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.     

Fertilization in the two-spotted spider mite (Tetranychus urticae: Acari)

Different aspects of the fertilization of the two-spotted spider mite (Tetranychus urticae Koch) were studied. It appeared that after the mating act of virgin females, haploid eggs were produced first before the appearance of diploid eggs. From this and other facts, it was concluded that fertilization of the oocytes occurs in a very early stage of egg development. The fertilization is assumed to take place in the ovary and not in the oviduct.By using genetic markers, it could be proved that in most cases the first mating is effective. Later matings usually are ineffective. It is suggested that the sperm supply in the first mating determines the success of later matings.

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Zusammenfassung Die Befruchtung der Bohnenspinnmilbe wurde unter verschiedenen Gesichtspunkten untersucht. Nach der Begattung jungfräulicher Weibchen wurden zunächst haploide Eier abgelegt, bevor diploide Eier produziert wurden. Daraus und aus anderen Grüunden wird geschlossen, daß die Befruchtung der Oocyten in einem sehr frühen Stadium der Entwicklung erfolgt. Die Befruchtung findet vermutlicht im Ovar und nicht im Ovidukt statt.Unter Verwendung genetischer Markierung wurde nachgewiesen, daß in den meisten Fällen die erste Begattung ausschlaggebend ist. Spätere Kopulationen bleiben gewöhnlich wirkungslos. Es wird vermutet, daß die Spermafüllung bei der ersten Begattung den Erfolg späterer Begattungen bestimmt.

     

Wolbachia-induced 'hybrid breakdown' in the two-spotted spider mite Tetranychus urticae Koch

The most common post-zygotic isolation mechanism between populations of the phytophagous mite Tetranychus urticae is 'hybrid breakdown', i.e. when individuals from two different populations are crossed, F1 hybrid females are produced, but F2 recombinant male offspring suffer increased mortality. Two-spotted spider mites collected from two populations, one on rose and the other on cucumber plants, were infected with Wolbachia bacteria. These bacteria may induce cytoplasmic incompatibility in their hosts: uninfected (U) females become reproductively incompatible with infected (W) males. We report on the effect of Wolbachia infections in intra- and interstrain crosses on (i) F1 mortality and sex ratios (a test for cytoplasmic incompatibility), and (ii) the number of haploid offspring and mortality in clutches of F1 virgins (a test for hybrid breakdown). U x W crosses within the rose strain exhibited partial cvtoplasmic incompatibility. More interestingly, F2 males suffered increased mortality, a result identical to the hybrid breakdown phenomenon. The experiments were repeated using females from the cucumber strain. In interstrain U x W and U x U crosses, hybrid breakdown was much stronger in the former (80 versus 26%). This is the first report of a Wolbachia infection causing a hybrid breakdown phenotype. Our results show that Wolbhachia infections can contribute to reproductive incompatibility between populations of T. urticae.     

Demographic examination of sex ratio in the two-spotted spider mite, Tetranychus urticae

Primary sex ratio of arrhenotokous tetranychid mites depends on the age-specific schedule of ratios of progeny produced by individual females. Since primary sex ratio is conditional upon female age and since the proportion of females of a given age in a population is influenced by the rate of increase, both the agespecific ratio of males to females and the overall ratio of males to females depends on the rate of increase. We given an expression for this dependence and compare it to experimental results for the two-spotted spiter mite. These results imply that there is no standard sex ratio of spider mites because of interpopulation variation in rates of increase.

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Résumé Le taux sexuel des acariens tétranychidés arrhénotoques est défini comme le rapport de la somme des nombres de mâles sur la somme des nombres de femelles pour chaque âge de la distribution en âges. Comme le taux sexuel primaire dépend de l'âge de la femelle et comme la proportion de femelles d'un âge donné dans la population est influencée par le taux d'accroissement, alors le rapport mâle/femelle spécifique d'un âge déterminé et le rapport global mâle/femelle dépendent du taux d'accroissement. Nous avons cherché à exprimer cette dépendance et à la comparer aux résultats expérimentaux pour Tetranychus urticae. Ces résultats signifient qu'il n'y a pas de taux sexuel caractéristique de Tétranyques par suite des variations du taux d'accroissement suivant les populations.

     

Genetics of diapause supression in the two-spotted spider mite,Tetranychus urticae Koch

Starting from the Sambucus strain of the two-spotted spider miteTetranychus urticae Koch, the strains ND₁ and ND₂ were selected for lack of diapause under a short-day regimen (LD 1014, 18°C). The genetic basis of the suppression of diapause in ND₁ and ND₂ was analysed by means of mendelian crosses and backcrosses, using the photoperiodic cycle LD 1014 as diagnostic for the distinction of diapausing and non-diapausing phenotypes. Suppression of diapause is inherited as a recessive trait. From backcross analysis it was concluded that the suppression of diapause in ND₁ and ND₂ is under monogenic control. The major genes for suppression of diapause in ND₁ and ND₂ are alleles at a locus designated asd. A mutation for albinism of the locusa-p is pleiotropic for suppression of diapause. It was shown from crosses between ND₁ and the albino strain, thatd anda-p are distinct loci.     

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.     

Digestive proteases in bodies and faeces of the two-spotted spider mite,Tetranychus urticae

Digestive proteases of the phytophagous mite Tetranychus urticae have been characterised by comparing their activity in body and faecal extracts. Aspartyl, cathepsin B- and L-like and legumain activities were detected in both mite bodies and faeces, with a specific activity of aspartyl and cathepsin L-like proteases about 5- and 2-fold higher, respectively, in mite faeces than in bodies. In general, all these activities were maintained independently of the host plant where the mites were reared (bean, tomato or maize). Remarkably, this is the first report in a phytophagous mite of legumain-like activity, which was characterised for its ability to hydrolyse the specific substrate Z-VAN-AMC, its activation by DTT and inhibition by IAA but not by E-64. Gel free nanoLC–nanoESI-QTOF MS/MS proteomic analysis of mite faeces resulted in the identification of four cathepsins L and one aspartyl protease (from a total of the 29 cathepsins L, 27 cathepsins B, 19 legumains and two aspartyl protease genes identified the genome of this species). Gene expression analysis reveals that four cathepsins L and the aspartyl protease identified in the mite faeces, but also two cathepsins B and two legumains that were not detected in the faeces, were expressed at high levels in the spider mite feeding stages (larvae, nymphs and adults) relative to embryos. Taken together, these results indicate a digestive role for cysteine and aspartyl proteases in T. urticae. The expression of the cathepsins B and L, legumains and aspartyl protease genes analysed in our study increased in female adults after feeding on Arabidopsis plants over-expressing the HvCPI-6 cystatin, that specifically targets cathepsins B and L, or the CMe trypsin inhibitor that targets serine proteases. This unspecific response suggests that in addition to compensation for inhibitor-targeted enzymes, the increase in the expression of digestive proteases in T. urticae may act as a first barrier against ingested plant defensive proteins.     

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.     

Effects of triflumuron on the two-spotted spider mite,Tetranychus urticae (Acari: Tetranichydae)

The effects of triflumuron on the mortality, fecundity, and fertility of the two-spotted spider mite, Tetranychus urticae, were evaluated in the laboratory. No differences in toxicity for larvae, protonymphs and deutonymphs were observed, but immature stages were 3.8-times more susceptible than adults at the LC₅₀. The compound exhibited a direct contact ovicidal activity influenced by eggs age. 48–72 hold eggs were significantly more sensitive than eggs of the other age classes. No hatch inhibition was observed in eggs laid by treated adult females using a sublethal dose at two physiological times (<12 and 48–72h old). However, fecundity decreased in younger treated females, but it increased in the older-ones. The toxicity for immatures and eggs, and the sublethal effects described suggest that triflumuron could be an interesting incorporation in integrated pest programs of T. urticae. This revised version was published online in July 2006 with corrections to the Cover Date.     

Aerial dispersal of the two-spotted spider mite (Tetranychus urticae) from field corn

Field data from 48 plots monitored during diverse weather conditions in two separate years were subjected to multiple regression analysis to determine which factors were related to spider-mite aerial dispersal. With the number of aerially dispersing mites as the dependent variable, partial regression coefficients (b) for mite population density and percent corn-leaf area infested with mites were positive, while those for percentNeozygites-infected mites and hours per week 90% r.h. were negative. When an aerial dispersal index (number of aerially dispersing mites/mites per plant) was used as the dependent variable, the partial regression coefficient for percent leaf area infested was positive, while coefficients for hours per week 90% r.h. and percentNeozygites-infected mites were negative. Mite aerial dispersal was greatest in predator-suppressed field plots under dry weather conditions. Mite aerial dispersal was substantially reduced in plots where moist weather conditions induced epizootics ofNeozygites floridana before corn plants became entirely infested with mites.     

Genome wide gene-expression analysis of facultative reproductive diapause in the two-spotted spider mite Tetranychus urticae

Background

Diapause or developmental arrest, is one of the major adaptations that allows mites and insects to survive unfavorable conditions. Diapause evokes a number of physiological, morphological and molecular modifications. In general, diapause is characterized by a suppression of the metabolism, change in behavior, increased stress tolerance and often by the synthesis of cryoprotectants. At the molecular level, diapause is less studied but characterized by a complex and regulated change in gene-expression. The spider mite Tetranychus urticae is a serious polyphagous pest that exhibits a reproductive facultative diapause, which allows it to survive winter conditions. Diapausing mites turn deeply orange in color, stop feeding and do not lay eggs.

Results

We investigated essential physiological processes in diapausing mites by studying genome-wide expression changes, using a custom built microarray. Analysis of this dataset showed that a remarkable number, 11% of the total number of predicted T. urticae genes, were differentially expressed. Gene Ontology analysis revealed that many metabolic pathways were affected in diapausing females. Genes related to digestion and detoxification, cryoprotection, carotenoid synthesis and the organization of the cytoskeleton were profoundly influenced by the state of diapause. Furthermore, we identified and analyzed an unique class of putative antifreeze proteins that were highly upregulated in diapausing females. We also further confirmed the involvement of horizontally transferred carotenoid synthesis genes in diapause and different color morphs of T. urticae.

Conclusions

This study offers the first in-depth analysis of genome-wide gene-expression patterns related to diapause in a member of the Chelicerata, and further adds to our understanding of the overall strategies of diapause in arthropods.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-14-815) contains supplementary material, which is available to authorized users.     

Vertical dispersal of the two-spotted spider mite Tetranychus urticae, and the predatory mite Phytoseiulus persimilis on dwarf hops

1 The pattern of dispersion within plants of the two-spotted spider mite, Tetranychus urticae, and its predator, the phytoseiid Phytoseiulus persimilis, was studied on the dwarf hop variety First Gold from May to September in 1997 and 1998. 2 Spider mite populations developed on the lower leaves initially but, by late July, as the numbers of mites increased, most were found towards the top of plants. From early August, the numbers of spider mites decreased most rapidly on the upper parts of plants. 3 Where P. persimilis was released, the predator maintained the numbers of T. urticae below those found on non-release plots throughout the season. 4 By early August, the predator’s pattern of dispersion was similar to that of the pest. 5 Predators spread to non-release plots by 20 June in 1997 and 24 July in 1998 and eventually became more numerous than on the plots where they had been released.     

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.     

Sequence of a cDNA encoding acetylcholinesterase from susceptible and resistant two-spotted spider mite,Tetranychus urticae

Acetylcholinesterase (AChE) from two-spotted spider mites, Tetranychus urticae was compared between an organophosphate susceptible (TKD) and a resistant (NCN) strain. The AChE of TKD had lower affinity to acetylthiocholine and propionylthiocholine than that of NCN, and the inhibition of AChE by DDVP, ambenonium, eserine and n-methyl-eserine showed that NCN was more insensitive than TKD. AChE cDNA sequence was determined, and the 687 amino acids of primary structure were deduced. There were six replacements of amino acid residues in TKD and two in NCN. #F331(439)C was the only substitution unique to NCN, however, this mutation existed homozygously in only two out of nine mites. This residue is one of the gorge lining components, and #F331(439)C might act an important role in the sensitivity of AChE to the inhibitors.     

Functional response of Nesidiocoris tenuis (Hemiptera: Miridae) to the two-spotted spider mite,Tetranychus urticae

We examined the functional response of Nesidiocoris tenuis to varying densities of the two-spotted spider mite, Tetranychus urticae and found that the data fit a Type II response, with the attack rate (a) increasing with increased predator size. N. tenuis can be considered as a potential biological control agent of this pest in tomato.     

Inter- and intra-population variations in diapause attribute of the two-spotted spider mite,Tetranychus urticae Koch,in Japan

Populations of the two-spotted spider mite, Tetranychus urticae Koch collected from various localities and from various host plants in Japan showed wide variations in diapause attribute. Diapause percentages at 18°C/9L15D varied from nearly 100% in the north to 0% in the south-west. At intermediate latitudes the mites showed wide inter-population variations. Populations on herbaceous hosts in vinyl- or glass-houses gave significantly lower incidence of diapause than those on roses and deciduous fruit trees. Presence of winter hosts and better host quality under protected environments seemed to favour non-diapausing mites. The temperature threshold for diapause expression also varied widely among local populations. Northern populations consistently had higher and less variable thresholds than populations at intermediate latitudes with thresholds between 15 and 18°C. Inbred lines derived from a population in Kyoto exhibited a wide variation in diapause percentage at 18°C. These results show that diapause in T. urticae is a quantitative threshold trait and that populations in central Japan consist of a variety of genotypes with different diapause traits. This might provide a genetic source for adaptation to local and temporal variations in environmental conditions.     

Expression of pair-rule gene homologues in a chelicerate: early patterning of the two-spotted spider mite Tetranychus urticae

Embryo segmentation has been studied extensively in the fruit fly, DROSOPHILA: These studies have demonstrated that a mechanism acting with dual segment periodicity is required for correct patterning of the body plan in this insect, but the evolutionary origin of the mechanism, the pair-rule system, is unclear. We have examined the expression of the homologues of two Drosophila pair-rule genes, runt and paired (Pax Group III), in segmenting embryos of the two-spotted spider mite (Tetranychus urticae Koch). Spider mites are chelicerates, a group of arthropods that diverged from the lineage leading to Drosophila at least 520 million years ago. In T. urticae, the Pax Group III gene Tu-pax3/7 was expressed during patterning of the prosoma, but not the opisthosoma, in a series of stripes which appear first in even numbered segments, and then in odd numbered segments. The mite runt homologue (Tu-run) in contrast was expressed early in a circular domains that resolved into a segmental pattern. The expression patterns of both of these genes also indicated they are regulated very differently from their Drosophila homologues. The expression pattern of Tu-pax3/7 lends support to the possibility that a pair-rule patterning mechanism is active in the segmentation pathways of chelicerates.     

Chlorophyll and its degradation products in the two-spotted spider mite, Tetranychus urticae: observations using epifluorescence and confocal laser scanning microscopy

Chlorophyll and chlorophyll degradation products were observed in the two-spotted spider mite (Tetranychus urticae) using epifluorescence microscopy (EFM) and confocal laser scanning microscopy (CLSM). A clear red fluorescence (EFM) and a fluorescence induced by a laser wavelength of 650 nm (CLSM) were observed. In the lateral caeca, in the ventriculus and in the excretory organ, a bright light blue fluorescence was observed in close association with chlorophyll by using EFM. The same material can be localized with CLSM by using a laser with a wavelength of 488 nm. By comparison with synthetic guanine, this bright fluorescence is supposed to be guanine. The presence of guanine fluorescence in the mite pellets confirms this hypothesis. A possible mechanism for guanine formation is discussed.