|
Pest Thysanoptera of Mediterranean Fruit TreesConcerted Action: AIR3 PL94 1938 FUNDING: CEC, DIRECTORATE GENERAL FOR AGRICULTURE DGVI-FII-3 FINAL REPORT Contents: 1 Summary The general objective of this concerted action was to co-ordinate the ongoing research on Western Flower Thrips (WFT), Frankliniella occidentalis (Perg.), of taxonomists and those working on its biological control. WFT has an American origin and was introduced to Europe during the first half of the 1980s, through the horticulture trade in living plants. WFT may be controlled by the biological control agent Ceranisus menes (a hymenopteran endoparasite, or parasitoid), and by entomopathogenic fungi. This concerted action was planned so that we could lay the basis for future studies into the possibility of targeting particular biotypes of pest thrips with relevant parasitoids or entomopathogenic fungi, in order to enhance the biological control possibilities for these damaging pests that develop insecticide resistance so readily. Specific objectives addressed were: 1. the characterization of the biology of populations of WFT with respect to life histories, host plants, host-specificity of hymenopteran parasitoids and fungal pathogens - with the aim of identifying strains or 'biotypes' for use in biological control; 2. the characterization of WFT by molecular means, in order to permit the unambiguous identification of the species and of any crop biotypes or locality strains; to provide training and establish experimental protocols so that all participating laboratories are capable of utilizing the defined methods of molecular identification; 3. the transfer of expertise by means of a series of technical workshops in all participating laboratories; 4. the diffusion of results through specific publications and a final 'open doors' workshop. The partners in this concerted action observed no significant biological differences between any stocks and, therefore, found no support for the recognition of distinctive strains or biotypes of WFT in Europe. Compatible with this conclusion, DNA typing (RAPD-PCR) failed to provide markers diagnostic for 2 or more stocks of WFT that might represent biological strains. We discovered minimal polymorphism in mitochondrial DNA (COI) sequences from WFT stocks as widely distributed as Europe and Australia and, taken with the RAPD-PCR results, we conclude that recent introductions to Europe have mostly come from just one natural genetic lineage (originating in the Americas). This result is important for agriculture because those studying WFT and related species were unclear about the extent and nature of natural variation among WFT populations introduced into Europe at different times and from different overseas localities and to what extent any such infra-specific variation might be correlated with the success or failure of control measures. Prof. Laurence Mound, The Natural History Museum (NHM), London, U.K. (Thrips taxonomist; organizer of workshop no.1; co-ordinator of the concerted action; assisted by Mr David Hollis, Associate Keeper of Entomology, who acted as financial co-ordinator when Prof. Mound was overseas) Dr Rita Marullo, Universita degli Studi della Basilicata, Potenza, Italy (Thrips taxonomist: organizer of workshop no.5) Mr Arturo Goldarazena Lafuente, University of Navarra, Pamplona, Spain (Thrips specialist; organizer of workshop no.2) Mr Antoon J.M. Loomans, Wageningen Agricultural University, Netherlands (Thrips parasitoid specialist: organizer of workshop no.3) Prof. Gerald Moritz, Martin-Luther University, Halle-Wittenberg, Germany (Specialist in thrips and their fungal pathogens; organizer of workshop no.4) Dr Paul D. Ready, NHM, London, U.K. (Molecular Systematics Laboratory, Dept. Entomology) Miss Johann M. Testa, NHM, London, U.K. (Research assistant/laboratory manager, Molecular Systematics Laboratory, Dept. Entomology) Thysanoptera (thrips) are small, herbivorous insects of economic importance in warmed glasshouses and in field conditions in the south of the European Communities. They include a number of quarantine organisms, such as Thrips palmi. The introduced Western Flower Thrips (WFT, Frankliniella occidentalis (Perg.)) has an American origin and was introduced to Europe during the first half of the 1980s, through the horticulture trade in living plants. WFT and other thrip pests may be controlled by biological control agents such as predators, parasitoids (eg Ceranisus menes, a hymenopteran endoparasite), and by entomopathogenic fungi. Biological control does not always work, and this may be related to natural variation among WFT populations introduced into Europe at different times and from different overseas localities. In its area of origin (western U.S.A.), WFT is variable in colour and size, with three sympatric colour forms readily interbreeding in the laboratory (Bryan & Smith, 1956), with both the pale and the dark forms being similarly able to transmit tomato spotted wilt virus (TSWV) (Sakimura, 1962). The third, intermediate colour form is the one believed to have spread around the world since the 1980s, and it is highly resistant to various pesticides (Brodsgaard, 1994). A number of European scientists are studying WFT and related species, but they were unclear about the extent and nature of infra-specific variability and to what extent it might be correlated with the success or failure of control measures. The concerted action was planned to co-ordinate the work on thrips of different specialists over an 18-month period. The overall objective was to co-ordinate the research on WFT (and related species) of taxonomists and those working on the biological control of these species. Specific objectives addressed during this concerted action were: 4.1 To characterize the biology of populations of WFT with respect to life histories, host plants, host-specificity of hymenopteran parasitoids and fungal pathogens - in order to identify strains or 'biotypes' of WFT 4.2 To characterize WFT by molecular means, in order to permit the unambiguous identification of the species and any crop biotypes or locality strains; to provide training and establish experimental protocols so that all participating laboratories are capable of utilizing the defined methods of molecular identification 4.3 To transfer expertise between specialists by means of a series of technical workshops 4.4 To diffuse results through specific publications and a final 'open doors' workshop. All workshops involved presentations of results and progress towards meeting the objectives. The main focus of each meeting is stated below. Workshop no.1, NHM, London, U.K., 15-26 May 1995 Participants: A.G. Lafuente, R. Marullo, G. Moritz, L.A. Mound, P.D. Ready, J.M. Testa; and Prof. C. de Miguel (Dept. Biochemistry, University of Navarra, Spain) Main focus: RAPD-PCR techniques (see 5.3.2) were used by the participants to characterize populations of WFT and other thrip species, and protocols were distributed. Workshop no.2, Pamplona, Spain, 15-21 September 1995 Participants: A.G. Lafuente, A.J.M. Loomans, R. Marullo, G. Moritz; and Prof. Dr R. Jordana Butticaz, Prof. Dr. C. de Miguel (University of Navarra, Spain). Main focus: polyacrylamide gel electrophoretic separation of thrips proteins was demonstrated, and biological experiments were planned. Workshop no.3, Wageningen, Netherlands, 1-3 April 1996 Participants: A.G. Lafuente, A.J.M. Loomans, R. Marullo, G. Moritz, P.D. Ready; and members of the host university and allied research institutes. Main focus: biological control (parasitoids; pathogenic fungus, Verticillium lecanii) and comparative life-history studies were discussed. Workshop no.4, Halle-Wittenberg, Germany, 21-24 August 1996 Participants: A.G. Lafuente, A.J.M. Loomans, R. Marullo, G. Moritz, L.A. Mound; and members of the host university. Main focus: comparative life-history and scanning electron microscopy (SEM) studies were discussed. Workshop no.5, Potenza, Italy, 27-30 October 1996 Participants: A.G. Lafuente, A.J.M. Loomans, R. Marullo, G. Moritz, L.A. Mound, P.D. Ready, J.M. Testa; and Dr G. del Bene (Italy), Dr D. Grasselly (France), Dr T. Lewis (U.K.), Dr G. Schreiter (Halle-Wittenberg, Germany), as well as members of the host university. Main focus: practical workshop on RAPD-PCR DNA characterization techniques, and round-up of all topics. 5.2 Biological characterization of WFT populations The objective of this work was to obtain a large number of contrasted populations from defined biotopes, so as to provide a common working basis for all the partners. The detailed methodology and results are as follows: Field and glasshouse collections Thrips were collected (for biological and molecular analyses) from chrysanthemums and other flowers, as well as from strawberries, table grapes and other fruits from Germany (glasshouses), Italy, Netherlands (glasshouses) and Spain. No WFT populations could be distinguished by colour, all being intermediate-pale. In this species, colour variation is partly phenotypic, being affected by temperature, host plant etc. (A.J.M. Loomans). Dark forms were collected from strawberries near Potenza, but DNA sequencing clearly showed them not to be WFT (see 5.3.3). Standardization of rearing and experimental methods Experimental and rearing conditions were standardized (Loomans & Murai, 1996). In experimental set-ups, 12 cell Greiner multiwell plates (bean leave discs on agar) were used for individual biological assays and experimental chambers were held at 24+2oC, LD 16:8. 30-50 thrips were tested per population. Comparative life history studies of WFT populations In Halle and Potenza, the following biological parameters of local WFT populations were compared to those of an Australian (Perth) population: longevity, fecundity, sex ratio, mortality (Italy) and developmental time. No reproducible, statistically significant differences were found in any of these parameters. Intraspecific variation in thrips and its relevance to biocontrol In Wageningen, host-specificity studies were performed for Ceraniscus menes (Walker) and Ceraniscus americensis (Girault) (Hymenoptera: Eulophidae) using different strains of WFT and the onion thrips, Thrips tabaci, as hosts (A.J.M. Loomans). For a Dutch and an Australian (Perth) population of WFT (both collected on chrysanthemums), parasitoids did not differ in the number of parasitized offspring, but there were small differences in the size of pupae and developmental times. Larger samples are needed to test the significance of these differences. Classical morphotaxonomy In addition to the use of light microscopy for routine identifications of sampled specimens, SEM was employed to view classical morphological characters of different ontogenetic stages of WFT populations from Germany and Australia (Perth), but variation provided no support for recognizing strains (Halle). 5.3 Molecular characterization of WFT populations Table 1 details the species and populations most frequently investigated. 5.3.1 Comparative DNA sequencing to demarcate species boundaries 360 nucleotides of the cytochrome oxidase I gene (COI) of the mitochondrial genome were amplified by the polymerase chain reaction (PCR) for 2 samples (of 1-3 thrips) of independent laboratory stocks (populations) of WFT from Germany (stock 16), Netherlands (stocks 1, 2) and Australia (Perth, stock 15) (Table 1). The same fragment was amplified from other thrips species: Frankliniella schultzei (stock 10), Frankliniella intonsa (stock 11) and two stocks of Thrips tabaci (stocks 6, 7) (Table 1). PCR amplification was achieved by using degenerate primers, which we designed based on an alignment of COI sequences from various insects; they amplified a fragment stretching from the second internal loop to the sixth membrane-spanning helix of the gene (Lunt et al., 1996; modified by P.D. Ready). The PCR products were directly cycle-sequenced on each strand using each of the PCR primers, and the sequences were read and analysed using an ABI automated sequencing system (see: Ready et al., 1997). These sequences will be deposited in GenBank at the time of publication. These original sequences were aligned with those obtained from GenBank (submitted by: Crespi et al., 1996) for the following species: Taeniothrips inconsequens, Thrips trehernei and Frankliniella tritici. Both the nucleotide and the deduced amino acid sequence alignments were analysed using the software 'Phylogenetic Analysis Using Parsimony' (PAUP; Swofford, 1993), with T. inconsequens as the designated outgroup. In both cases, F. intonsa was shown to be the sister taxon to F. occidentalis in the resolved phylogeny, but the genetic distance between these two species was quite high (5.1% and 1.7% pairwise differences between nucleotide and amino acid sequences, respectively). This mtDNA sequence analysis unambiguously characterized WFT, the 4 stocks of which displayed only 1 nucleotide difference: stock 2 from the Netherlands had 1 substitution compared with the other stocks from Germany, Netherlands and Australia. 5.3.2 RAPD-PCR DNA characterization of WFT populations The COI mtDNA sequencing demonstrated that thrips populations recognized by morpho-taxonomists as the intermediate-pale form of WFT (F. occidentalis) comprise one genetic lineage, but COI sequences have a relatively slow rate of sequence divergence (giving rise to a pairwise sequence dissimilarity of not more than 2.5% per million years) and, therefore, would not be expected to provide any diagnostic markers for populations isolated following their introduction to Europe 10-15 years ago. For fine-scale (strain) characterization we chose to use Random Amplified Polymorphic DNA (RAPD)-PCR. This reveals sequence polymorphisms in genomic DNA (isolated from individuals) by using single, simple-sequence primers to anneal in PCR to inverted homologous target sites. Polymorphic length variations in the amplified intervening sequences are detected by agarose gel electrophoresis and result from a variety of causes, including recombination, substitutions or deletions of the target sequence, and natural insertions/deletions in the amplified fragment. The distance of electrophoretic migration of PCR fragments (obtained from individual thrips) depends on their length and, after such fractionation in agarose gels stained with ethidium bromide, the DNA fragments (or 'bands') can be visualized with ultraviolet light. RAPD-PCR was used to characterize single-specimen samples of all the populations listed in Table 1, as well as many others collected by the participants in Italy, Spain, Hungary and Switzerland. Primer sequences tested and results are listed in Tables 2 & 3. Despite much work, we could detect no bands reproducibly diagnostic for any population or group of populations of WFT, although some primers did amplify species-specific bands. In Halle alone, primer OPA-07 was diagnostic for one German population, but the significance of this result is unclear. Quantitative methods of analysis were not attempted, because there was no indication that a combination of bands might place samples into 2 or more well-differentiated groups, or 'strains'. RAPD-PCR can produce bands diagnostic for sibling species or strains of some insects (Black et al., 1992; Cenis et al., 1993; Haymer, 1995; Heckel et al., 1995; Wilkerson et al., 1993), but there are literature reports of failures to find reproducible differences between biological clones or strains of some agricultural pests, eg the peach-potato aphid Myzus persicae (Al-Aboodi et al., 1995). The technique is sensitive to small changes in experimental procedures, but a failure to find any diagnostic band with our large range of primers strongly suggests that there is little strain differentiation in the WFT populations we studied from Europe and Australia. The small size of WFT presents some problems for RAPD-PCR, because quick DNA extraction protocols provide DNA (ca. 10 ng) sufficient only for one or a small number of tests. 5.3.3 Misidentifications of WFT Thrips are very small and, therefore, the mixing of laboratory stock cultures can easily go unnoticed for short periods, as can the sudden appearance of other species amongst WFT field populations. Black 'WFT' collected in southern Italy were discovered to have a COI sequence 10+% different to that of all laboratory stocks of WFT and, therefore, represent another species, probably from another genus. One sample of 'WFT' from Netherlands was shown by RAPD-PCR, and then by the sequencing of COI, to contain specimens of T. tabaci. The difficulties of identifying all species of thrips within samples is a point that should be noted by State plant health services. 5.4 Molecular training and experimental protocols An important objective of this concerted action was to share knowledge of the latest procedures and to diffuse technologies to all partners. All members of the concerted action group had the opportunity to learn the RAPD-PCR technique chosen for strain typing. Thanks to this concerted action, Dr R. Marullo now has RAPD-PCR facilities in Potenza, in the laboratory used for the final workshop, and now Prof. G. Moritz also has all resources for RAPD-PCR in Halle, where his team has routinely used the technique with thrips. Workshop no.1, NHM, London, U.K., 15-26 May 1995 All participants carried out DNA extractions, PCR, RAPD-PCR and agarose gel electrophoresis. Bilateral exchange, NHM, London, U.K., March 1996 Mr A.G. Lafuente spent 1 month practising all steps of the RAPD-PCR protocols with WFT and other thrips he brought from Spain. Bilateral exchange, NHM, London, U.K., 10-21 June 1996 Dr G. Schreiter practised all steps of the RAPD-PCR protocols with WFT and other thrips he brought from Germany. Workshop no.5, Potenza, Italy, 27-30 October 1996 Participants carried out all steps of the RAPD-PCR protocols with WFT and other thrips brought from their countries. Bilateral exchange, NHM, London, U.K., November 1996 Dr R. Marullo spent 2 weeks practising all steps of the RAPD-PCR protocols with WFT and other thrips she brought from Italy. Experimental protocols Ranges of optimal PCR conditions, defined thanks to this concerted action, are listed in Table 3. WFT is already established as an important pest of European agriculture that is frequently being introduced without its natural enemies. Biological control programmes of exotic pests try to use predators, parasitoids or pathogens naturally associated with the pest in its region of origin. For this reason, strain identification of pest and control agent is often important. Strains of WFT are known: 3 sympatric colour forms were described from the western U.S.A. (Bryan & Smith, 1956) and a biotype associated with lupin flowers (Lupine) in New Zealand is susceptible to pesticides and has a significantly lower reproductive potential compared with European stocks (A.J.M. Loomans). All the European stocks seen by us are of the intermediate-pale form that is now believed to be cosmopolitan and resistant to insecticides. We observed no significant biological differences between any of these stocks and, therefore, have found no support for the recognition of distinctive strains or biotypes of WFT in Europe. This is regarded as an important and novel result, the more so because it has been arrived at by a multi-state research group. The recognition of distinctive biological strains usually precedes the search first for morphological characters and then for molecular markers to permit diagnostic identification. In the circumstances it is not surprising that RAPD-PCR failed to provide markers diagnostic for 2 or more stocks of WFT that might represent biological strains. If one adds to this our discovery that there is minimal polymorphism in COI sequences from WFT stocks as widely distributed as Europe and Australia, then it is reasonable to conclude that recent introductions to Europe have mostly come from just one natural genetic lineage originating in the Americas. A review of recent literature showed that Gillings and colleagues used RAPD-PCR to detect 2 different populations of WFT in Australia (Gillings et al., 1995), but the biological relevance of this preliminary finding is not clear. Following a personal communication, Gillings kindly made known to us the primer used to distinguish the Australian strains, as well as the experimental protocols employed, but using his methods we were unable to separate our European and Australian stocks into strains with diagnostic RAPD markers. Details of these results will be reported following the publication of Gillings' findings. We are glad to have been able to work in this way with our Australian opposite numbers. Al-Aboodi, A. & ffrench-Constant, R.H. (1995) RAPD PCR confirms absence of genetic varaiation between insecticide resistant variants of the green peach aphid, Myzus persicae (Homoptera: Aphididae). Great Lakes Entomologist 28, 127-133. Black, W.C. et al. (1992) Use of random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) to detect DNA polymorphisms in aphids (Homoptera: Aphididae). Bulletin of Entomological Research 82, 151-159. Brodsgaard, H.F. (1994) Insecticide resistance in European and African starins of the western flower thrips (Thysanoptera: Thripidae) tested in a new residue-on-glass test. Journal of Economic Entomology 87, 1141-1146. Bryan, D.E. & Smith, R.F. (1956) The Frankliniella occidentalis (Pergande) complex in California (Thysanoptera: Thripidae). University of California Entomological Publications 10, 359-410. Cenis, J.L., Perez, P. & Fereres, A. (1993) Identification of aphid (Homoptera: Aphididae) species and clones by random amplified polymorphic DNA. Annals of the Entomological Society of America. 86, 545-550. Crespi, B. et al. (1996) Molecular phylogenetics of Thysanoptera. Systematic Entomology 21, 79-87. Gillings, M.R., Rae, D., Herron, G.A. & Beatti, G.A.C. (1995) Tracking thrips populations using DNA based methods. Proceedings of 1995 Australian & New Zealand Thrips Workshop, 97-103. Haymer, D.S. (1995) Genetic analysis of laboratory and wild strains of the melon fly (Diptera: Tephritidae) using random amplified polymorphic DNA-polymerase chain reaction. Annals of the Entomological Society of America. 88, 705-710. Heckel, D.G. et al. (1995) Randomly amplified polymorphic DNA differences between strains of Diamondback moth (Lepidoptera: Plutellidae) susceptible or resistant to Bacillus thuringiensis. Annals of the Entomological Society of America. 88, 531-537. Lunt, D.H. et al. (1996) The insect cytochrome oxidae 1 gene: evolutionary patterns and conserved primers for phylogenetic studies. Insect Molecular Biology 5, 153-165. Ready, P.D., Day, J.C., de Souza, A.A., Rangel, E.F. & Davies, C.R. (1997) Mitochondrial DNA characterization of populations of Lutzomyia whitmani (Diptera: Psychodidae) incriminated in the peri-domestic and silvatic transmission of Leishmania species in Brazil. Bulletin of Entomological Research 87, 187-195. Sakimura, K. (1962) Frankliniella occidentalis (Thysanoptera: Thripidae), a vector of the tomato spotted wilt virus, with special reference to the color forms. Annals of the Entomological Society of America 55, 387-389. Swofford, D.L. (1993) Phylogenetic Analysis Using Parsimony (PAUP), version 3.1.1. Illinois Natural History Survey, Urbana, Illinois. Wilkerson, R.C. et al. (1993) Random amplified polymorphic DNA (RAPD) markers readily distinguish cryptic mosquito species (Diptera: Culicidae: Anopheles). Insect Molecular Biology 1, 205-211. Author: Paul
Ready 25-Nov-2002-2002 Dr B R Pitkin
|
- Back to:
- Projects
