A critical review on some closely related species of <Emphasis Type="Italic">Tetranychus</Emphasis> sensu stricto (Acari: Tetranychidae) in the public DNA sequences databases

Taxonomic misidentification of the specimens used to obtain DNA sequences is a growing problem reported for different groups of organisms, which threatens the utility of the deposited sequences in public DNA databases. This paper provides new evidence of misidentifications in molecular DNA public databases in phytophagous mites of the Tetranychidae family belonging to the group Tetranychus (Tetranychus). Several species in this group are of economic and quarantine importance in agriculture and among them Tetranychus urticae, a highly polyphagous mite causing outbreaks in many crops worldwide, is certainly the most studied. We analyzed and evaluated the identity of 105 GenBank accessions of ITS2 rDNA and 138 COI mtDNA sequences which were deposited as T. urticae and those of 14 other taxa morphologically closely related to Tetranychus sensu stricto. In addition, ITS2 and COI sequences of 18 T. urticae samples collected for this study and identified by morphological criteria, were generated and included in the analyzed dataset. Among the deposited sequences in the GenBank database, numerous cases of apparently mistaken identities were identified in the group Tetranychus s. str., especially between T. urticae, T. cinnabarinus, T. kanzawai and T. truncatus. Unreliable sequences (misidentified or dubious) were estimated at nearly 30%. In particular the analysis supports the invalidity of the controversial species status of T. cinnabarinus. More generally, it highlights the need of using combined morphological and molecular approaches to guarantee solid species diagnostics for reliable sequence accessions in public databases.     

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.     

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.     

Sublethal effects of esfenvalerate residues on pyrethroid resistant Typhlodromus pyri (Acari: Phytoseiidae) and its prey Panonychus ulmi and Tetranychus urticae (Acari: Tetranychidae)

The effect of residues of esfenvalerate on oviposition of the resistant strain of the predatory mite Typhlodromus pyri and its main prey, European red mite Panonychus ulmi and two-spotted spider mite Tetranychus urticae, were investigated. T. pyri showed a significant linear reduction in oviposition after 24h in the presence of increasing levels of esfenvalerate residue applied at the field rate. Furthermore, when given a choice, T. pyri preferred to lay eggs on residue-free surfaces. Of the two prey species, only P. ulmi showed significant avoidance of increasing levels of residues of the field rate concentration of esfenvalerate, as measured by runoff mortality, however both P. ulmi and T. urticae, when given a choice, showed a preference for esfenvalerate-free surfaces. As with the predatory mite T. pyri, both prey species showed a significant linear reduction of oviposition with increasing esfenvalerate residues and a preference to lay eggs on esfenvalerate-free surfaces. Esfenvalerate residues as high as 15X field rate were not repellent to pyrethroid-resistant T. pyri. The possible effects of these sublethal effects on predator-prey dynamics and implications for integrated mite control programmes in apple orchards are discussed.     

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.

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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.

     

Molecular approach in spider mites (Acari: Tetranychidae): Preliminary data on ribosomal DNA sequences

DNA sequence data were used to examine phylogenetic relationships between six species of economically important Tetranychidae mites:Eotetranychus carpini (Oudemans),E. pruni (Reck),Tetranychus pacificus McGregor,T. mcdanieli McGregor,T. turkestani Ugarov & Nikolski andT. urticae Koch. With primers directed toward conserved elements flanking the target region, the Polymerase Chain Reaction was used to amplify the ITS2 spacer of the ribosomal DNA molecule. The nucleotide sequence of a 300-bp fragment of the ITS2 was determined by direct sequencing and nucleotide divergence used for intra-generic comparison in mites. The resulting phylogenetic tree expressing interspecific relationships in genusTetranychus agrees with morphological data. The study demonstrates the usefulness of the approach in the assessment of the systematics and evolution of the group.     

Nuclear ribosomal DNA monophyly versus mitochondrial DNA polyphyly in two closely related mite species: the influence of life history and molecular drive

In two very closely related but reproductively isolated mite species, Tetranychus urticae and T. turkestani, we found nucleotide diversity to be extensive for mitochondrial DNA (mtDNA) cytochrome oxidase 1 (COI) (3-4%) but extremely reduced for nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS2) (less than 0.5%). By contrast, ITS2 was shown to evolve much faster than COI between species of this genus. Furthermore, we found that these two species are polyphyletic for mtDNA but monophyletic for rDNA. Thus it appears that despite its biparental transmission and multiplicity of copies in the genome, nuclear rDNA has a smaller effective population size than mtDNA in these species. The conjunction of efficient concerted evolution and/or gene conversion in the rDNA cluster, the haplodiploidy of these species and their female-biased sex ratio could account for this apparent contradiction.     

Life-history of predatory mites Neoseiulus californicus and Phytoseiulus persimilis (Acari: Phytoseiidae) on four spider mite species as prey, with special reference to Tetranychus evansi (Acari: Tetranychidae)

The commercially available strains of Phytoseiulus persimilis Athias-Henriot, the biological control agent of Tetranychus urticae Koch, perform poorly in the Western Mediterranean, probably because they are not well adapted to local climatic conditions. For that reason, efforts are being focused on the development of a biological control programme using native phytoseiid mites. Four species of red spider mites can be found in vegetable crops in eastern Spain: T. urticae, Tetranychus turkestani Ugarov and Nikolski, Tetranychus ludeni Zacher and the recently introduced Tetranychus evansi Baker and Pritchard. To evaluate their potential role as biological control agents, the present study evaluates the life-history of local populations of Neoseiulus californicus (McGregor) and P. persimilis when fed on T. urticae, T. turkestani, T. evansi, and T. ludeni in the laboratory. Results indicate that N. californicus and P. persimilis are able to feed and complete their development on the four tested red spider mite species. The predators may exhibit a particularly high capacity for population increase when fed on T. urticae, T. turkestani, and T. ludeni, thus may be able to provide effective control of these species in the field. When fed T. evansi, however, predator performance was poor; significant increase in development and preoviposition times, and a reduction in oviposition period and fecundity were recorded. The resultant low capacity for population growth suggests poor ability of the two tested predators to suppress T. evansi populations on commercial crops. It is unlikely therefore that P. persimilis and N. californicus, now being widely used to control T. urticae in greenhouse crops in Central Europe, will be able to halt any spread of T. evansi to greenhouse crops in temperate areas.     

Herbivore performance and plant defense after sequential attacks by inducing and suppressing herbivores

It is well known that herbivore-induced plant defenses alter host plant quality and can affect the behavior and performance of later arriving herbivores.Effects of sequential attacks by herbivores that either suppress or induce plant defenses are less well studied.We sequentially infested leaves of tomato plants with a strain of the phytophagous spider mite Tetranychus urticae that induces plant defenses and the closely related Tetranychus evansi, which suppresses plant defenses.Plant quality was quantified through oviposifion of both spider mite species and by measuring proteinase inhibitor activity using plant material that had been sequentially attacked by both herbivore species.Spider-mite oviposifion data show that T.evansi could suppress an earlier induction of plant defenses by T.urticae,and T.urticae could induce defenses in plants previously attacked by T.evansi in 1 day.Longer attacks by the second species did not result in further changes in oviposifion.Proteinase inhibitor activity levels showed that T.evansi suppressed the high activity levels induced by T.urticae to constitutive levels in 1 day,and further suppressed activity to levels similar to those in plants attacked by T.evansi alone.Attacks by T.urticae induced proteinase inhibitor activity in plants previously attacked by T.evansi,eventually to similar levels as induced by T.urticae alone.Hence,plant quality and plant defenses were significantly affected by sequential attacks and the order of attack does not affect subsequent performance,but does affect proteinase inhibitor activity levels.Based on our results,we discuss the evolution of suppression of plant defenses.     

The effect of groundcover and herbicide treatment on twospotted spider mite density and dispersal in southern Oregon pear orchards

A total of 49 groundcover plant species representing 47 genera in 22 families were identified from a survey of 5 pear orchards. Density of twospotted spider mite (Tetranychus urticae Koch) inhabiting these plants was estimated visually several times during the field season. Plants were ranked in 1 of 3 categories depending on mite densities found on these plants during the summer. T. urticae was found to be highly abundant (category 3) on 26 species, at lower densities on 10 species (category 2), and was rarely or never found on the remaining 12 species (category 1). Dispersal of mites from groundcover plants into trees was found to be highly variable within and between orchards. Within orchard dispersal appeared to be related to the distribution and abundance of category 3 host plants in the orchard. Variability between orchards may also be affected by groundcover management techniques and levels of acaricide resistance in T. urticae. The use of herbicides to control groundcover plants significantly increased the dispersal of T. urticae into the orchard trees.     

Mite species identification in the production of allergenic extracts for clinical use and in environmental samples by ribosomal DNA amplification

The identification of allergy‐causing mites is conventionally based on morphological characters. However, molecular taxonomy using ribosomal DNA (rDNA) may be particularly useful in the analysis of mite cultures and purified mite fractions in the production of allergenic extracts. Full‐length internal transcribed spacers (ITS1 and ITS2) were obtained from Dermatophagoides farinae, Dermatophagoides pteronyssinus, Dermatophagoides microceras and Euroglyphus maynei (Astigmata: Pyroglyphidae), Glycyphagus domesticus and Lepidoglyphus destructor (Astigmata: Glycyphagidae), Tyrophagus fanetzhangorum, Tyrophagus putrescentiae, Tyrophagus longior, Tyrophagus neiswanderi, Acarus farris and Acarus siro (Astigmata: Acaridae), and Blomia tropicalis (Astigmata: Echymopodidae), using mite‐specific primers. Polymerase chain reaction (PCR) products were digested with HpaII and RsaI restriction enzymes in order to produce species‐specific PCR restricted fragment length polymorphism (RFLP) profiles. A semi‐nested re‐amplification step was introduced before the RFLP in order to apply the method to environmental samples. Results demonstrate that rDNA sequences can be used for the unambiguous identification of mite species. The PCR–RFLP system allows the identification of species in purified mite fractions when the availability of intact adult mite bodies for morphological identification is limited. This reliable and straightforward PCR–RFLP system and the rDNA sequences obtained can be of use in the identification of allergy‐causing mite species.     

Predator avoidance in phytophagous mites: response to present danger depends on alternative host quality

We studied whether volatiles released by putative host plants affect the antipredator response of an herbivorous mite, Tetranychus urticae, when the patch was invaded by Phytoseiulus persimilis. Tetranychus urticae laid a lower number of eggs on tomato leaves than on lima bean leaves, suggesting that lima bean is a preferred host food source for T. urticae. In addition, T. urticae preferred lima bean plant volatiles to tomato plant volatiles in a Y-tube olfactometer test. To investigate the antipredator response of T. urticae, we examined the migration of T. urticae from a lima bean leaf disc to a neighbouring leaf disc (either a tomato or lima bean leaf disc) when ten predators were introduced into the original lima bean disc. A Parafilm bridge allowed for migration between the leaf discs. No migrations occurred between leaf discs when there were no predators introduced to the original leaf disc. However, when predators were introduced migrations did occur. When the neighbouring leaf disc was upwind of the original disc, the migration rate of the mite from original lima bean leaf disc to a neighbouring tomato leaf disc was significantly lower than that to a neighbouring lima bean leaf disc. By contrast, when the neighbouring leaf disc was downwind of the original leaf disc, there was no difference in the migration rates between lima bean leaf discs and tomato leaf discs. The number of T. urticae killed by P. persimilis for each treatment was not different, and this clearly shows that the danger was the same in all treatments regardless of the decision made by T. urticae. From these results, we conclude that T. urticae change their antipredator response by evaluating the difference in host plant volatiles in the patch they inhabit.     

Dispersal of diapausing Tetranychus urticae and T. kanzawai

The spider mites Tetranychus urticae Koch and Tetranychus kanzawai Kishida (Acari: Tetranychidae) overwinter mostly as mated adult diapausing females. Their overwintering survival depends in part on their dispersal towards suitable habitats. We investigated the dispersal behaviour of diapausing females of T. urticae and T. kanzawai with respect to factors known to affect the dispersal of non‐diapausing mites: light, population density, gravity, and humidity. In general, diapausing females of T. urticae showed a stronger tendency to disperse than did those of T. kanzawai under all test conditions. High population density promoted the dispersal of diapausing T. urticae, but not of T. kanzawai. Dispersal of diapausing females of both species was not significantly affected by gravity, humidity, or whether feeding damage was caused by conspecifics or heterospecifics. On plants, more T. urticae than T. kanzawai moved downward. We propose that dispersal after the onset of diapause may be an important life‐history strategy in T. urticae, but not in T. kanzawai.     

Isolation and characterization of polymorphic microsatellite markers in Tetranychus urticae and cross amplification in other Tetranychidae and Phytoseiidae species of economic importance

Tetranychus urticae Koch is a cosmopolitan phytophagous mite considered as the most polyphagous species among spider mites. Population genetic studies using molecular markers such as microsatellites have proven to be extremely informative to address questions about population structure, phylogeography and host preferences. The aim of this study was to increase the available molecular tools to gain insight into the genetic structure of T. urticae populations of citrus orchards, which might help in their management. Five microsatellite DNA libraries were developed using probes with the motifs CT, CTT, GT and CAC following the FIASCO protocol. Positive clones, those that included the insert with the microsatellite, were detected using the PIMA-PCR technique. Combinations of primers were designed on 22 out of 32 new microsatellites loci and their polymorphism was tested in four populations sampled along the eastern coast of Spain. Eleven successful amplifications were obtained. Cross amplification was tested in the tetranychids Aphlonobia histricina, Eutetranychus banksi, E. orientalis, Oligonychus perseae, Panonychus citri, Tetranychus evansi, T. okinawanus and T. turkestani, and the phytoseiids Amblyseius swirskii, A. cucumeris, A. andersoni, Euseius stipulatus, Neoseiulus barkeri, N. californicus, Phytoseiulus persimilis and Typhlodromus phialatus. Eight successful cross amplifications were obtained.     

Which predatory mite can control both a dominant mite pest,Tetranychus urticae,and a latent mite pest,Eotetranychus asiaticus,on strawberry?

After biological control of Tetranychus urticae using Phytoseiulus persimilis, a latent mite pest, Eotetranychus asiaticus, was found on strawberries growing in a plastic greenhouse in western Japan. To determine whether the release ofP. persimilis, an exotic natural enemy of T.urticae, enhanced the occurrence of the latent pest, the efficiency of P. persimilis in controlling E.asiaticus was compared with the efficiency of two indigenous phytoseiid mites, Amblyseius californicus and A. womersleyi, under laboratory conditions. Phytoseiuluspersimilis and A. californicus attacked not onlyT. urticae but also E. asiaticus. However, the predators produced very few eggs and their offspring rarely reached adulthood when fed on E. asiaticus, so their potential as control agents for the latent mite appears to be low. In contrast, A.womersleyi feeding on E. asiaticus reproduced as well as when fed on T. urticae, and exterminated both species of spider mite. This suggests that A. womersleyihas greater potential as a biological control agent in crops where both species may occur. This revised version was published online in August 2006 with corrections to the Cover Date.     

Intraguild Predation and Feeding Preferences in Three Species of Phytoseiid Mite Used for Biological Control

The ecological impact of introduced biological control agents on native species of arthropods is a matter of considerable debate. This study investigated the ability of the non-native predatory mite Neoseiulus californicus to feed on the native Typhlodromus pyri and vice versa, as both species now co-occur in UK orchards. Typhlodromips montdorensis is a candidate for introduction into the UK as a glasshouse biological control agent. The ability of T. montdorensis to feed on the widely used N. californicus was investigated to identify possible intraguild predation, which might influence the effectiveness of either or both species as predators of Tetranychus urticae. Both N. californicus and T. pyri consumed larval stages of each other, but in choice experiments both showed a preference for T. urticae. Both N. californicus and T. montdorensis also fed on each other, but whereas N. californicus again showed a preference for T. urticae, T. montdorensis fed equally on T. urticae and N. californicus. Interactions between N. californicus and T. pyri and N. californicus and T. montdorensis are discussed in relation to their effectiveness as biological control agents in the glasshouse and the natural control of spider mite in the field.     

The effect of pyrethroid lambdacyhalothrin applications on the spatial distribution of phytophagous and predatory mites in apple orchards

The spatial distribution of three phytophagous mites,Panonychus ulmi (Koch),Tetranychus urticae Koch andAculus schlechtendali (Nalepa), and two predacious mites,Zetzellia mali (Ewing) andAmblyseius fallacis (Garman), and the effect of pyrethroid lambdacyhalothrin applications on mite spatial dispersion were investigated over a 3-year period in an apple orchard in Ontario. The index of dispersion and the slope of Taylor's power law were used to evaluate dispersion patterns of mites. Panonychus ulmi showed that between-tree spatial variation decreased with an increase of population densities, whereas between-leaf variation increased with population densities. With all other four species it appeared that between-tree variation is much greater than between-leaf variation at all field population density levels. The values ofb by Taylor's power law suggested that all five species of mites are aggregated, but that in generalP. ulmi andT. urticae (b=1.427–1.872) are more aggregated than their predators (b=1.254–1.393). Taylor's regression technique suggests that pyrethroid applications causedP. ulmi, T. urticae, Z. mali andA. fallacis to be less aggregated whileA. schlechtendali was more aggregated. The impact of changes in mite spatial distribution following pyrethroid applications on sampling plans is discussed.     

Effects of <Emphasis Type="Italic">Euseius stipulatus</Emphasis> on establishment and efficacy in spider mite suppression of <Emphasis Type="Italic">Neoseiulus californicus</Emphasis> and <Emphasis Type="Italic">Phytoseiulus persimilis</Emphasis> in clementine

The two-spotted spider mite, Tetranychus urticae, is one of the most problematic phytophagous pests in Spanish clementine orchards. The most abundant predatory mites in this ecosystem are Euseius stipulatus, Phytoseiulus persimilis and Neoseiulus californicus. Euseius stipulatus is dominant but poorly adapted to utilize T. urticae as prey. It mainly persists on pollen and citrus red mite, Panonychus citri. A recent study suggested that the more efficacious T. urticae predators P. persimilis and N. californicus are negatively affected by lethal and non-lethal intraguild interactions with E. stipulatus. Here, we investigated the potential of N. californicus and P. persimilis to colonize and thrive on young clementine trees infested by T. urticae in presence and absence of E. stipulatus. Presence of E. stipulatus interfered with establishment and abundance of P. persimilis and negatively affected the efficacy of N. californicus in T. urticae suppression. In contrast, the abundance of E. stipulatus was not affected by introduction of a second predator. Trait-mediated effects of E. stipulatus changing P. persimilis and N. californicus behavior and/or life history were the most likely explanations for these outcomes. We conclude that superiority of E. stipulatus in intraguild interactions may indeed contribute to the currently observed predator species composition and abundance, rendering natural control of T. urticae in Spanish clementine orchards unsatisfactory. Nonetheless, stronger reduction of T. urticae and/or plant damage in the predator combination treatments as compared to E. stipulatus alone indicates the possibility to improve T. urticae control via repeated releases of N. californicus and/or P. persimilis.     

The distribution of herbivores between leaves matches their performance only in the absence of competitors

Few studies have tested how plant quality and the presence of competitors interact in determining how herbivores choose between different leaves within a plant. We investigated this in two herbivorous spider mites sharing tomato plants: Tetranychus urticae, which generally induces plant defenses, and Tetranychus evansi, which suppresses them, creating asymmetrical effects on coinfesting competitors. On uninfested plants, both herbivore species preferred young leaves, coinciding with increased mite performance. On plants with heterospecifics, the mites did not prefer leaves on which they had a better performance. In particular, T. urticae avoided leaves infested with T. evansi, which is in agreement with T. urticae being outcompeted by T. evansi. In contrast, T. evansi did not avoid leaves with the other species, but distributed itself evenly over plants infested with heterospecifics. We hypothesize that this behavior of T. evansi may prevent further spread of T. urticae over the shared plant. Our results indicate that leaf age determines within‐plant distribution of herbivores only in absence of competitors. Moreover, they show that this distribution depends on the order of arrival of competitors and on their effects on each other, with herbivores showing differences in behavior within the plant as a possible response to the outcome of those interactions.     

Intraspecific invariability of the internal transcribed spacer region of rDNA of the truffleTerfezia terfezioides in Europe

ITS regions (internal transcribed spacers—ITS1 andITS2—with the 5.8S gene of the nuclear rDNA) of 25 fruit body samples ofTerfezia terfezioides, originating from Hungary and Italy, were compared. The amplification and sequencing of the ITS region was successful with both theITS1-ITS4 andITSIF-ITS4 primer pairs. No differences of the restriction fragment length polymorphism profiles were detected among 19 samples collected in one place at the same time. The sequences of the ITS region of 9 samples collected in different localities were highly invariable, differing in only two bases. Thus the intraspecific homogeneity of the ITS region seems to be an important species-specific characteristic ofT. terfezioides in contrast to otherTerfezia species. As the samples of the species were collected from different and distant localities of Europe, the ITS sequence ofT. terfezioides can be considered a very conservative, reliable molecular marker of the fungus. *** DIRECT SUPPORT *** A00EN076 00008