Restriction fragment length polymorphism catalog for molecular identification of Japanese Tetranychus spider mites (Acari: Tetranychidae)

Species identification is a basic issue in biosecurity. Polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) is a useful molecular diagnostic tool for species identification. However, the lack of transferability of data has been a serious shortcoming of this method. A RFLP catalog, i.e., a graph of PCR-RFLP patterns expected from sequence data, was devised as a tool to facilitate PCR-RFLP data sharing among laboratories. Twelve species of Tetranychus spider mites have been recorded in Japan to date. In this study, we analyzed DNA sequences of the internal transcribed spacer (ITS) region in nuclear ribosomal DNA of 11 Tetranychus species. For the species identification using PCR-RFLP, we chose six candidates from 131 restriction endonucleases and developed an RFLP catalog of all known Japanese Tetranychus species except Tetranychus neocaledonicus André. The RFLP catalog revealed that most Tetranychus species had diagnostic restriction fragments. The RFLP catalog is transferable and simple molecular diagnostic tool, and it has the ability to add more species and newly found intraspecific variations. Therefore, we believe that the RFLP catalog will contribute to biosecurity as a practical diagnostic tool for species identification of spider mites.     

Non-destructive direct polymerase chain reaction (direct PCR) greatly facilitates molecular identification of spider mites (Acari: Tetranychidae)

Spider mites (Acari: Tetranychidae) include serious agricultural pests and some species have spread globally as invasive species.. For this reason, rapid and simple identification of spider mite species is necessary for agricultural field and plant quarantine inspection. DNA sequence-based molecular techniques can rapidly identify spider mites. However, extracting DNA from minute invertebrates is difficult, expensive and time consuming. Here, we describe a polymerase chain reaction (PCR) technique in which the whole body of a spider mite adult is non-destructively soaked in PCR solution. The mite is then removed intact and can be used as a voucher specimen, leaving the PCR solution as the template and avoiding the need for a DNA extraction kit. For this study, we used six common spider mite species from four genera [Tetranychus urticae Koch (red form), Tetranychus kanzawai Kishida, Tetranychus parakanzawai Ehara, Oligonychus gotohi Ehara, Eotetranychus smithi Pritchard & Baker and Panonychus citri (McGregor)]. A portion of the mitochondrial cytochrome c oxidase subunit I (COI) gene was amplified with a universal primer pair from all individuals examined and sequenced. This method shortened the time for molecular identification from 9 to 5 h and eliminated the cost of commercial kits for DNA extraction [ca. 600 yen (~5.3 USD) per sample].     

Cardinium symbionts cause cytoplasmic incompatibility in spider mites

Intracellular symbiotic bacteria belonging to the Cytophaga-Flavobacterium-Bacteroides lineage have recently been described and are widely distributed in arthropod species. The newly discovered bacteria, named Cardinium sp, cause the expression of various reproductive alterations in their arthropod hosts, including cytoplasmic incompatibility (CI), induction of parthenogenesis and feminization of genetic males. We detected 16S ribosomal DNA sequences similar to those of Cardinium from seven populations of five spider mite species, suggesting a broad distribution of infection of Cardinium in spider mites. To clarify the effect of Cardinium on the reproductive traits of the infected spider mites, infected mites were crossed with uninfected mites for each population. In one of the populations, Eotetranychus suginamensis, CI was induced when infected males were crossed with uninfected females. The other six populations of four species showed no reproductive abnormalities in the F(1) generation, but the possibility of CI effects in the F(2) generation remains to be tested. One species of spider mite, Tetranychus pueraricola, harbored both Cardinium and Wolbachia, but these symbionts seemed to have no effect on the reproduction of the host, even when the host was infected independently with each symbiont.     

Biogenic amines in the two-spotted spider mite

Whole body extracts of the two-spotted spider mite (Tetranychus urticae Koch) were analyzed using a 16-channel electrochemical array high performance liquid chromatography (HPLC)-based detection system that allows the simultaneous isolation and identification of a variety of biogenic amines. The spider mite extracts were found to contain the biogenic amines octopamine, dopamine, and 5-hydroxytryptamine (5-HT), as well as several precursors and metabolites including tyrosine, tyramine, tryptophan, and N-acetyl octopamine. Differences in the levels of biogenic amines were observed between eggs and the adult stages and between males and females. This is the first direct determination of biogenic amines in the Tetranychidae and the first demonstration of 5-HT in any mite species. © 1994 Wiley-Liss, Inc.     

Survey of natural enemies of spider mites (Acari: Tetranychidae) in citrus orchards in eastern Spain

Field surveys were conducted from 2004 to 2007 to determine the species composition and relative abundance of natural enemies associated with colonies of either the citrus red mite, Panonychus citri, or the two spotted spider mite, Tetranychus urticae, in Valencian citrus orchards (eastern Spain). Fourteen species were recorded, six phytoseiid mites and eight insect predators. Two of them are reported for the first time on citrus in Spain and two more are first reports as predators associated with T. urticae. The community of predators associated with T. urticae and P. citri was almost identical, and the Morisita–Horn index of similarity between both natural enemy complexes was close to one, suggesting that predators forage on both pest species. Quantifying the presence of many known spider mites predators in Valencian citrus orchards is an important first step towards spider mite control. A challenge for future studies will be to establish conservation and/or augmentation management strategies for these predators, especially to improve T. urticae biological control.     

Intra-specific rDNA-ITS restriction site variation and an improved protocol to distinguish species and hybrids in the Daphnia longispina complex

A collaborative research effort was undertaken to evaluate the robustness of a recently developed genetic tool for species identification of members in the morphologically variable Daphnia longispina species complex. This genetic method, based on restriction fragment length polymorphism (RFLP) of the internal transcribed spacer region (ITS) of nuclear ribosomal DNA (rDNA) with restriction enzymes Mwo I and Sau96 I [Billiones et al., 2004. Hydrobiologia 526: 43–53], was applied to many different European populations. Results were compared with two or more independently obtained characters (morphology, allozymes, mitochondrial DNA (mtDNA), or cloned rDNA-ITS sequences). Individuals of most taxa were readily identified, but unexpected ITS-RFLP patterns were found in many individuals indicated by other markers to be D. galeata or one of its hybrids. Among 43 investigated D. galeata populations (902 specimen analysed by ITS-RFLP), deviant RFLP fragment patterns occurred in 26 (i.e., more than half) of the populations. The deviant patterns could be attributed to the loss of one single restriction site in the ITS2 region. This loss made the distinction of D. galeata from other species unreliable, and F1 hybrids could not be identified. Future users should be aware of this shortcoming of the Billions et al. [2004. Hydrobiologia 526: 43–53] protocol. As a solution to this problem, we present an improved genetic identification protocol based on a simple double digestion of the rDNA-ITS region with the restriction enzymes BsrB I and EagI. Sequence analyses of rDNA-ITS clones and preliminary testing indicate that the new protocol is unaffected by the rDNA variation which troubled the Mwo I/Sau96 I protocol. Further, the new protocol identifies all European species of the D. longispina complex, as well as their F1 hybrids. However, a wider screening is required to verify its general utility for all species, since yet unknown variation may occur. Guest editor: Piet Spaak Cladocera: Proceedings of the 7th International Symposium on Cladocera     

Evidence of a high level of gene flow among apple trees in <Emphasis Type="Italic">Tetranychus urticae</Emphasis>

The dispersal mechanism of the two-spotted spider mite Tetranychus urticae Koch (Acari: Tetranychidae) could affect predator–prey population dynamics and the spread of acaricide resistance. To investigate the propensity for spider mite migration in the field, the genetic structure of spider mite populations was studied in two apple orchards using five microsatellite markers. Adult female mites were collected from trees separated by approximately 10–24 m along a line covering a distance of about 100 m. The genetic data suggested that a high population density increased the migration rate among the breeding colonies within a single tree. Spatial autocorrelation analysis suggested a positive genetic structure in the first distance class within the two orchards, which might have been caused by crawling or short-distance aerial dispersal. Meanwhile, mites may also have a large-scale migration system that could cause a high level of gene flow and constrained isolation-by-distance or genetic clines within the approximately 100-m range of the study sites. Therefore, mites might aerially disperse over long distances on a scale of <100 m while also taking shorter trips among nearby trees within a distance of 10–24 m in the apple orchards.     

Sequence analysis of the ribosomal internal transcribed spacers region in spider mites (Prostigmata: Tetranychidae) occurring in citrus orchards in Eastern Spain: use for species discrimination

Tetranychus urticae is a polyphagous mite which is an important pest of citrus worldwide. This mite can be found feeding on many plant species occurring in the citrus agrosystem moving from weeds to trees. Because field samples consist of a mixture of different Tetranychidae species, as a first step necessary to further implement population characterisation of T. urticae, species‐discriminating criteria based on molecular techniques are needed. In this study, the nucleotide variation of the internal transcribed spacers (ITS) 1 and 2 and the intergenic 5.8S fragment of nuclear rDNA of T. urticae, Tetranychus turkestani, Tetranychus evansi, Tetranychus ludeni and Panonychus citri have been determined. Results demonstrate that for these species, the rDNA ITS2 regions are much more conserved than the corresponding rDNA ITS1. The high homogeneity of the ITS2 sequence observed among the specimens of T. urticae obtained from the same ecoregion makes this DNA sequence an excellent tool for species discrimination. ITS sequences differentiate not only species but also specimens from different geographical origin. Furthermore, polymerase chain reaction–restriction fragment length polymorphism analysis of the ITS2 proved adequate for a quick screening of high numbers of field samples.     

DNA barcoding gap: reliable species identification over morphological and geographical scales

The philosophical basis and utility of DNA barcoding have been a subject of numerous debates. While most literature embraces it, some studies continue to question its use in dipterans, butterflies and marine gastropods. Here, we explore the utility of DNA barcoding in identifying spider species that vary in taxonomic affiliation, morphological diagnosibility and geographic distribution. Our first test searched for a ‘barcoding gap’ by comparing intra‐ and interspecific means, medians and overlap in more than 75 000 computed Kimura 2‐parameter (K2P) genetic distances in three families. Our second test compared K2P distances of congeneric species with high vs. low morphological distinctness in 20 genera of 11 families. Our third test explored the effect of enlarging geographical sampling area at a continental scale on genetic variability in DNA barcodes within 20 species of nine families. Our results generally point towards a high utility of DNA barcodes in identifying spider species. However, the size of the barcoding gap strongly depends on taxonomic groups and practices. It is becoming critical to define the barcoding gap statistically more consistently and to document its variation over taxonomic scales. Our results support models of independent patterns of morphological and molecular evolution by showing that DNA barcodes are effective in species identification regardless of their morphological diagnosibility. We also show that DNA barcodes represent an effective tool for identifying spider species over geographic scales, yet their variation contains useful biogeographic information.     

Pest species diversity enhances control of spider mites and whiteflies by a generalist phytoseiid predator

To test the hypothesis that pest species diversity enhances biological pest control with generalist predators, we studied the dynamics of three major pest species on greenhouse cucumber: Western flower thrips, Frankliniella occidentalis (Pergande), greenhouse whitefly, Trialeurodes vaporariorum (Westwood), and two-spotted spider mites, Tetranychus urticae Koch in combination with the predator species Amblyseius swirskii Athias-Henriot. When spider mites infested plants prior to predator release, predatory mites were not capable of controlling spider mite populations in the absence of other pest species. A laboratory experiment showed that predators were hindered by the webbing of spider mites. In a greenhouse experiment, spider mite leaf damage was lower in the presence of thrips and predators than in the presence of whiteflies and predators, but damage was lowest in the presence of thrips, whiteflies and predators. Whitefly control was also improved in the presence of thrips. The lower levels of spider mite leaf damage probably resulted from (1) a strong numerical response of the predator (up to 50 times higher densities) when a second and third pest species were present in addition to spider mites, and (2) from A. swirskii attacking mobile spider mite stages outside or near the edges of the spider mite webbing. Interactions of spider mites with thrips and whiteflies might also result in suppression of spider mites. However, when predators were released prior to spider mite infestations in the absence of other pest species, but with pollen as food for the predators, we found increased suppression of spider mites with increased numbers of predators released, confirming the role of predators in spider mite control. Thus, our study provides evidence that diversity of pest species can enhance biological control through increased predator densities.     

PCR-based identification of the pathogenic bacterium, Acaricomes phytoseiuli, in the biological control agent Phytoseiulus persimilis (Acari: Phytoseiidae)

The predatory mite, Phytoseiulus persimilis is an important biological control agent of herbivorous spider mites. This species is also intensively used in the study of tritrophic effects of plant volatiles in interactions involving plants, herbivores, and their natural enemies. Recently, a novel pathogenic bacterium, Acaricomes phytoseiuli, has been isolated from adult P. persimilis females. This pathogen causes a characteristic disease syndrome with dramatic changes in longevity, fecundity, and behavior. Healthy P. persimilis use spider mite-induced volatiles to locate prey patches. Infection with A. phytoseiuli strongly reduces the attraction to herbivore-induced plant volatiles. The loss of response to herbivore-induced plant volatiles along with the other disease symptoms can have a serious impact on the success of biological control of spider mites. In this study, we have developed a molecular tool (PCR) to detect the pathogenic bacterium in individual predatory mites. PCR primers specific for A. phytoseiuli were developed based on 16S ribosomal DNA of the bacterium. The PCR test was validated with DNA extracted from predatory mites that had been exposed to A. phytoseiuli. A survey on different P. persimilis populations as well as other predatory mite species from several companies that rear predatory mites for biological control revealed that the disease is widespread in Europe and is restricted to P. persimilis. The possibility that the predatory mites get infected via their prey Tetranychus urticae could be eliminated since the PCR test run on prey gave a negative result.     

Predatory mirids of the green apple aphidAphis pomi,the two-spotted spider miteTetranychus urticae and the European red mitePanonychus ULMI in apple orchards in Québec

The incidence of predation of eight species of predacious mirids (Hemiptera: Miridae) present in an apple orchard of Québec on the green apple aphid, two-spotted spider mite and European red mite were investigated. The daily consumption rates varied from 1–2 green apple aphids forHyaliodes vitripennis Say andCampylomma verbasci Meyer to 7–9 aphids forDeraeocoris fasciolus Knight andLepidopsallus minisculus Knight.H. vitripennis consumed significantly more mites than the other mirid species with 26 and 18 mites per day for the two-spotted spider mite and the European red mite respectively. The combined use ofH. vitripennis andL. minisculus is suggested for the control of phytophagous mites. This paper is contribution No. 335/91.06.02R, Research Station, Agriculture Canada, Saint-Jean-sur-Richelieu, Québec, Canada.     

Evaluation of airborne methyl salicylate for improved conservation biological control of two-spotted spider mite and hop aphid in Oregon hop yards

The use of synthetic herbivore-induced plant volatiles (HIPV) to attract natural enemies has received interest as a tool to enhance conservation biological control (CBC). Methyl salicylate (MeSA) is a HIPV that is attractive to several key predators of two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), and hop aphid, Phorodon humuli (Schrank) (Homoptera: Aphididae). A 2-year study was conducted to evaluate the recommended commercial use of MeSA in hop yards in Oregon. Slow-release MeSA dispensers were stapled to supporting poles in 0.5 ha plots and these plots were compared to a paired non-treated plot on each of three farms in 2008 and 2009. Across both years, there was a trend for reduced (range 40–91%) mean seasonal numbers of T. urticae in five of the six MeSA-baited plots. Stethorus spp., key spider mite predators, tended to be more numerous in MeSA-baited plots compared to control plots on a given farm. Mean seasonal densities of hop aphid and other natural enemies (e.g., Orius spp. and Anystis spp.) were similar between MeSA-treated and control plots. Variability among farms in suppression of two-spotted spider mites and attraction of Stethorus spp. suggests that the use of MeSA to enhance CBC of spider mites in commercial hop yards may be influenced by site-specific factors related to the agroecology of individual farms or seasonal effects that require further investigation. The current study also suggests that CBC of hop aphid with MeSA in this environment may be unsatisfactory.     

Effect of African nightshade species (Solanaceae) on developmental time and life table parameters of <Emphasis Type="Italic">Tetranychus evansi</Emphasis> (Acari: Tetranychidae)

The effect of five African nightshade (Solanum sp.) species on the biological and demographic parameters of the tomato spider mite, Tetranychus evansi Baker and Pritchard, was examined in the laboratory at 25 ± 1°C, 70–80% RH and 12L:12D photoperiod. Duration of each development stage, reproduction rate, longevity, intrinsic rate of natural increase (r _m), and doubling time (DT) of the tomato spider mite on the five nightshade species were calculated. The results indicated that S. villosum, S. scabrum, S. tarderemotum and S. americanum are more suitable for T. evansi due to a shorter developmental period, longer adult longevity, higher reproduction and intrinsic rate of natural increase ranging between 0.180 and 0.196 females/female/day compared with S. sarrachoides which cannot support T. evansi populations as the r _m (−0.063 females/female/day) and DT were negative on this host. Differences in developmental time and life table parameters among the other host plants were not significant.     

Microsatellite markers reveal spatial genetic structure of Tetranychus urticae (Acari: Tetranychidae) populations along a latitudinal gradient in Europe

The genetic structure of populations of the two-spotted spider mite Tetranychus urticae was investigated along a south–north European transect spanning from southern France to The Netherlands. Mites were collected on Urtica dioica in 6 sampling zones. Microsatellite variation at 5 loci revealed considerable genetic variation with an average heterozygozity of 0.49. Significant heterozygote deficiency was found in 7 populations out of the 18 samples analyzed and one of them was completely monomorphic. Tetranychus urticae populations show some level of genetic structuring. First, genetic differentiation between localities (F _ST estimates) was significant for all comparisons. Second, the analysis of molecular variance, AMOVA, indicates that there is an effect, albeit low (9%), of the locality in accounting for allele frequency variance. Geographic distance emerges as a factor responsible for this genetic structure. The results are discussed in relation to the biological features of the species and the known patterns of migration. Related agronomical issues are addressed.     

Migration of specialist insect predators to exploit patchily distributed spider mites

It is believed that specialist predators of spider mites often migrate by flight or aerial transport to exploit patchily distributed prey. The migration is an important factor in determining the seasonal occurrence of the predators in a field. Several species of specialist insect predators, such as Oligota kashmirica benefica (Coleoptera: Staphylinidae) and Scolothrips takahashii (Thysanoptera: Thripidae), migrate between orchard trees and plants near the trees (e.g., groundcover, weeds, or windbreaks) to exploit abundant spider mites. This migration is at least partly triggered by prey scarcity in the original local habitats. Although these predators are tiny insects, they have flight abilities. For example, adult O. kashmirica benefica (body length, ∼1 mm) could move at least 5–16 m in one flight. Presumably, migration of the insect predators between prey-infested plants occurs mainly by flights. Predatory mites, such as Amblyseius fallacis and Amblyseius womersleyi (Acari: Phytoseiidae), migrated to spider mite-infested plants outside an orchard by aerial transport when they suffered from prey scarcity in the orchard. S. takahashii can use plant volatiles from lima bean plants induced by the spider mite Tetranychus urticae as cues for prey location during migration in Satsuma mandarin groves. However, it remains unknown how far from the trap boxes S. takahashii could respond to herbivore-induced plant volatiles in the groves. Received: August 25, 2000 / Accepted: February 1, 2001     

Diapause incidence in the two-spotted spider mite increases due to predator presence, not due to selective predation

We recently reported evidence for increased diapause incidence in the spider mite Tetranychus urticae in presence of the predatory mite Typhlodromus pyri. This effect may arise from (1) selective predation on non-diapause spider mites, (2) predator-induced diapause in spider mites, or (3) both. Using a different strain of T. urticae, we first recovered increased diapause incidence in association with predators. Then, we tested for selective feeding in two-choice experiments with equal numbers of non-diapause and diapause spider mites. We found that the predatory mite had a significant preference for the latter. This indicates that increased diapause incidence in association with predatory mites is not due to selective predation. Therefore, predator-mediated physiological induction of diapause seems a more likely explanation. The cues leading to induction appear to relate to the predators, not their effects, since predation simulated by spider-mite removal or puncturing did not significantly affect diapause incidence. Why spider mites benefit from this response, remains an open question.This revised version was published online in May 2005 with a corrected cover date.     

A DNA barcoding approach for identification of hidden diversity in Parmeliaceae (Ascomycota): Parmelia sensu stricto as a case study

Accurate specimen identification is challenging in groups with subtle or scarce taxonomically diagnostic characters, and the use of DNA barcodes can provide an effective means for consistent identification. Here, we investigate the utility of DNA barcode identification of species in a cosmopolitan genus of lichen‐forming fungi, Parmelia (Parmeliaceae). Two hundred and two internal transcribed spacer (ITS) sequences generated from specimens collected from all continents, including Antarctica, were analysed, and DNA barcodes of 14 species of Parmelia s.s. are reported. Almost all species show a barcode gap. Overall, intraspecific divergence values were lower than the threshold previously established for Parmeliaceae. However, the mean and range were elevated by deep barcode divergences in three species, indicating the likely occurrence of overlooked species‐level lineages. Here, we provide a DNA barcode reference library with well‐identified specimens sampled worldwide and sequences from most of the type material to enable easy and fast accurate sample identification and to assist in uncovering overlooked species in Parmelia s.s. Further, our results confirm the efficiency of the ITS region in the identification of species of Parmelia s.s. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180 , 21–29.     

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.     

Odour-mediated responses of a predatory mirid bug and its prey, the two-spotted spider mite

It has been shown that many natural enemies of herbivorous arthropods use herbivore induced plant volatiles (HIPVs) to locate their prey. Herbivores can also exploit cues emitted by plants infested with heterospecifics or conspecifics. A study was conducted to test whether green bean HIPVs as well as odours emitted directly by spider mites influenced the orientation behaviour of the predatory mirid bug, Macrolophus caliginosus and its prey, Tetranychus urticae in a Y-tube olfactometer. Our results show that both spider mites and M. caliginosus preferred spider mite infested green bean plants to uninfested plants. For M. caliginosus this response was mediated by HIPVs whereas for T. urticae it was mediated through a composite response to both HIPVs and odours emitted directly by the conspecifics (and their associated products). The results may be of use in practical biocontrol situations, through e.g., plant breeding for improved HIPV production, conditioning of mass-reared predators to appropriate cues, and employment of “push–pull-strategies” by using HIPVs.