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International Symposium on Biological Control of Arthropods 424 Poster Presentations ______ POSTER PRESENTATIONS ______________________________________________________________ Poster Presentations 423 IMPROVEMENT OF RELEASE METHOD FOR APHIDOLETES APHIDIMYZA (DIPTERA: CECIDOMYIIDAE) BASED ON ECOLOGICAL AND BEHAVIORAL STUDIES Junichiro Abe and Junichi Yukawa Entomological Laboratory, Kyushu University, Japan ABSTRACT. In many countries, Aphidoletes aphidimyza (Rondani) has been used effectively as a biological control agent against aphids, particularly in greenhouses. In Japan, A. aphidimyza was reg- istered as a biological control agent in April 1999, and mass-produced cocoons have been imported from The Netherlands and United Kingdom since mass-rearing methods have not yet been estab- lished. In recent years, the effect of imported A. aphidimyza on aphid populations was evaluated in greenhouses at some Agricultural Experiment Stations in Japan. However, no striking effect has been reported yet from Japan. The failure of its use in Japan seems to be caused chiefly by the lack of detailed ecological or behavioral information of A. aphidimyza. Therefore, we investigated its ecological and behavioral attributes as follows: (1) the survival of pupae in relation to the depth of pupation sites; (2) the time of adult emergence in response to photoperiod during the pupal stage; (3) the importance of a hanging substrate for successful mating; and (4) the influence of adult size and nutrient status on adult longev- ity and fecundity. (1) A commercial natural enemy importer in Japan suggests that users divide cocoons into groups and put each group into a plastic container filled with vermiculite to a depth of 100 mm. However, we believe this is too deep for A. aphidimyza pupae, since under natural conditions mature larvae spin their cocoons in the top few millimeters to a maxmum depth of 30 mm. Our experiments indicated that the deeper the pupation site in the vermiculite, the higher the pupal and adult mortality. (2) In nature emergence of A. aphidimyza adults peaks soon after sunset. From the imported cocoons, however, most adults emerged near midnight. This means that the imported individuals were apparently no longer in synch with local time after transportation to Japan. (3) Unlike many other cecidomyiids, A. aphidimyza mate by hanging from spider webs and facing each other. Our experiment showed that A. aphidimyza could not copulate without a hanging substrate. (4) The females of A. aphidimyza reared with honey solution lived significantly longer and had significantly more eggs than those reared with water alone. Based on these results, we suggest the following ideas to improve the release methods of use of imported cocoons: (1) keep the cocoons in shallow containers to increase pupal survival; (2) keep cocoons under local photoperiod immediately after arrival in Japan to adjust synchrony to local time; (3) provide hanging substrate sites for adult mating near release locations using spider-web-like sub- strates such as fishing lines; and (4) provide females with artificial nutrient diet near the release locations. 1st International Symposium on Biological Control of Arthropods 424 Poster Presentations _____________________________________________________________________ DEVELOPMENT OF MOLECULAR MARKERS TO STUDY THE ROLE OF COLLEMBOLA AS AN ALTERNATIVE PREY SUSTAINING SPIDERS IN CEREAL CROPS N. Agustí and W.O.C. Symondson Cardiff University, Cardiff, Wales, United Kingdom ABSTRACT. The conservation and fostering of larger populations of native predator species can be effectively used to regulate crop pests. In order to manipulate generalist predators and use them for this purpose in a safe, environmentally friendly and sustainable manner, food webs in farmland eco- systems must be well understood. We focused on developing novel molecular techniques to analyze the gut contents of polypha- gous predators to investigate how prey species diversity and the availability of nonpest prey help to maintain predator populations within crops. The model system chosen was linyphiid spider predators and Collembola, a major nonpest prey. These spiders have been shown to be potentially important predators of aphids and other pest species in winter wheat crops, while Collembola are a numerically dominant alternative prey of linyphiids. Understanding the role of such nonpest prey in the spider- aphid system provides a model with much wider application to other predator-prey systems. Recent studies, based on the development of molecular markers as tools for the analysis of predator-prey interactions, have shown DNA techniques to be an exciting new approach with enormous potential for the study of complex food webs in farmland and other ecosystems. Here we report how relevant parts of the mitochondrial gene Cytochrome Oxidase I (COI) were amplified by PCR from the three most common species of Collembola in our area of study: Isotoma anglicana (Lubbock) (Collembola: Isotomidae), Lepidocyrtus cyaneus Tullberg, and Entomobrya multifasciata (Tullberg) (Collembola: Entomobrydae). The DNA from these Collembola was sequenced and aligned with sequences for other potential prey species present in the same crop and several species of spider. Specific primers were then designed for the target Collembola species. We used three pairs of these primers, one for each of the three different species of Collembola, all of which amplified short sequences (~200 bp). Specificity analyses were performed using these primers, in which all the species previously sequenced plus many others were tested. The primers proved to be species-specific, amplifying exclusively the target prey species and no other Collembola, other prey, or spiders, effectively eliminating the possibility that predation might be overestimated though the detection of false positives. Experiments are in progress to measure the detection period for Collembola DNA in the guts of these spiders following ingestion. Preliminary results have already shown that detection of Collembola ingested by the spiders is possible using our primers. Application of these methods will allow us in future to determine the prey range (pest and nonpest) of linyphiid spiders in the field, will help us to quantify the role of predation (by both indi- vidual species of predator and predator guilds) in prey population dynamics, and will help us to ana- lyze the effects of biodiversity on the natural regulation of pests. 1st International Symposium on Biological Control of Arthropods ______________________________________________________________ Poster Presentations 425 USING MOLECULAR MARKERS TO DETECT CACOPSYLLA PYRICOLA REMAINS IN ARTHROPOD PREDATOR GUTS N. Agustí,1 T.R. Unruh,2and S.C. Welter3 1Cardiff University, Wales, United Kingdom 2Yakima Agricultural Research Laboratory, U.S. Department of Agriculture, Agricultural Research Service, Wapato, Washington, U.S.A 3Department of Environmental Science, University of California, Berkeley, California, U.S.A. ABSTRACT. The pear psylla, Cacopsylla pyricola (Förster) (Homoptera: Psyllidae), one of the main pear pests in the United States, can severely affect the productivity of pear trees in three different ways: by acting as a vector for the pear decline disease, by producing honeydew blackening the foliage and provoking skin russets on the fruits, and by injecting a toxin into the tree causing death. To solve this problem, predators have been studied for use in Integrated Pest Management (IPM) programs against this species. In recent years, the main approach to studying predation in the field by predators that suck liquefied prey has been the use of protein-based techniques, particularly, the development of Monoclonal Antibodies (MAbs). An attractive alternative to the expensive and complex processes involved with the production of MAbs is the development of molecular markers using PCR-based techniques for detection and identification of prey DNA in predator guts. We developed DNA markers to detect pear psylla remains in arthropod predator guts using specific primers designed from mitochondrial Cytochrome Oxidase I (COI) gene sequences. COI gene segments 400 bp long from C. pyricola, other psyllid species, other potential prey species present in the same crop, and several predator species included within the Heteroptera, Neuroptera, Co- leoptera, and Arachnida were sequenced. Alignment and comparison of these sequences allowed the design of 12 pairs of primers potentially specific for C. pyricola. Here we show the results of one of these pairs of primers, which allow the amplification of a 271 bp fragment. When this pair of primers was tested for species specificity against DNA from other prey and predator species, the 271 bp fragment was detected exclusively in species within the Cacopsylla genus. These results indicate that primers with high specificity were designed from the COI gene to detect prey remains in predator guts. In order to study the ability of the primers to detect the psyllid sequence over time following ingestion, Anthocoris tomentosus Pericart (Heteroptera: Anthocoridae) adults were fed C. pyricola nymphs and were then either immediately frozen or were held for 2, 4, 6, 8, 16, 24 or 32 h at 22 ºC. Predators were then assayed for the presence of pear psylla DNA in their gut. These primers demonstrated successful detection of psyllid DNA in the predator gut at all digestion periods tested. 1st International Symposium on Biological Control of Arthropods 426 Poster Presentations _____________________________________________________________________ DEVELOPMENT
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