Traditional and modern methods for the identification of thrips (Thysanoptera) species

Transcription

1 J Pest Sci (2012) 85: DOI /s REVIEW Traditional and modern methods for the identification of thrips (Thysanoptera) species Nataša Mehle Stanislav Trdan Received: 8 September 2011 / Accepted: 24 February 2012 / Published online: 10 March 2012 Ó Springer-Verlag 2012 Abstract Many thrips are pests of commercial crops due to the damage they cause by feeding on developing flowers or vegetables. Thrips may also serve as vectors for plant diseases, such as tospoviruses. Their small size and predisposition towards enclosed places makes them difficult to detect by phytosanitary inspection. In this review, several methods available for identifying thrips, including their advantages and disadvantages, are discussed. A combination of different methods gives the most reliable identification. Relatively new morphometric, molecular and biochemical methods for identifying thrips species represent valuable alternatives for situations in which correct identification with classical morphological methods is very difficult, time consuming or virtually impossible. However, traditional morphological methods should not be neglected, especially because adequate identification using morphological keys is usually an indispensable first step in the development and validation of these new modern methods. In addition, modern systems may still require specimen identification to the genus level via morphological keys, or such keys may be recommended to confirm the results of modern identification methods. Keywords Thrips Thysanoptera Identification Morphological keys Molecular methods Communicated by M. Traugott. N. Mehle Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, 1000 Ljubljana, Slovenia S. Trdan (&) Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1111 Ljubljana, Slovenia stanislav.trdan@bf.uni-lj.si Introduction Thrips ( Thysanoptera) are very small insects (an average length is 1 2 mm), widespread throughout the world. They are divided into two subs, the Terebrantia, with a blunt or angled body end, and the Tubulifera, in which the abdomen forms a tube at the end (Moritz 1994; Moritz et al. 2000). Females of the common thrips lay eggs on plants, and eclosion is followed by two wingless larval stages that are either predatory or feed on plant leaves, flowering parts or fungi. The ensuing two or three pupal stages are non-feeding, and these stages occur either in the soil or on plants, depending on the thrips species. The resulting adults may be winged or wingless, depending on both the sex and the species (Palmer et al. 1989; Oetting et al. 1993; Mound and Kibby 1998; Whitfield et al. 2005; Pakyari et al. 2011). Of the more than 5,500 thrips species thus far identified (Mound 1997; zur Strassen 2003), some are beneficial as pollinators or biological control agents (Mound and Kibby 1998; Trdan et al. 2005); in contrast, some are considered pests in agriculture, horticulture and forestry (Lewis 1997; Trdan et al. 2007). Thrips inflict damage not only by feeding on plants but also by transmitting tospoviruses to the plants (Moritz et al. 2000; Pappu et al. 2009). Whitfield et al. (2005) reported that at least 10 polyphagous species of thrips can acquire a virus of the genus Tospovirus during their larval stages and later transmit the virus to other plant species, although the efficiency of transmission varies among thrips and tospovirus species (Wijkamp et al. 1995; Whitfield et al. 2005). In Europe, Thrips palmi Karny, Scirtothrips aurantii Faure, Scirtothrips dorsalis Hood, Scirtothrips citri (Moulton) and Frankliniella occidentalis (Pergande) are species of major quarantine concern, and they are listed in the European Union Plant Health

2 180 J Pest Sci (2012) 85: Directive (2000/29/EC) (T. palmi: I.A.I; S. aurantii, S. dorsalis and S. citri: II.A.I) and/or on European and mediterranean plant protection organization (EPPO) quarantine lists (T. palmi, S. aurantii and S. citri: EPPO A1; S. dorsalis and F. occidentalis: EPPO A2) (Council Directive, 2000; EPPO 2011). The minute size and cryptic behaviour of thrips make them difficult to detect in the field and on plants or plant products transported for international trade. In particular, fresh plant material transported around the world may readily spread these insects in the form of eggs, larvae or adults (Kirk and Terry 2003). Consequently, many species have now spread from their original natural habitats. After translocation to new areas outside of the home range, some species may reproduce rapidly and cause significant economic losses. Treatments against thrips are often very specific, especially for thrips that have acquired insecticide resistance (Bielza 2008), and as a fundamental first step, these treatments require the correct and unambiguous identification of species (Moritz 1994; Brunner et al. 2002; Rugman-Jones et al. 2006). In the identification of thrips, it is also important to understand their biology and to empower integrated pest management strategies (Moritz et al. 2007). In this review, different methods for identifying thrips, including the advantages and disadvantages of these methods, are discussed and presented in table form (Table 1). Traditional methods Traditionally, the identification of thrips has been based, as with other insects, on external morphology (appearance). Appearance can vary in many ways within species, such as in colour and size (Arnett 1993; Moritz 1994; Mound and Kibby 1998); on the other hand, the different species of thrips are generally similar in appearance, differing only in minute detail, so accurate determination of thrips species requires the use of diagnostic aids called keys. Keys usually require the user to make a choice between only two characters at a time, the so-called dichotomous keys. However, it is also possible to have keys where the user is asked to pick among several groups of characters simultaneously. The choices are usually numbered, and the user is referred to various sections of the key according to number. Keys are often based on written descriptions of contrasting characters. Illustrated keys have a major advantage in that they graphically display the characters of interest (Capinera 2008). Usually identification of species is difficult and requires expert knowledge of the genus. To identify thrips, the specimens need to undergo a process to clear and mount the specimens on microscope slides. Due to the small size of thrips, accurate identification requires the magnification of specimens with a microscope. Each thrips is glued to a microscope slide, and then the features used to identify the thrips are examined under high magnification. Morphological methods of identification require the examination of various features of the thrips, including the number of antennal segments and the distribution and number of setae across the body and along the forewings. Without fully cleared and expertly slide-mounted specimens, the minute structural details used to diagnose thrips species cannot be studied with accuracy (Palmer et al. 1989; Moritz 1994; Mound and Kibby 1998; Rugman-Jones et al. 2006). Identification of specimens to (Thysanoptera), family and genus via morphological methods are possible with several different keys (Mound and Kibby 1998; Triplehorn and Johnson 2005). In 1928, Priesner published the first key for thrips species present in Europe. This key is based on written descriptions of contrasting characters. However, there are problems when identifying most species with Priesner s keys because thrips are characterised by only one or a few characters, with a limited number of pictures. In 1979, Schliephake and Klimt published a key that has an advantage over the Priesner key in that it includes tables and more drawings for better illustration of the text. Later, several other keys were published to identify thrips species, with plenty of drawings included to clarify the text (Jenser 1982; Palmer et al. 1989; Oetting et al. 1993; Bhatti 1999a, b, c; zur Strassen 2003). Moritz (1994) published a pictorial key that allows, through a sequence of simple decisions, the determination of 70 economically of the Thysanoptera in Central Europe. A similar visual identification system for thrips species was also published by Mound and Kibby (1998). Mound s great contribution to the thrips taxonomy, including several morphological keys not mentioned in this paper, is clearly evident from a recent biographical article written by Funderburk and Hoddle (2011). In 2006, Moritz upgraded his pictorial key with colour photomicrographs of thrips on slides and some new species (Moritz 2006). Those pictorial keys are much more user-friendly than keys in text form only. When identifying adult thrips species using traditional, printed dichotomous keys, a series of questions must be answered in a fixed, and this sequential approach can limit identification if an early character is not identifiable. For example, if the first question concerns the number of antennal segments, and analysed specimens are damaged and have lost their antennae, there will be a serious problem. Moritz et al. (2000, 2007, 2011) developed modern computerised keys for economically of thrips, with colour photomicrographs of thrips on slides and also video clips. For example, in one of their interactive identification and information systems, 180 fully illustrated species of thrips from all over the world are

3 J Pest Sci (2012) 85: Table 1 Identification methods for thrips species, including advantages and disadvantages Methods Basis for key Population studied a Adults species identifications Immature stages species identifications Other advantages Other disadvantages References Morphological keys (advantages: relatively inexpensive laboratory equipment and reagents; disadvantages: require taxonomic expertise, tedious diagnosis and intact specimens, usually impossible to identify the immature stages to the species level): b Dichotomous key characters? drawings (only a few at the end of the book) characters? drawings? tables characters? drawings characters? drawings characters? drawings characters? drawings characters? drawings characters? colour photographs characters? colour photographs characters? black & white photographs Europe Many species from all of the families known at that time Europe Many species from four families Hungarian Many species from four families World Economically North America 11 thrips species on greenhouse ornamentals Descriptions for larval and pupal stages, but almost useless for identification to the species level Australia No First- and secondinstar larvae of Granite Belt stonefruit thrips and F. occidentalis Europe, Mediterranean 399 thrips species from five families Worldwide 5500 known species The Western Palaearctic Region (? some quarantine species) Poland and other Central European countries No Second instar larvae of 130 species No Second instar larvae of 35 species of the Thrips genus No Quite a good illustration of the text No Quite a good illustration of the text No Good illustration of the text No Good illustration of the text Overview of that time in German, the lack of illustrations, answer a series of questions in a fixed Good illustration of the text No Good illustration of the text No Precise description of families and sub-families Good illustration of the text by colour photographs Good illustration of the text by black & white photographs in German, answer a series of questions in a fixed in Hungarian, answer a series of questions in a fixed In German, answer a series of questions in a fixed Photographs of selected species only Priesner (1928) Schliephake and Klimt (1979) Jenser (1982) Palmer et al. (1989) Oetting et al. (1993) Milne et al. (1997) zur Strassen (2003) Mound et al. (2009) Vierbergen et al. (2010) Kucharczyk (2010)

4 182 J Pest Sci (2012) 85: Table 1 continued Methods Basis for key Population studied a Adults species identifications Immature stages species identifications Other advantages Other disadvantages References Pictorial key Drawings of contrasting characters Drawings of contrasting characters Drawings and colour photographs of contrasting characters Computerized colour photographs? description of contrasting characters Computerized colour photographs? description of contrasting characters Computerized colour photographs? description of contrasting characters Central Europe 70 economically from three families World To the 60 genus, but only to some species of four genus (economically significant) Central Europe More than 80 economically from four families World 180 economically California 214 species found, another 53 species not yet reported but probably introduced Africa Over 90 species, predators and pests Larva, propupa and pupa stages only to the family level Identification through a sequence of simple decisions No Identification through a sequence of simple decisions Larva, propupa and pupa stages only to the family level Morphometrics (advantages: less subjective than morphological keys; disadvantages: still require taxonomic expertise): Quantitative feature? principal component analysis Thrips. atratus and T. montanus Identification through a sequence of simple decisions, colour photographs No It is possible to address to any character at any time No It is possible to address any character at any time No Any character can be addressed at any time, also key of thrips parasitoids, biogeographic information Larvae of T. atratus and T. montanus in German, answer a series of questions in a fixed Moritz (1994) Mound and Kibby (1998) Moritz (2006) Moritz et al. (2001) Hoddle et al. (2008a, b) Moritz et al. (2011) Kucharczyk and Kucharczyk (2009)

5 J Pest Sci (2012) 85: Table 1 continued Methods Basis for key Population studied a Adults species identifications Immature stages species identifications Other advantages Other disadvantages References Quantitative feature? artificial neural networks Europe 101 economically important thrips species Semiautomated identification of biological objects protein analysis (advantages: all stages of insects can be used, there are no needs for intact specimen; disadvantages: more expensive laboratory reagents): Isozyme analysis Enzyme profiles (esterase zymograms) ELISA Proteins as antigens Six thrips species (T. palmi from two sources) SDS-PAGE Protein profiles One five populations from different countries (many individuals) Asian population Seven species Seven species (second instar larva, pupa) Rapid detection method for immature stages Thrips palmi Thrips palmi High diagnostic throughput, screening method eliminating the vast majority of negative samples Heliothrips haemorrhoidalis, Frankliniella occidentalis, Thrips tabaci Heliothrips haemorrhoidalis, Frankliniella occidentalis, Thrips tabaci Enzyme activity may quickly deteriorate Limited cross reactivity study (any positive results require confirmation by other methods) Technically difficult method, different protein profiles for different stages of the life cycle Fedor et al. (2009) Murai (1993, 1994) Banks et al. (1998) Reboredo et al. (2003) Molecular methods (advantages: all stages of insects can be used, there is no need for intact specimen, highly reproducible, robust, specific and sensitive; disadvantages: relatively expensive laboratory reagents and equipment): PCR-RFLP RFLP (two enzymes) patterns of portion of the COI gene Differences in size of the PCR products of the ITS2 regions? RFLP patterns (5 enzymes) Differences in size of the PCR products of the ITS2 regions? RFLP patterns (one enzymes) Differences in size of the multiplex PCR products of the ITS1 and ITS2 regions? RFLP patterns (max 2 enzymes) For each species: individuals from different populations or countries 10 Economically 10 Economically Multiple-step protocols Brunner et al. (2002) No data 19 Species 19 Species Multiple-step protocols Moritz et al. (2002) For each species: two five specimens from different locality in japan For each species: one two specimens from different continents Nine species on Japanese fruit trees Many economically important pest species of the genus Scirothrips Nine species on Japanese fruit trees Many economically important pest species of the genus Scirothrips Multiple-step protocols Toda and Komazaki (2002) Still requires identification of specimens to genus via morphological methods, multiplestep protocols Rugman- Jones et al. (2006)

6 184 J Pest Sci (2012) 85: Table 1 continued Methods Basis for key Population studied a Adults species identifications Immature stages species identifications Other advantages Other disadvantages References PCR Differences in size of the PCR products of the COI gene (two sets of primers) Size of the PCR products of ITS2 regions Real time PCR PCRsequencing Morphological and molecular methods One Indian population for each species 10 thrips species (15 S. dorsalis populations) Species specific amplicon 21 thrips species (many individuals from different populations) Species specific amplicon (within the COI gene) 24 thrips species (76 specimens commonly occuring in Europe) Species specific amplicon Seven thrips species (for F. occidentallis: many individuals from different origins) Sequence of portion of the COI gene For each species: individuals from different populations or countries Sequence of portion of COI gene For each species: individuals from different localities in southern Africa Computerized colour photographs? description of contrasting characters? video clips? RFLP patterns (5 enzymes) of ITS regions Thrips palmi, Thrips tabaci Scirtothrips dorsalis Thrips palmi, Thrips tabaci Less steps than PCR- RFLP Scirtothrips dorsalis Less steps than PCR- RFLP Targeted only at specific taxa, no data for other populations Targeted only at specific taxa Thrips palmi Thrips palmi One-step protocol Targeted only at specific taxa Thrips palmi Thrips palmi One-step protocol Targeted only at specific taxa Frankliniella occidentalis 10 economically important thrips species 9 Economically World Economically important thrips species Frankliniella occidentalis 10 economically important thrips species 9 Economically Economically important thrips species One-step protocol Targeted only at specific taxa Maximal information content of the PCR product Maximal information content of the PCR product It is possible to address any character at any time Not all laboratories possess an automated sequencer Not all laboratories possess an automated sequencer Asokan et al. (2007) Farris et al. (2010) Walsh et al. (2005) Kox et al. (2005) Huang et al. (2010) Brunner et al. (2002) Timm et al. (2008) Moritz et al. (2000) The size of the studied populations is particularly important in initiating molecular and other diagnostic assays (for example to study the specificity and efficacy of the assay). For morphological keys, geographical coverage is given a B Only the morphological keys available to us are included

7 J Pest Sci (2012) 85: included (Moritz et al. 2001). Hoddle et al. (2008b) combines an interactive, matrix-based computer diagnostic and information system with current taxonomic methods that facilitate confident and rapid identification of California s large and complex thrips (Thysanoptera) fauna. The advantage of this type of key is that it allows a user to address any character at any time. Morphological keys usually focus on a target group of thrips species, either those important for greenhouse ornamentals (Oetting et al. 1993), economically important species (Palmer et al. 1989; Moritz 1994, 2006) or species present within a certain geographic range, as constrained as one country (Jenser 1982), or as wide as Europe and surrounding areas (Priesner 1928; Schliephake and Klimt 1979; zur Strassen 2003). Some of those keys are written in less commonly used languages, such as German (Priesner 1928; Schliephake and Klimt 1979; zur Strassen 2003; Moritz 2006) or Hungarian (Jenser 1982), so they are useless for people unfamiliar with those languages. For European quarantine of thrips species, adjusted morphological keys are included in official diagnostic protocols published by EPPO (EPPO standards 2002, 2005, 2006). Furthermore, many small keys to a few thrips species have been published as the result of studies of small populations of thrips species in a given area or when new morphological characters are discussed for some species (Bhatti 1999a, b, c; Minaei et al. 2007; Zhang et al. 2011). Alternatively, Mound et al. (2009) created World Thysanoptera, a website established to provide a base for the development of taxonomic research and dissemination of information on the Thysanoptera. It is being developed as a focal point for information on the 5,500 known species of thrips from around the world, with particularly emphasis on systems of identification. Thrips are commonly found on imported plants as larvae with no adults present, partly because the smaller life stages are harder to detect. For example, T. palmi is commonly found on imported Momordica fruit as a first instar larvae, suggesting that undetected eggs hatch as the fruit comes out of cold-freight storage (Walsh et al. 2005). Similarly, western flower thrips (F. occidentalis) are often intercepted on plants or plant products in international trade and are commonly found on imported perishable plant materials as larvae without the presence of adults (Huang et al. 2010). Therefore, samples sent to the laboratory for identification often consist entirely of larvae. The identification of larval, propupal and pupal stages can be performed using some morphological keys, but only at the family level (Moritz 1994, 2006). Descriptions of the second larval instar for a limited number of thrips species can be found in the literature (Priesner 1928; Milne et al. 1997; Kucharczyk 2010; see also other references in Vierbergen et al. 2010). Recently, a dichotomous key (with colour photographs) to the second larval instar of 130 species of Thripidae occurring in the Western Palaearctic region (40% of the total number of Thripidae species from this region), including some species of quarantine interest, was published (Vierbergen et al. 2010). However, in practice, many immature stages of thrips cannot be identified with certainty to the species level using morphological methods (Banks et al. 1998; Brunner et al. 2002; Reboredo et al. 2003; Walsh et al. 2005; Huang et al. 2010). Therefore, for morphological identification at species level, these immature thrips must be raised to adulthood, resulting in a critical delay before results can be reported (Brunner et al. 2002; Walsh et al. 2005; Huang et al. 2010). Modern methods Morphometric analysis Sometimes the specimens are very similar to each other, such that only the complex combination of characters, both qualitative and quantitative (morphometric), may distinguish them. For example, the characteristic of long or short setae may be treated as qualitative, but for an inexperienced person it may be difficult to determine which characteristic a given thrips has; morphometric features are much clearer. The statistical methods for taxonomic studies, and in particular principal component analysis, have been shown to be useful for discriminating between thrips. Principal component analysis compares sample groups (taxa or specimens), taking into consideration the value of their characters. All of the characters, but especially the morphometric ones, must be based on numerous samples because of high variation among specimens. Although the morphometric results are subject to measurement errors, these data could play a large role in the discrimination of similar species, e.g., T. atratus and T. montanus (Kucharczyk and Kucharczyk 2009). Principal component analysis allows for the selection of the most significant characters in the discrimination of researched taxa (Kucharczyk 2010). Artificial neural networks, defined under artificial intelligence, suggest possible practical methods for semiautomated identification of biological objects (Fedor et al. 2008). Together with other statistical tools, such as principal component analysis or classification trees, such networks incorporate digital age science. An optimum architecture, established and supervised by experts, transforms metadata through multilayer system processing using an artificially intelligent apparatus. For instance, Fedor et al. (2009) evaluated 101 economically important

8 186 J Pest Sci (2012) 85: European thrips species: extrapolation of the verification test data indicated 95% reliability. For the most part, quantitative morphometric characters, such as head, clavus, wing and ovipositor length and width, formed the input variable computation set in a Trajan neural network simulator. Reliable species distinctions would not be possible if only a single character only were considered. However, in appropriate combinations (relative values), character states can be unique and specific. In addition to morphometrics, molecular and biochemical assays also offer tools that might be used to help alleviate the limitation of morphological analysis. Those methods are based either on DNA or protein analysis. DNA and protein-based analyses are very discriminating and can be used regardless of the life stages available. Methods based on protein analysis Over the past 20 years, the isozyme electrophoretic discrimination method has been applied to thrips identification (Murai 1993, 1994). The main drawback with enzyme electrophoresis is that enzyme activity may quickly deteriorate if samples are not kept alive or deep frozen immediately after collection (Toda and Komazaki 2002). In 1998, monoclonal antibodies were developed for use in an enzymelinked immunosorbent assay (ELISA) to detect T. palmi (Banks et al. 1998), but in a limited cross-reactivity study, only T. tabaci Lindeman and F. occidentalis were confirmed to not give false positive results. Thus, any positive results obtained by means of ELISA will therefore still require confirmation by examining individuals from the same sample under the microscope. Regardless, the method is useful, especially for immature stages of thrips, as a high throughput screening assay that is very effective in eliminating the vast majority of negative samples (Banks et al. 1998). Reboredo et al. (2003) investigated the suitability of using sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) to characterise immature stages of Thysanoptera. The low intraspecific variability shown among populations and the stability of the protein profiles obtained from the developmental stages make this technique promising for characterising the immature life stages of thrips. The authors observed clear differences between the patterns of F. occidentalis and T. tabaci larvae. There are technical limitations to this methodology, because clear patterns with sharp bands, which are essential for correct identification, are obtained consistently only with careful control of critical aspects of electrophoresis and staining procedures. Methods based on DNA analysis The use of molecular tools to identify organisms is a relatively new science. This process requires tissue from the specimen to be crushed, damaging the cell structure and releasing the tightly coiled DNA strands. However, nodestructive DNA extraction of insects is possible, if preserving its outer body surface for later morphological examination is needed (Staudacher et al. 2011). The DNA is then replicated through a molecular process to increase the number of DNA strands within the sample. In a conventional polymerase chain reaction (PCR), replicated DNA is then placed under gel electrophoresis, where a charge is passed through a gel-like substance and causes the DNA to move through the gel, separating the DNA strands, based on size, into bands that can be observed using DNA dyes. These size bands and the patterns they make allow scientists to determine the species present (Newton and Graham 1994). For successful identification of thrips species, PCR is usually combined with restriction fragment-length polymorphism (RFLP). In RFLP analysis, the amplified DNA sample is broken into pieces by restriction enzymes, and the resulting restriction fragments are separated according to their lengths by gel electrophoresis. PCR-based typing methods are widely used because they are highly reproducible and technically simple, requiring only basic laboratory skills and minimal amounts of DNA; further, once established, they are sensitive and specific (Brunner et al. 2002; Rugman-Jones et al. 2006). Several PCR-based protocols have been developed to separate selected groups of thrips species, including species difficult to recognise by morphological characters. Some of them are based on the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA (rdna) (Moritz et al. 2002; Toda and Komazaki 2002; Rugman-Jones et al. 2006; Farris et al. 2010) and the other protocols are based on a portion of the mitochondrial cytochrome oxidase I (COI) coding gene (Brunner et al. 2002; Asokan et al. 2007). Both the ITS and COI regions show both high interspecific variability and extremely low intraspecific variability (Brunner et al. 2002; Moritz et al. 2002). The mitochondrial COI gene has also been confirmed to be a suitable marker for thrips species identification by Frey and Frey (2004). While Asokan et al. (2007) developed a species-specific PCR for T. tabaci and T. palmi and Farris et al. (2010) did so for Scirtothrips dorsalis, most other PCR assays need further analysis, e.g., by RFLP or by sequencing PCR products. There are characteristic patterns of the RFLP analysis for each species, although in a few cases similar patterns are produced from several species with one restriction enzyme; this similarity can be compensated for using two or more restriction enzymes. For example, two restriction enzymes are necessary to analyse PCR products of the mitochondrial COI coding gene to allow unambiguous identification of 10 economically important thrips species

9 J Pest Sci (2012) 85: (Brunner et al. 2002), and five restriction enzymes are needed for the analysis of the ITS2 PCR product to accurately identify several thrips species (Moritz et al. 2000, 2002). In contrast, nine thrips species from Japanese fruit trees can be identified in both adult and immature stages when using PCR-RFLP of the ITS2 region of rdna with only one restriction enzyme (Toda and Komazaki 2002). Not only the RFLP patterns but also the size of the PCR products are species-specific (Moritz et al. 2002; Toda and Komazaki 2002; Rugman-Jones et al. 2006). Rugman-Jones et al. (2006) provide a molecular key using diagnostic characters obtained by multiplex PCR of the ITS1 and ITS2 regions of rrna and restriction of the subsequent products with two enzymes. With this key, they have identified many economically important pest species of the genus Scirothrips. It is necessary to note that this system still requires the identification of specimens to genus via morphological methods, which may be difficult for workers not familiar with thrips identification keys and slide preparation techniques. The design of a set of primers that would result in a genus-specific PCR product would complete their key. However, such a goal is unrealistic because it would mean sequencing thousands of species from other genera to ensure that the primer set used was truly unique to the genus Scirtothrips (Rugman-Jones et al. 2006). Based on nucleotide sequence analysis of the mitochondrial COI gene, a system of identifying economically important thrips species present in Europe (Brunner et al. 2002) and in southern Africa (Timm et al. 2008) was developed. Brunner et al. (2002) used sequencing instead of RFLP to analyse the PCR products. With RFLP, only a fraction (i.e., the cutting sites) of the information present in the amplified DNA fragment is assessed, while sequencing makes use of the maximal information content (individual nucleotide sites) (Brunner et al. 2002). However, at this moment, not all laboratories possess or have access to an automated sequencer, and sending PCR products to a commercial firm for sequencing could result in a critical delay before the results can be reported. PCR-RFLP is a useful tool for identifying thrips; however, a post-pcr restriction digest and the need to run gels produce time-consuming, multiple-step protocols and increase the chance of post-pcr contamination. A relatively new method, real-time PCR, offers many advantages over gel-based PCR methods. Detection is performed in a closed-tube format with a simple one-step protocol, negating the need for post-pcr manipulations, shortening the assay time and significantly reducing the risk of both post-pcr contamination and operator error. Although the cost of instruments capable of performing real-time PCR is greater than that of standard thermocyclers, the cost of reagents is very similar to that of standard PCR; in addition, time can be saved substantially because electrophoresis is unnecessary (Walsh et al. 2005; Dorak 2006). To the best of our best knowledge, only three real-time PCR assays have been developed for the identification of thrips species thus far: two that are specific for T. palmi (Kox et al. 2005; Walsh et al. 2005) and one specific to F. occidentalis (Huang et al. 2010). All these assays are based on TaqMan chemistry. Real-time PCR is a very sensitive method, so it could detect the DNA of one thrips larvae (Kox et al. 2005; Huang et al. 2010). Real-time PCR is also very robust and can be used with material collected from plants or caught on sticky traps. Adult T. palmi from the English outbreak were detected even though they had been on a sticky trap (wrapped in polythene) stored at room temperature for over 4 years (Walsh et al. 2005). Real-time PCR assays could be extended to other thrips species, even without prior sequence information, by using random amplified polymorphic DNA (RAPD) analysis to identify putative markers and by screening potential probes with Southern blotting (Walsh et al. 2005). RAPD analysis has been used to highlight the inter- and intra-specific polymorphisms of thrips (e.g. Kraus et al. 1999; Bayar et al. 2002). A future goal could be to search for possibilities of simplifying the entire molecular diagnostic procedure to enable easy confirmations of the presence of the thrips onsite. Such a tool would reduce the time from sampling to result and facilitate the adoption of fast measures to prevent potential risks. To adapt the developed methods to on-site requirements, special emphasis must be given to reducing analysis time and to the use of portable devices. The realtime PCR detection method using the portable Smart Cycler real-time PCR thermocycler was successfully adapted to meet field requirements (Gutierrez-Aguirre et al. 2011). Although it was used to detect rotaviruses in water samples, it could also be adapted to detect thrips. The next method development steps should lead to designing a userfriendly on-site adapted device for molecular detection. Isothermal amplification procedures, such as loop-mediated isothermal amplification (LAMP) (James et al. 2010; Nemoto et al. 2010; Tsutsumi et al. 2010), enable target amplification without the need for expensive thermocyclers. In addition, LAMP amplification products can be easily detected in a lateral flow device (James et al. 2010), simplifying the methodology even more. Approaches based on PCR and real-time PCR principles are designed to detect one or a few thrips species at a time, but it would be ideal to have multiplex detection of numerous different thrips species in a single step. DNA barcoding proposes a solution to this limitation. DNA barcoding requires conventional PCR, using degenerate or universal primer sequences, and sequencing. The mitochondrial COI coding gene has been shown to provide

10 188 J Pest Sci (2012) 85: sufficient variation to be used in future DNA barcoding efforts within the genus Thrips, and for that purpose, some researchers are creating a reference database of thrips species (Collins et al. 2010; Glover et al. 2010). Traditional and modern methods for identification do not always match Molecular methods can indicate surprising differences compared to the traditional methods of identification based on structural characters. For example, different patterns were observed with PCR-RFLP of ITS2 regions of T. hawaiiensis (Morgan) (Toda and Komazaki 2002). Rugman-Jones et al. (2006), based on sizes and sequences of the ITS1 and ITS2 regions, suggested that Indian and South African specimens of S. dorsalis (or specimens that key out to S. dorsalis on the basis of morphological characters) are indeed not the same species. Furthermore, S. dorsalis has been found to consist of at least three separable groups identifiable at the molecular level based on the sequence of the COI gene, but indistinguishable morphologically (Hoddle et al. 2008a). Similarly, the sequences of the D2 domain of the 28S and COI genes revealed the existence of two different species of F. occidentalis in California (Rugman-Jones et al. 2010). Using molecular methods to discriminate cryptic species (species which satisfy the biological definition of species, but whose morphology is very similar) is indispensable. However, we still do not know if all such molecular species are equally important as pests or tospovirus vectors. These new molecular data demonstrate that we have a lot to learn about the species concept, and what we have thought to traditionally be a biological species may actually be more than one. In contrast, the analysis of ITS1 and ITS2 regions by Rugman- Jones et al. (2006) did not confirm the findings of five new species in the genus Scirtothrips on avocados in Mexico (Johansen and Mojica-Guzman 1998); this classification had also previously been questioned because the species designations were made according to morphological characters that exhibit high intraspecific variation (Mound and zur Strassen 2001). Furthermore, two long-established species, T. fuscipennis Haliday and T. sambuci Heeger, could not be separated using analysis of sequences of the COI gene (Collins et al. 2010). Combining traditional and modern methods for identification is necessary Because of the many advantages offered by molecular diagnostic tools, the traditional morphological methods are sometimes neglected by young diagnosticians. However, we must keep in mind that adequate identification by traditional morphological keys is usually indispensable as the first step in the development and introduction of new molecular methods. In addition, molecular methods may still require identification of specimens to genus via morphological methods (Rugman-Jones et al. 2006). Because of these limitations of molecular methods, and because of the inability of morphological keys to distinguish cryptic species and most immature stages, each modern diagnostic laboratory should have an expert for both traditional and molecular methods. In routine diagnosis of quarantine thrips species, it is suggested (EPPO standard 2006) that molecular biological methods can be used to confirm morphological examination of adults. In the case of eggs, larvae or pupae, molecular methods are the only tool for reliable identification. In this case, it is recommended that at least two different molecular biological assays, preferably using different target sequences, are performed (EPPO standard 2006). The combination of both morphological and molecular keys was suggested also by Moritz et al. (2000, 2007). Their modern computerised key for economically of thrips partly solved the problem regarding the incapacity to morphologically identify immature stages by including one of the first developed molecular keys, based on ITS-RFLP analysis. Conclusions Ideally, identification should be based on two methods, i.e. a morphological and a molecular identification method. A combination of different methods gives the most reliable identification. Molecular methods for identifying thrips species represent a valuable alternative for situations in which correct identification using classical morphological methods is very difficult, time consuming or virtually impossible. For identifying thrips species, modern methods, used carefully and in conjunction with morphological keys, offer the possibility of a significant increase in species resolution across all life stages of thrips. Acknowledgments This study was carried out within Professional Tasks from the Field of Plant Protection, a programme funded by the Ministry of Agriculture, Forestry, and Food of the Phytosanitary Administration of the Republic of Slovenia. The authors thank Dr. Halina Kucharczyk and Dr. Peter Fedor for kindly providing us with the basic data on morphometric analysis that they use in thrips identification. The authors thank three anonymous reviewers whose comments and suggestions helped us to improve the manuscript. References Arnett RH (1993) American insects: a handbook of the insects of America North of Mexico. The Sandhill Crane Press, Gainsville, pp 20 23

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