Cloning and different expression of ATP synthase genes between propargite resistant and susceptible strains of Tetranychus cinnabarinus (Acarina: Tetranychidae)

The carmine spider mite, Tetranychus cinnabarinus (Boisduval), is a serious phytophagous mite damaging important crops and can rapidly develop resistance to acaricides. Mitochondrial ATP synthase (F1F0 ATP synthase) is an important target site of acaricides. The role of ATP synthase in acaricide resistance remains unclear at the molecular level. In this study, twelve full-length cDNAs of ATP synthase genes were cloned and characterized from T. cinnabarinus and their expression levels were determined for both progargite-resistant and susceptible strains. The effect of propargite exposure on gene expression was also evaluated. Analyses of gene expression revealed that TcATPsynU-2, TcATPsynF0-2 and TcATPsynF0-4 were significantly down-regulated in the progargite-resistant strain. TcATPsynF0-2 and TcATPsynF0-4 had a strong response to progargite exposure. The results suggest that lower levels of TcATPsynU-2, TcATPsynF0-2 and TcATPsynF0-4 expression might be related to propargite-resistance observed in the resistant T. cinnabarinus. This is the first attempt to identify specific ATPase genes involved in propargite resistance in T. cinnabarinus.     

Phenotypic- and Genotypic-Resistance Detection for Adaptive Resistance Management in Tetranychus urticae Koch

Rapid resistance detection is necessary for the adaptive management of acaricide-resistant populations of Tetranychus urticae. Detection of phenotypic and genotypic resistance was conducted by employing residual contact vial bioassay (RCV) and quantitative sequencing (QS) methods, respectively. RCV was useful for detecting the acaricide resistance levels of T. urticae, particularly for on-site resistance detection; however, it was only applicable for rapid-acting acaricides (12 out of 19 tested acaricides). QS was effective for determining the frequencies of resistance alleles on a population basis, which corresponded to 12 nonsynonymous point mutations associated with target-site resistance to five types of acaricides [organophosphates (monocrotophos, pirimiphos-methyl, dimethoate and chlorpyrifos), pyrethroids (fenpropathrin and bifenthrin), abamectin, bifenazate and etoxazole]. Most field-collected mites exhibited high levels of multiple resistance, as determined by RCV and QS data, suggesting the seriousness of their current acaricide resistance status in rose cultivation areas in Korea. The correlation analyses revealed moderate to high levels of positive relationships between the resistance allele frequencies and the actual resistance levels in only five of the acaricides evaluated, which limits the general application of allele frequency as a direct indicator for estimating actual resistance levels. Nevertheless, the resistance allele frequency data alone allowed for the evaluation of the genetic resistance potential and background of test mite populations. The combined use of RCV and QS provides basic information on resistance levels, which is essential for choosing appropriate acaricides for the management of resistant T. urticae.     

Novel and selective acetylcholinesterase inhibitors for Tetranychus cinnabarinus (Acari: Tetranychidae)

The carmine spider mite, Tetranychus cinnabarinus (Acari: Tetranychidae), is an economically important and extremely polyphagous herbivorous pest, with the title of “resistance champion” among arthropods. Anticholinesterase insecticides such as organophosphate and carbamate account for more than one-third of global insecticide sales. The non-target toxicity and resistance problem of organophosphate and carbamate have become of growing concern, which may be due to the fact that they target the ubiquitous catalytic serine residue of acetylcholinesterase (AChE) in mammals, birds, and beneficial insects. In this study, the structural differences between T. cinnabarinus AChE and human AChE, at or near the catalytic pocket, were illustrated. From the SPECS chemical lead-compound database, 55 AChE inhibitor candidates were screened for high affinity for T. cinnabarinus AChE, but low affinity for human AChE, using the DOCK 6 and AutoDock Vina software. Three of the fifty-five candidates had inhibitory activity greater than that of the reversible AChE inhibitor eserine, with no observed inhibitory activities against human AChE. Two of the three had toxicity to T. cinnabarinus comparable to that of natural insecticidal pyrethrins. However, their potency is low compared with that of etoxazole, and further work is needed to optimize their potency. The selectivity of the three compounds over human and mite AChE may be due to their interaction with the mite-specific residues, as analyzed by Cyscore. The three compounds are potential lead compounds for development of novel acaricides against T. cinnabarinus with reduced toxicity to non-target species and a low propensity for resistance.     

Adaptation of acaricide stress facilitates Tetranychus urticae expanding against Tetranychus cinnabarinus in China

The two‐spotted spider mite, Tetranychus urticae, and the carmine spider mite, Tetranychus cinnabarinus, are invasive and native species in China, respectively. Compared with T. cinnabarinus, T. urticae has expanded into most parts of China and has become the dominant species of spider mite since 1983, when it was first reported in China. However, the mechanism of the demographic conversion has not been illuminated. In this study, one T. urticae field population and one T. cinnabarinus field population were isolated from the same plant in the same field, and the toxicological characteristics were compared between these two species. Laboratory bioassays demonstrated that T. urticae was more tolerant to commonly used acaricides than T. cinnabarinus. The activities of detoxification enzymes were significantly greater in T. urticae, and the fold changes of enzymes activities in T. urticae were also greater following exposure to acaricides. Furthermore, more metabolism‐related genes were upregulated at a basal level, and more genes were induced in T. urticae following exposure to acaricides. The comparison of proteins and genes between both species led credence to the hypothesis that T. urticae was more resistant to acaricides, which was the reason explaining the expansion of invasive T. urticae against native T. cinnabarinus. Laboratory simulation experiments demonstrated that following the application of acaricides, the composition of a mixed T. urticae/T. cinnabarinus population would change from a T. cinnabarinus‐dominant to a T. urticae‐dominant population. This study not only reveals that T. urticae possesses stronger detoxification capacity than its sibling species T. cinnabarinus, which facilitated its persistent expansion in China, but also points to the need to accurately identify Tetranychus species and to develop species‐specific management strategies for these pests.     

Prey Preference and Life Table of Amblyseius orientalis on Bemisia tabaci and Tetranychus cinnabarinus

Amblyseius orientalis (Ehara) (Acari: Phytoseiidae) is a native predatory mite species in China. It used to be considered as a specialist predator of spider mites. However, recent studies show it also preys on other small arthropod pests, such as Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae). Experiments were conducted to investigate (1) prey preference of A. orientalis between Tetranychus cinnabarinus (Boisd.) (Acari: Tetranychidae) and B. tabaci, and (2) development, consumption and life table parameters of A. orientalis when reared on T. cinnabarinus, B. tabaci or a mix of both prey species. When preying on different stages of T. cinnabarinus, A. orientalis preferred protonymphs, whereas when preying on different stages of B. tabaci, A. orientalis preferred eggs. When these two most preferred stages were provided together (T. cinnabarinus protonymphs and B. tabaci eggs), A. orientalis randomly selected its prey. Amblyseius orientalis was able to complete its life cycle on B. tabaci eggs, T. cinnabarinus protonymphs, or a mix of both prey. However, its developmental duration was 53.9% and 30.0% longer when reared on B. tabaci eggs than on T. cinnabarinus and a mix of both prey, respectively. In addition, it produced only a few eggs and its intrinsic rate of increase was negative when reared on B. tabaci eggs, which indicates that B. tabaci is not sufficient to maintain A. orientalis population. The intrinsic rates of increase were 0.16 and 0.23 when A. orientalis was fed on the prey mix and T. cinnabarinus, respectively. These results suggest that although B. tabaci is a poor food resource for A. orientalis in comparison to T. cinnabarinus, A. orientalis is able to sustain its population on a mix of both prey. This predatory mite may thus be a potential biological control agent of B. tabaci when this pest co-occurs with the alternative minor pest T. cinnabarinus.     

Resistance status of the carmine spider mite,Tetranychus cinnabarinus and the twospotted spider mite,Tetranychus urticae to selected acaricides on strawberries

The carmine spider mite, Tetranychus cinnabarinus (Boisduval) and the twospotted spider mite, Tetranychus urticae Koch, are serious pests of strawberries and many other horticultural crops. Control of these pests has been heavily dependent upon chemical acaricides. Objectives of this study were to determine the resistance status of these two pest species to commonly used acaricides on strawberries in a year‐round intensive horticultural production region. LC₉₀ of abamectin for adult carmine spider mites was 4% whereas that for adult twospotted spider mites was 24% of the top label rate. LC_90s of spiromesifen, etoxazole, hexythiazox and bifenazate were 0.5%, 0.5%, 1.4% and 83% of their respective highest label rates for carmine spider mite eggs, 0.7%, 2.7%, 12.1% and 347% of their respective highest label rates for the nymphs. LC_90s of spiromesifen, etoxazole, hexythiazox and bifenazate were 4.6%, 11.1%, 310% and 62% of their respective highest label rates for twospotted spider mite eggs, 3%, 13%, 432,214% and 15% of their respective highest label rates for the nymphs. Our results suggest that T. cinnabarinus have developed resistance to bifenazate and that the T. urticae have developed resistance to hexythiazox. These results strongly emphasize the need to develop resistance management strategies in the region.     

Establishment of an acaricide-susceptible Tetranychus urticae strain and its species confirmation based on morphological and molecular characters

Acquisition of a reference Tetranychus strain that is completely susceptible to acaricides and retains an identical genetic background to acaricide-resistant strains is an essential step in elucidating mechanisms of resistance. To establish a strain completely susceptible to various acaricides, we collected Tetranychus mite populations from several regions in South Korea, including both remote and heavily cultivated regions. We tested their suitability as a susceptible reference strain by determining baseline susceptibility to six acaricides and by determining species identity as Tetranychus urticae. The UD strain, originally collected from a remote island region, was found to be most susceptible to all five major acaricides tested and was confirmed to as T. urticae on the basis of both morphological and molecular evidence. Moreover, molecular phylogenetic analysis revealed that the UD strain is an ancestral strain of other prevalently collected green-type strains. Taken together, we propose that the UD strain can be used as a susceptible reference strain for T. urticae resistance studies as it provides baseline susceptibility to acaricides and possesses a common genetic background with most other acaricide-resistant strains.     

二斑叶螨对七种杀螨剂的抗药性测定及其机理研究

室内测定了相对敏感种群(S)和抗性种群(R)对常用7种杀螨剂的敏感性,并测定了羧酸酯酶、谷胱甘肽S-转移酶和乙酰胆碱酯酶3种酶的比活力。结果表明:二斑叶螨Tetranychus urticae Koch R种群已对甲氰菊酯和哒螨灵产生了抗性,抗性倍数分别为5.45和105.47。其中,甲氰菊酯对雌成螨的毒力最低(>3000mg/L),已远远超过田间推荐剂量,不宜继续使用。酶活测定结果表明:谷胱甘肽S-转移酶解毒活性的提高是二斑叶螨对甲氰菊酯产生抗性的原因之一;二斑叶螨对哒螨灵抗性的增强可能与羧酸酯酶有关。     

Identification of microRNAs in Macaca fascicularis (Cynomolgus Monkey) by Homology Search and Experimental Validation by Small RNA-Seq and RT-qPCR Using Kidney Cortex Tissues

MicroRNAs (miRNAs) present in tissues and biofluids are emerging as sensitive and specific safety biomarkers. MiRNAs have not been thoroughly described in M. fascicularis, an animal model used in pharmaceutical industry especially in drug safety evaluation. Here we investigated the miRNAs in M. fascicularis. For Macaca mulatta, a closely related species of M. fascicularis, 619 stem-loop precursor miRNAs (pre-miRNAs) and 914 mature miRNAs are available in miRBase version 21. Using M. mulatta miRNAs as a reference list and homology search tools, we identified 604 pre-miRNAs and 913 mature miRNAs in the genome of M. fascicularis. In order to validate the miRNAs identified by homology search we attempted to sequence miRNAs expressed in kidney cortex from M. fascicularis. MiRNAs expressed in kidney cortex may indeed be released in urine upon kidney cortex damage and be potentially used to monitor drug induced kidney injury. Hence small RNA sequencing libraries were prepared using kidney cortex tissues obtained from three naive M. fascicularis and sequenced. Analysis of sequencing data indicated that 432 out of 913 mature miRNAs were expressed in kidney cortex tissues. Assigning these 432 miRNAs to pre-miRNAs revealed that 273 were expressed from both the -5p and -3p arms of 150 pre-miRNAs and 159 miRNAs expressed from either the -5p or -3p arm of 176 pre-miRNAs. Mapping sequencing reads to pre-miRNAs also facilitated the detection of twenty-two new miRNAs. To substantiate miRNAs identified by small RNA sequencing, 313 miRNAs were examined by RT-qPCR. Expression of 262 miRNAs in kidney cortex tissues ware confirmed by TaqMan microRNA RT-qPCR assays. Analysis of kidney cortex miRNA targeted genes suggested that they play important role in kidney development and function. Data presented in this study may serve as a valuable resource to assess the renal safety biomarker potential of miRNAs in Cynomolgus monkeys.     

Molecular analysis of resistance to acaricidal spirocyclic tetronic acids in Tetranychus urticae: CYP392E10 metabolizes spirodiclofen,but not its corresponding enol

Spirodiclofen is one of the most recently developed acaricides and belongs to the new family of spirocyclic tetronic acids (ketoenols). This new acaricidal family is an important chemical tool in resistance management strategies providing sustainable control of spider mites such as Tetranychus urticae. Spirodiclofen targets lipid biosynthesis mediated by direct inhibition of acetyl coenzyme A carboxylase (ACCase). In this study, we investigated two genetically distant spider mite strains with high resistance to spirodiclofen. Despite the strong resistance levels to spirodiclofen (up to 680-fold), only limited cross-resistance with other members of this group such as spiromesifen and spirotetramat could be detected. Amplification and sequencing of the ACCase gene from resistant and susceptible strains did not reveal common non-synonymous mutations, and expression levels of ACCase were similar in both resistant and susceptible strains, indicating the absence of target-site resistance. Furthermore, we collected genome-wide expression data of susceptible and resistant T. urticae strains using microarray technology. Analysis of differentially expressed genes revealed a broad response, but within the overlap of two resistant strains, several cytochrome P450s were prominent. Quantitative PCR confirmed the constitutive over-expression of CYP392E7 and CYP392E10 in resistant strains, and CYP392E10 expression was highly induced by spirodiclofen. Furthermore, stage specific expression profiling revealed that expression levels were not significantly different between developing stages, but very low in eggs, matching the age-dependent resistance pattern previously observed. Functional expression of CYP392E7 and CYP392E10 confirmed that CYP392E10 (but not CYP392E7) metabolizes spirodiclofen by hydroxylation as identified by LC–MS/MS, and revealed cooperative substrate binding and a K_m of 43 μM spirodiclofen. CYP392E10 also metabolizes spiromesifen, but not spirotetramat. Surprisingly, no metabolism of the hydrolyzed spirodiclofen-enol metabolite could be detected. These findings are discussed in the light of a likely resistance mechanism.     

Resistance to binapacryl and tetradifon, and the genetic background, in fruit tree red spider mite, Panonychus ulmi, from English apple orchards

Assays on eggs of Panonychus ulmi held on leaf discs of Myrobalan plum were used to characterise strains (1) multi-resistant to organophosphates (OPs) and binapacryl (strain BR), (2) multi-resistant to OPs and tetradifon (strain TR), and (3) susceptible to OPs, binapacryl and tetradifon (strain EM). By crosses of EM females and BR males, and of EM females and TR males, and by repeated backcrossing with EM males under selection pressure of the acaricides concerned, two strains were produced in which the specific genes for resistance to binapacryl and tetradifon were isolated in the genome of the EM strain. Reciprocal crosses of the resulting backcrossed strains with EM, assays on the F₁ eggs and the F₂ haploid eggs, confirmed the presence of single major genes for resistance to binapacryl and tetradifon, and the absence of maternal effects. The expression of gene BR was incompletely dominant in response to binapacryl, whilst gene TR was effectively dominant in response to tetradifon. Eggs of the field-derived strain BR showed c. 50× resistance to binapacryl, 8–19 × to dinobuton, but only c. 2× to dinocap and dinoseb. The backcrossed strain was less resistant (26 ×) to binapacryl implying that resistance in strain BR was augmented by the effects of minor genes. Resistance to tetradifon (150–200 × on eggs) involved cross-resistance to tetrasul (23 ×) and to chlorfensulphide (27×); resistance in the field-derived strain appeared to depend on the major gene alone. Resistance mechanisms were highly specific in their effects, so that there was no cross-resistance to unrelated acaricides, including amitraz, dicofol, cyhexatin, formetanate, chlordimeform, and quinomethionate. Fecundity of strain BR was 44% less than that of EM; laboratory and field evidence suggest that multi-resistant mites tend to be less fit than susceptible mites. The practical implications are discussed.     

Apple ring rot-responsive putative microRNAs revealed by high-throughput sequencing in Malus × domestica Borkh.

MicroRNAs (miRNAs) are small non-coding RNAs, which silence target mRNA via cleavage or translational inhibition to function in regulating gene expression. MiRNAs act as important regulators of plant development and stress response. For understanding the role of miRNAs responsive to apple ring rot stress, we identified disease-responsive miRNAs using high-throughput sequencing in Malus × domestica Borkh.. Four small RNA libraries were constructed from two control strains in M. domestica, crabapple (CKHu) and Fuji Naga-fu No. 6 (CKFu), and two disease stress strains, crabapple (DSHu) and Fuji Naga-fu No. 6 (DSFu). A total of 59 miRNA families were identified and five miRNAs might be responsive to apple ring rot infection and validated via qRT-PCR. Furthermore, we predicted 76 target genes which were regulated by conserved miRNAs potentially. Our study demonstrated that miRNAs was responsive to apple ring rot infection and may have important implications on apple disease resistance.     

Differential Distribution of Genes Encoding the Virulence Factor Trans-Sialidase along Trypanosoma cruzi Discrete Typing Units

Trypanosoma cruzi the agent of Chagas disease is a monophyletic but heterogeneous group conformed by several Discrete Typing Units (DTUs) named TcI to TcVI characterized by genetic markers. The trans-sialidase (TS) is a virulence factor involved in cell invasion and pathogenesis that is differentially expressed in aggressive and less virulent parasite stocks. Genes encoding TS-related proteins are included in a large family divided in several groups but only one of them contains TS genes. Two closely related genes differing in a T/C transition encode the enzymatically active TS (aTS) and a lectin-like TS (iTS). We quantified the aTS/iTS genes from TcII and TcVI aggressive and TcI low virulent strains and found variable aTS number (1–32) per haploid genome. In spite of being low TS enzyme-expressers, TcI strains carry 28–32 aTS gene copies. The intriguing absence of iTS genes in TcI strains together with the presence of aTS/iTS in TcII and TcVI strains (virulent) were observed. Moreover, after sequencing aTS/iTS from 38 isolates collected along the Americas encompassing all DTUs, the persistent absence of the iTS gene in TcI, TcIII and TcIV was found. In addition, the sequence clustering together with T/C transition analysis correlated to DTUs of T. cruzi. The consistence of TS results with both evolutionary genome models proposed for T. cruzi, namely the “Two Hybridization” and the “Three Ancestor” was discussed and reviewed to fit present findings. Parasite stocks to attempt genetic KO or to assay the involvement of iTS in parasite biology and virulence are finally available.     

On gene flow between Tetranychus urticae Koch, 1836 and Tetranychus cinnabarinus (Boisduval) Boudreaux, 1956 (Acari: Tetranychidae): Sononomy between the two species

It was found that the reproductive separation between the two-spotted spider mite (Tetranychus urticae Koch) and the carmine spider mite (Tetranychus cinnabarinus (Boisduval) is far from complete. Hybrid strains were established which possessed the morphological characteristics of T. cinnabarinus, but which were homozygous for a marker gene originating from T. urtica. In addition, short-day treatment leads to diapause not only in the species from cold climates (T. urticae), but also in a population of T. cinnabarinus. It is questioned whether T. cinnabarinus deserves ranking at species level.

---

Résumé Ce travail montre que l'isolement reproductif entre Tetranychus uriticae Koch et Tetranychus cinnabarinus (Boisduval), est loin d'être complet. L'auteur a obtenu des souches hybrides présentant les caractéristiques morphologiques de T. cinnabarinus, mais homozygotes pour un gêne marqueur provenant de T. urticae. De plus, une diminution de la photopériode entraîne l'entrée en diapause des espèces provenant de régions à climat froid (T. urticae), mais aussi d'une population de T. cinnabarinus.Il est finalemant proposé de considérer Tetranychus cinnabarinus (Boisduval) Boudreaux, 1956, comme un synonyme de Tetranychus urticae Koch, 1836.