentiation between the morphs. This pattern was reflected in the incomplete, but strong postzygotic incompatibility in crosses between colour morphs, while crosses within morphs from different geographical locations were largely compatible. In addition, our results suggest recent/on-going gene flow between green-coloured T. urticae and T. turkestani. By screening the sequences of 10 resistance genes, we found evidence for multiple independent origins and for single evolutionary origins of target-site resistance mutations. Our results indicate that target-site mutations mostly evolve independently in populations on different geographical locations, and that these mutations can spread due to incomplete barriers to gene flow within and between populations.

Acaricide application remains an integral component of integrated pest management (IPM) for the two-spotted spider mite Tetranychus urticae. Species and strains of phytoseiid predatory mites vary significantly in their response to acaricides. For the success of IPM, it is imperative to identify the determinants of selectivity and molecular stress responses of acaricides in predatory mites. The three classical acaricides bifenazate, cyflumetofen, and fenbutatin oxide did not affect the survival and fecundity of Phytoseiulus persimilis regardless of the route of exposure. Selectivity of the orange oil and terpenoid blend-based botanical acaricides was low via a combination of direct exposure, acaricide-laced diet, and residual exposure but improved when limiting exposure only to diet. To gain insights into the molecular stress responses, the transcriptome of P. persimilis was assembled. Subsequent gene expression analysis of predatory mites orally exposed to fenbutatin oxide and orange oil yielded only a limited xenobiotic stress response. In contrast, P. persimilis exhibited target-site resistance mutations, including I260M in SdhB, I1017M in CHS1, and kdr and super-kdr in VGSC. Extending the screen using available Phytoseiidae sequences uncovered I136T, S141F in cytb, G119S in AChE, and A2083V in ACC, well-known target-sites of acaricides. Selectivity of the tested botanical acaricides to P. persimilis was low but could be enhanced by restricting exposure to a single route. Differential gene expression analysis did not show a robust induced stress response after sublethal exposure. In contrast, this study uncovered target-site mutations that may help to explain the physiological selectivity of several classical acaricides to phytoseiid predators.

In the State of Mexico, Mexico, Rosa sp. production is one of the most important economic activities. However, the two-spotted mite, Tetranychus urticae Koch, is a pest that limits this crop's productivity, and its control is carried out mainly with acaricides. Therefore, we estimated the levels of resistance to six acaricides in three field-collected populations of T. urticae from greenhouses of this growing region, and compared their response with a susceptible population of the same species. The products tested were abamectin, fenbutatin oxide, chlorfenapyr, amitraz, bifenthrin, and propargite. Significant differences were found in the response to the applied concentrations of acaricides. The levels of resistance were high to all tested acaricides, except fenbutatin oxide, in the Villa Guerrero and Tenancingo populations. The Coatepec population was susceptible to fenbutatin oxide and abamectin and had higher levels of tolerance to bifenthrin, amitraz, and chlorfenapyr. The RR50 values in resistant populations, ranged from 10 (propargite in Tenancingo population) to 6.400-fold (amitraz in Villa Guerrero population), in comparison to the susceptible reference. Amendments to both pest management strategies and the use of acaricides against T. urticae in Rosa sp. are proposed.

The two-spotted spider mite Tetranychus urticate is an important agricultural pest worldwide. It is extremely polyphagous and has developed resistance to many pesticides. Here, we assessed the pesticide resistance of seven field populations of T. urticae in China, their target site mutations and the activities of their detoxification enzymes. The results showed that abamectin and the traditional pesticides pyridaben, profenofos and bifenthrin had higher resistance or lower toxicity than more recently developed pesticides including chlorfenapyr, spinetoram, cyflumetofen, cyenopyrafen, bifenazate and B-azolemiteacrylic. The frequency of point mutations related to abamectin resistance, G314D in the glutamate-gated chloride channel 1 (GluCl1) and G326E in GluCl3, ranged 47%-70% and 0%-97%, respectively. The frequency of point mutations in A1215D and F1538I of the voltage-gated sodium channel gene (VGSC), which may increase resistance to pyrethroids, ranged 88%-100% and 10%-100%, respectively. For target sites related to organophosphate resistance, mutation frequencies ranged 25%-92% for G119S and 0%-23% for A201S in the acetycholinesterase gene (Ace). Mutation G126S in the bifenazate resistance-related cytochrome b gene (Cytb) was observed in three of the seven T. urticae populations. Higher activities of detoxification enzymes (P450, GST, CarEs and UGTs) were observed in two T. urticae populations, with significant difference in the XY-SX population. These results provide useful information on the status of pesticide resistance of T. urticae in China and suggest that T. urticae field populations may have multiple resistance mechanisms.

This study evaluated the acaricidal effects of biosurfactants produced by Serratia ureilytica against the two-spotted spider mite Tetranychus urticae and their compatibility with the predatory mite Ambliseus swirski. The biosurfactants were obtained via liquid cultures of the bacterial strains. In the laboratory, T. urticae was exposed via acaricide-immersed leaves and A. swirskii via acaricide-coated glass vials. In the greenhouse, mite-infested plants were sprayed with the biosurfactants. In the laboratory, biosurfactants produced by S. ureilytica NOD-3 and UTS exhibited strong acaricidal activity, causing 95% mortality in adults and reducing egg viability by more than 60%. In the greenhouse trial, all biosurfactants significantly suppressed T. urticae populations at all evaluated periods (7, 14, and 21 days post-application). Gas chromatography–mass spectrometry (GC-MS) analysis of the biosurfactants identified several fatty acids, including hexadecanoic acid, pentanoic acid, octadecanoic acid, decanoic acid, and tetradecanoic acid, as well as the amino acids L-proline, L-lysine, L-valine, and glutamic acid. These fatty acids and amino acids are known structural components of lipopeptides. Furthermore, the bioinformatic analysis of the genomes of the three S. ureilytica strains revealed nonribosomal peptide synthetase (NRPS) gene clusters homologous to those involved in the biosynthesis of lipopeptides. These findings demonstrate that S. ureilytica biosurfactants are promising eco-friendly acaricides, reducing T. urticae populations by >95% while partially sparing A. swirskii.

High frequency of ryanodine receptor and cytochrome P450 CYP9A186 mutations in insecticide-resistant field populations of Spodoptera exigua from China

Several genetic variants of the cd1- and ef-helices of the Qo site of mitochondrial cytochrome b have been associated with bifenazate resistance in the spider mite Tetranychus urticae, an important crop pest around the world. Maternal inheritance of bifenazate resistance has provided strong evidence for the involvement of many of these mutations alone or in combination. A number of populations highly resistant to bifenazate were uncovered that carried the G126S substitution in combination with other target-site mutations. This G126S mutation has therefore been investigated in several studies in the context of resistance evolution and the development of diagnostic markers. However, experimental data that link bifenazate resistance with the presence of the G126S mutation without additional cd1- and ef-helices mutations, remain very limited. Here, we genotyped 38T. urticae field populations for cytochrome b and uncovered nine field populations with a fixed or segregating G126S substitution without other target-site mutations in the conserved cd1- and ef-helices of the cytochrome b Qo pocket. Toxicity bioassays showed that all nine field populations were very susceptible to bifenazate, providing strong evidence that G126S alone does not confer bifenazate resistance. These findings also implicate that previous T. urticae populations with G126S found to be low to moderately resistant to bifenazate, evolved alternative mechanisms of resistance, and more importantly, that this mutation cannot be used as a molecular diagnostic for bifenazate resistance.

Identification and characterization of abamectin resistance in Tetranychus urticae Koch populations from greenhouses in Turkey