Geographical distribution and origin of acetylcholinesterase mutations conferring acaricide resistance in Tetranychus urticae populations from Turkey

Two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), is a cosmopolitan pest species that can feed on more than 1000 host plant species. Historically, organophosphate (OP) and carbamate insecticides have been used to control this extremely polyphagous pest. However, its ability to develop acaricide resistance rapidly has led to failure in control. Mutations in acetylcholinesterase gene (ace), the target-site of OP and carbamate insecticides, have been reported to be one of the major mechanisms underlying this developing resistance. In this study, mutations previously associated with resistance (G119S, A201S, T280A, G328A, F331W/Y) in ace have been screened in 37 T. urticae populations collected across Turkey. All mutations were found in various populations, except G119S. Almost all populations had F331W/Y mutation (being fixed in 32 populations), whereas only two populations harboured A201S mutation, but not fixed. On the other hand, more than half of the populations contained T280A and G328A mutations. In addition, the presence of same haplotypes in populations originating from distinct geographic locations and a wide variety of ace haplotypes might indicate multiple origins of F331W and F331Y mutations; however, this needs further investigation. The results of area-wide screening showed that ace mutations are widely distributed among T. urticae populations. Therefore, the use of this group of insecticides should be limited or only rotational use might be regarded as a resistance management tool due to its different mode of action from other main acaricide groups in T. urticae control across Turkey.

     

Rapid development of 36 polymorphic microsatellite markers for Tetranychus truncatus by transferring from Tetranychus urticae

Tetranychus truncatus Ehara is a phytophagous spider mite that is now one of the most important pests of agricultural and economic crops in East and Southeast Asia. However, population genetics and other studies of T. truncatus have been impeded by the lack of microsatellite markers, which are expensive and time-consuming to identify. Previous studies indicated a high potential of cross-amplification of microsatellites in Tetranychus species, meaning that the microsatellite flanking sequences are sufficiently homologous among Tetranychus species that the primers for one species may work in another species. Here, we tested 205 primer pairs designed from the whole genome sequence of Tetranychus urticae Koch, a sister species of T. truncatus, for microsatellite markers in three populations of T. truncatus in China (N = 94). About half (102) of these primer pairs yielded the desired PCR products, 36 of which revealed polymorphism in T. truncatus. Each of the 36 markers harbored between 2 and 23 alleles, with a mean polymorphic information content of 0.589 (0.119–0.922 range). The mean observed and expected heterozygosity across loci and the three populations were 0.468 and 0.628, respectively. Of the 36 primer pairs, 22 also worked in Tetranychus piercei, but only a few of them worked in T. ludeni and T. phaselus. Cross-amplification is thus a cost-effective way to develop microsatellite markers, which can be of great value in population genetics studies.     

二斑叶螨对阿维菌素、哒螨灵和甲氰菊酯的抗性选育及其解毒酶活力变化

在室内模拟田间药剂的选择压力,用阿维菌素、哒螨灵和甲氰菊酯对二斑叶螨Tetranychuc urticae逐代处理,以选育其抗性种群。选育至12代,对阿维菌素抗性增长到6.72倍,对哒螨灵抗性增长到12.1倍,对甲氰菊酯抗性增长到19.9倍。酶抑制剂和离体酶活性的测定结果表明,阿维菌素抗性种群的多功能氧化酶和谷胱甘肽S-转移酶的活性均有所提高;二斑叶螨对哒螨灵的抗性可能与多功能氧化酶、羧酸酯酶的活性增强有关;而羧酸酯酶、多功能氧化酶和谷胱甘肽S-转移酶活性的增强可能是二斑叶螨对甲氰菊酯产生抗性的主要原因。     

The formation of collective silk balls in the spider mite Tetranychus urticae Koch

Tetranychus urticae is a phytophagous mite that forms colonies of several thousand individuals. These mites construct a common web to protect the colony. When plants become overcrowded and food resources become scarce, individuals gather at the plant apex to form a ball composed of mites and their silk threads. This ball is a structure facilitating group dispersal by wind or animal transport. Until now, no quantitative study had been done on this collective form of migration. This is the first attempt to understand the mechanisms that underlie the emergence and growth of the ball. We studied this collective behaviour under laboratory conditions on standardized infested plants. Our results show that the collective displacement and the formation of balls result from a recruitment process: by depositing silk threads on their way up to the plant apex, mites favour and amplify the recruitment toward the balls. A critical threshold (quorum response) in the cumulative flow of mites must be reached to observe the emergence of a ball. At the beginning of the balls formation, mites form an aggregate. After 24 hours, the aggregated mites are trapped inside the silk balls by the complex network of silk threads and finally die, except for recently arrived individuals. The balls are mainly composed of immature stages. Our study reconstructs the key events that lead to the formation of silk balls. They suggest that the interplay between mites' density, plant morphology and plant density lead to different modes of dispersions (individual or collective) and under what conditions populations might adopt a collective strategy rather than one that is individually oriented. Moreover, our results lead to discuss two aspects of the cooperation and altruism: the importance of Allee effects during colonization of new plants and the importance of the size of a founding group.     

Differences among plant species in acceptance by the spider mite Tetranychus urticae Koch

Abstract:  The spider mite Tetranychus urticae Koch has a broad range of host plants. However, the spider mite does not accept all plants to the same degree because of differences in nutritive and toxic constituents. Other factors, such as the induction of secondary metabolites, the morphology of a leaf surface and the presence of natural enemies, also play an important role in plant acceptance. We compared plants from various families in their degree of acceptance by the spider mite, to get an indication of the plant's direct defence. Glycine max (soybean), Humulus lupulus (hop), Laburnum anagyroides (golden chain) and Nicotiana tabacum (tobacco) were highly accepted by the spider mites. Different glandular hair densities among tobacco cultivars did not affect their suitability towards spider mites significantly. Solanum melalonga (eggplant), Robinia pseudo-acacia (black locust), Vigna unguiculata (cowpea) and Datura stramonium (thorn apple) were accepted by the spider mites to a lesser degree. Vitis vinifera (grapevine) was poorly accepted by the spider mite. It might be that the food quality of the leaves was not high enough to arrest the spider mites. Also, Capsicum annuum (sweet pepper) and especially Ginkgo biloba (ginkgo) were poorly accepted by the spider mite, probably because of the presence and concentration of certain of the secondary metabolites in the leaves. The spider mites accepted all the plants belonging to the Fabaceae for feeding, but those belonging to the Solanaceae showed a larger variance in spider mite acceptance varying from well accepted (tobacco) to poorly accepted (sweet pepper).     

Ultrahistology of oogenesis and vitellogenesis in the spider mite Tetranychus urticae

Histology of the ovary of the spider mite Tetranychus urticae is described light and electron microscopically with special reference to oogenesis and vitellogenesis of this mite. Morphology of the ovary is comparable to the typical sac-like chelicerate ovary with oocytes protruding from the ovarian surface, thus resulting in a grape-like appearance. According to different oogenetic stages, a germ, pre-vitellogenic and vitellogenic region can be observed. Oogonia and primary oocytes characterized by extranuclear material or 'yolk nuclei' are situated in the germ region. Primary oocytes develop into three-nucleated nurse cells situated in the periphery of the pre-vitellogenic region, and into pre-vitellogenic oocytes protruding from the ovarian surface. Growth of oocytes is performed while they are in ovarian pouches by uptake of nurse cell cytoplasm and following extraovarian yolk precursors. Intraoocyte yolk synthesis interpreted from altered cytoplasmic organelles also occurs. Processes taking place during oogenesis and vitellogenesis in T. urticae are compared to published information on yolk synthesis of other animal species.     

Geographic variation of diapause intensity in the spider mite Tetranychus urticae

Abstract. Eight strains of the spider mite Tetranychus urticae, originating from different localities in western and central Europe, with latitudes ranging from 40.5 to 60^oN, displayed marked differences in the period of chilling at 4^oC required for diapause termination under a diapause-maintaining short-day photoperiodic regime at 19^oC, to which the mites were transferred after the cold period. The higher the latitude from which the strains originated the longer was the period of chilling required for diapause termination, suggesting the presence of a gradient in diapause intensity, diapause being deeper the more northern the origin of the strains. Two strains originating from higher altitudes appeared to have a much deeper diapause than expected from their latitudinal origin. In addition, these two mountain strains showed mutual differences in diapause intensity, notwithstanding the fact that they originated from similar latitudes and altitudes; local climatic conditions probably act as strong selective forces with regard to diapause depth. All strains appeared to be sensitive to photoperiod during the period of diapause development. Diapause was quickly completed by a long-day photoperiod (LD 17:7 h), but was maintained by a short-day photoperiod (LD 10:14h). However, even under the latter regime sensitivity to photoperiod gradually diminished and eventually disappeared, thus leading to ‘spontaneous’ termination of diapause. The length of the period of diapause development, as measured by the sensitivity to photoperiod of diapausing mites, varied between strains; it was shorter in the southern strains and longer in the northern strains. The results indicate great variation in diapause intensity between strains, which is probably genetically determined and may have adaptive significance for this widespread species. When young females which had just entered diapause were kept for ever longer periods of time under the diapause inducing short-day regime at which they had been reared, before being transferred to the cold room, the duration of the period of chilling required for diapause termination was found to decrease proportionally in all three strains tested. These results suggest that intensification of diapause does not occur in T. urticae; diapause intensity seems to be highest at the beginning of diapause and to diminish gradually during diapause development.     

The Effects of Clofentezine on Life-table Parameters in Two-spotted Spider Mite Tetranychus urticae

Sublethal effects of the growth inhibitor, clofentezine, on life-table parameters of Tetranychus urticae Koch females treated at different developmental stages with a concentration causing >90% mortality were investigated. Females which survived treatment as 'early' (0-24 h old) eggs produced 12% more offspring than the untreated females during the first five days of oviposition. This resulted in a significant rise in the intrinsic rate of increase (rj): 0.324, compared to 0.299 in the untreated females. This effect may be interpreted as hormoligosis. Clofentezine treatment at any other developmental stage of T. urticae significantly decreased both longevity and fertility of female survivors. Females which survived treatment either as 'late' (72-96 h old) eggs or larvae had 2.6 times lower net reproductive rate (R0) than the untreated females, and the rj values were significantly lower: 0.242 and 0.215, respectively (0.285 in the untreated females). Females which survived treatment either as protonymphs or deutonymphs had 3.9 times and 6 times lower R0, respectively. Corresponding rj values were 0.178 and 0.146, respectively (0.247 in the untreated females). The clofentezine treatment at all stages influenced the age distribution of survivors. The sublethal effects of clofentezine and their impact on T. urticae management are discussed.     

UV radiation elevates arylalkylamine N-acetyltransferase activity and melatonin content in the two-spotted spider mite, Tetranychus urticae

Ultraviolet (UV) radiation produces reactive oxygen species (ROS) in mammals, where melatonin plays the role of a ROS scavenger. The melatonin synthetic enzyme arylalkylamine N-acetyltransferase (NAT) is a significant element in a possible ROS removal system. Changes in NAT activity and melatonin content were determined in the two-spotted spider mite Tetranychus urticae by irradiating it with monochromatic light using the Okazaki Large Spectrograph at the National Institute for Basic Biology, Okazaki, Japan. The NAT activity and melatonin content were suppressed by blue light (450nm). No effects of red light (650nm) on the NAT activity and melatonin content were observed. UV radiation had intensity-dependent dual effects on the NAT activity and melatonin content. In the UV-B (300nm) treatment, the NAT activity and melatonin content were suppressed at the intensity below 1mumolm(-2)s(-1) but elevated when the intensity was as high as 10mumolm(-2)s(-1). In the UV-A (350nm) treatment, the melatonin content was elevated when the intensity was as high as 10mumolm(-2)s(-1), though the NAT activity and melatonin content were suppressed at the intensity below 10 and 1mumolm(-2)s(-1), respectively. Elevation of the NAT activity and melatonin content by high intensity UV irradiation may indicate that the UV signals initiate melatonin synthesis for ROS removal in mites.     

The number of catalytic sites in acetylcholinesterase

By using two methods of titration, the number of active sites in acetylcholinesterase was determined. Either stepwise inhibition of the enzyme by an irreversible inhibitor, namely di-isopropyl phosphorofluoridate, or direct measurement of the concentration of active sites by titration with o-nitrophenyl dimethylcarbamate yielded an equivalent weight of approx. 130000 for an active site in acetylcholinesterase. This indicates two sites per molecule, since the native enzyme has a molecular weight of 260000.     

温度对二斑叶螨实验种群生长的影响

采用大樱桃叶片饲养二斑叶螨Tetranychus urticae Koch,组建其在15,20,25,30和35℃下的实验种群生命表,分析温度对二斑叶螨种群动态的影响。结果表明:在试验的温度范围内,二斑叶螨的发育速率随着温度的升高而加快,并符合Logistic模型。二斑叶螨全世代的发育起点温度和有效积温分别为11.85℃和170.39日.度。计算出在不同温度下的实验种群的内禀增长率(rm)、净增殖率(R0)、周限增长率(λ)、世代平均周期(T)和种群加倍时间(t)等种群参数。     

汤普森被毛孢对二斑叶螨致死率的建模分析

本文采用蒸汽压提供湿度的方法研究了在施用不同浓度的孢子悬液及不同温湿度下汤普森被毛孢对二斑叶螨Tetranychus urticae Koch的致死情况,并用灰色预测和回归分析建立了数学模型。结果表明:试验条件范围内,完全二次方程的拟合效果较好;在温度为28.09℃、湿度为100%、浓度为250万个/mL时,致死率达到81.66%,防效最佳;较湿度而言,温度对被毛孢的影响更为显著;在高浓度或高湿度下,被毛孢对环境的变化更为敏感。综合评价,汤普森被毛孢对二斑叶螨的防效欠佳,若欲推广,尚需进一步的改良。     

Identification of Wolbachia-responsive microRNAs in the two-spotted spider mite,Tetranychus urticae

Background

The two-spotted spider mite, Tetranychus urticae, is infected with Wolbachia, which have the ability to manipulate host reproduction and fitness. MicroRNAs (miRNAs) are small non-coding RNAs that are involved in many biological processes such as development, reproduction and host-pathogen interactions. Although miRNA was observed to involve in Wolbachia-host interactions in the other insect systems, its roles have not been fully deciphered in the two-spotted spider mite.

Results

Small RNA libraries of infected and uninfected T. urticae for both sexes (in total four libraries) were constructed. By integrating the mRNA data originated from the same samples, the target genes of the differentially expressed miRNAs were predicted. Then, GO and pathway analyses were performed for the target genes. Comparison of libraries showed that Wolbachia infection significantly regulated 91 miRNAs in females and 20 miRNAs in males, with an overall suppression of miRNAs in Wolbachia-infected libraries. A comparison of the miRNA and mRNA data predicted that the differentially expressed miRNAs negatively regulated 90 mRNAs in females and 9 mRNAs in males. An analysis of target genes showed that Wolbachia-responsive miRNAs regulated genes with function in sphingolipid metabolism, lysosome function, apoptosis and lipid transporting in both sexes, as well as reproduction in females.

Conclusion

Comparisons of the miRNA and mRNA data can help to identify miRNAs and miRNA target genes involving in Wolbachia-host interactions. The molecular targets identified in this study should be useful in further functional studies.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1122) contains supplementary material, which is available to authorized users.     

Behavioral aspects of dicofol resistance in the twospotted spider mite Tetranychus urticae

Behavioral analysis of near-isogenic dicofol-resistant and dicofol-susceptible spider mites did not uncover behavior patterns, present in resistant individuals yet absent in susceptible individuals, that would have reduced exposure to dicofol. On the contrary, homozygous resistant individuals were more likely than homozygous susceptible individuals to remain in contact with treated leaf areas. In the case of dicofol resistance in twospotted spider mites, resistance appears to have resulted in the loss, rather than the acquisition, of behavioral traits that lessen exposure to pesticide. Therefore, we conclude that behavioral resistance does not play a major role in twospotted spider mite resistance to dicofol.Deux génotypes de T. urticae Koch (un sensible et un résistant) ont servi à étudier les aspects comportementaux de la résistance aux pesticides: la durée et la fréquence des comportements de nutrition, de marche et d'attente ont été mesurés chez cet acarien. Dans toutes les expériences, des femelles de mêmes âges avaient été placées sur des feuilles jeunes de haricot (Phaseolus vulgaris) portant des résidus de dicofol.3 séries d'expériences ont été réalisées pour examiner:

Chemical investigation of aggregation behaviour in the two-spotted spider mite Tetranychus urticae

The silk produced by the group-living mite Tetranychus urticae provides group protection and is used as an informative material during habitat settlement, egg laying, mating, and dispersal events. In this context, cues contained in the silk and other materials produced by mites [eggs, black faeces (BF) and white faeces + silk (WFS)] were investigated. Chemical compounds were extracted by hexane or methanol, and choice tests were used to determine the individual attractiveness of each extract. For both solvents, individuals did not respond to the extract from eggs and WFS. BF extracts were attractive for both solvents. After separating the BF methanol extract into four different chemical components using thin layer chromatography, no component was determined to be responsible for mite attraction. This work supports the evidence that the faeces of T. urticae do contain substances that promote behavioural changes. Not particular chemical compounds but combinations of them seem to induce the mites’ preference. Moreover, the response of mites to chemicals seemed to be context dependent as mites belonging to populations with different densities differed in their attraction to BF extracts.     

Discrimination through silk recognition: The case of the two-spotted spider mite Tetranychus urticae

Choosing a suitable habitat is a main step in the settlement process, particularly for species having weak movement abilities. Reliable cues are thus needed for habitat selection. In silk-spinning arthropods, silk can be used as a social cue to select an appropriate location. Silk can also provide information on the presence of related or non-related individuals. In this article, we compare the settlement behaviour of two strains of the two-spotted spider mite, Tetranychus urticae, in response to the presence of silk woven by an individual of its own or another strain. We then examined how individuals behaved when confronted with both types of silk (own/another strain) simultaneously. Both strains were sensitive to related silk. Settlement decision for both strains did not differ according to the origin of the silk. Mites used the silk as a communication cue for habitat selection and strain discrimination. Our results provide experimental evidence for the use of multiple social cues in the settlement decision by weaving mites.     

The effect of ionic strength on reversible inhibition of catalytic activity of acetylcholinesterase by thione phosphonates

The effect of ionic strength was used to analyze the mechanism of reversible acetylcholinesterase inhibition by three alkoxymethylthionphosphonates. The most considerable realization of the hydrophobic interaction with the surroundings of the enzyme esteratic site was marked for n-butyl derivative (compound I). The replacement of piperidine by morpholine (compound II) resulted in a decrease of the anticholinesterase activity by an order due to enhancement of the inhibitor hydrophilicity. An increase of MgCl2 concentration promotes an enhancement of the uncompetitive component contribution for compound III contrast to compound II. Hydrophobicity of the phosphoryl part of the compound I molecule is balanced under hydrophobic interaction of the heterocyclic "cationic head" with the enzyme anionic site. The break of this equilibrium intensifies the allosteric regulation, on the one hand, and lowers the inhibitor efficiency, on the other hand.