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Doctor of Philosophy / Department of Entomology / David C. Margolies / Yoonseong Park / The predatory mite Phytoseiulus persimilis (Acari, Phytoseiidae) is one of the most frequently released natural enemies for biological control of spider mites in greenhouse and outdoors crops. In this research, I utilized Expresses Sequence Tags (ESTs), the most cost effective approach for transcriptome exploration, to study three different aspects of this arachnid species for which there is little genomic information. I combined two EST datasets from different whole body cDNA libraries and analyzed by bioinformatics means. Approximately 54% of 10,256 uniESTs were annotated based on the homology to sequences in the National Center for Biotechnological Information (NCBI) database. A list of these uniESTs, sorted from most to least likelihood based on the expected value from the blast search in public databases, was used to create tools for each of the three studies. First, I described sixty-one genes encoding products known to be important in pesticide metabolism and in endocrinology, including cytochrome P450s, glutathione-S-transferases, acetylcholinesterase homologs, neuropetides and neurohormones. Findings on arachnid specific esterases and neuropetides, and possible benefits to pest management programs, were discussed. Next, I inferred divergence time for Acari and the point of divergence of two lineages within anactinotrichid mites, Ixodes scapularis and Phytoseiulus persimilis. I used expresses sequence tags from the predatory mite P. persimilis to pull out 74 orthologous amino acid sequences of invertebrates species: nine insect species, Daphnia pulex, Ixodes scapularis, and Caenorhabditis elegans. I estimated a similar origin for Chelicerata (578.1 ± 38.2 - 482.2 ± 7.2 Mya) as in other recent studies. However, divergence dating using amino acid sequences suggested a Devonian origin of anactinotrichid mites (487.6 ± 32.2 - 410.1 ± 6.1 Mya) based on four reference dates (two fossil records and two molecular clocks) and four amino acid substitution methods; this estimate is much earlier that those in the current literature. This discrepancy of divergence times may be due to the use of a global clock. Finally, I developed molecular markers from the EST dataset to examine inheritance in the haplodiploid system in P. persimilis. Biparental contribution of chromosomes is required among the predatory mites but the paternal chromosome set seems to be eliminated or loss (Paternal genome loss, PGL) in male offspring. However, genetic studies in other two phytoseiid species were suggested diploid males with PGL only in the germ cells. In the present study, haploid adult males of P. persimilis have been observed using five independent EST-derived markers. Single mites derived from inter-population crosses were genotyped after whole genome amplification. The parahaploid genetic system in P. persimilis is supported by this study, in which both sexes arise from fertilized eggs but the paternal chromosome set is subsequently lost in males.

The spider mite predator Phytoseiulus persimilis Athias-Henriot and the thrips predator Amblyseius cucumeris (Oudemans) are both commercially mass-reared for use as biological control agents for greenhouse pests. The ability to stockpile these mites in cold storage would greatly facilitate economical mass-production and distribution. In addition, these two species provide an opportunity for comparative studies of cold hardiness because P. persimilis originated in subtropical Mediterranean type climates and is thought to be incapable of entering diapause whereas A. cucumeris is widespread in temperate zones where adult females enter a reproductive diapause for overwintering. Temperature/mortality curves confirmed that both species are freezing intolerant in the traditional sense that supercooling points (SCPs), at which freezing of body fluids occurs, represent absolute lower lethal temperatures. Both species were capable of moderate supercooling, into the range of-20°C to -30°C, and both showed a trend of increasing SCP temperatures during development from egg to adult that suggested an inverse relationship between supercooling capacity and body size within species. The only exception to this trend was adult female A. cucumeris, which may be significant because this is the only life stage that is capable of diapause or survival of temperate winters. On the other hand, diapause induction and low temperature acclimation had little or no effect on supercooling capacity, and survival of nonacclimated mites at subzero temperatures above their SCPs was limited to very short periods of exposure, suggesting that the SCP represents a physical property of the mites rather than an adaptation for survival of exposure to subzero temperatures. Cold-storage survival of both species was optimum at 7.5°C, was greatly enhanced when a source of moisture was provided, and was enhanced even further when food was provided even though the mites were held at temperatures below their theoretical temperature threshold for development. Under these conditions, survival of P. persimilis was 80% after six weeks whereas that of A. cucumeris was only 35% after the same period of time. Furthermore, longevity and fecundity of P. persimilis after eight weeks of cold storage were comparable to mites taken directly from rearing cultures whereas oviposition by A. cucumeris after six weeks of cold storage was low and irregular compared to previously reported values. Temperature data from areas where P. persimilis survives outdoors indicate that this species is capable of surviving prolonged exposure to temperatures below 10°C and occasional exposure to subzero temperatures, and apparently does so without diapausing. On the other hand, A. cucumeris may be capable of surviving prolonged exposures to subzero temperatures, but only as diapausing adult females. The results of the current studies are consistent with these ideas and further suggest that nondiapausing A. cucumeris are less cold hardy than P. persimilis. / Graduate

The twospotted spider mite, Tetranychus urticae Koch, is a serious pest of many ornamental plants (Johnson and Lyon, 1991). Pesticide resistance, the high cost of pesticides and loss of production time have raised interest by growers to introduce predatory phytoseiid mites to manage twospotted spider mites and reduce their need for acaricide applications (Sabelis, 1981). The predatory mite Phytoseiulus persimilis Athias-Henriot has been used successfully in integrated pest management programs for T. urticae suppression. Despite the success of P. persimilis in reducing populations of T. urticae, acaricide applications may still necessary due to limitations associated with the effectiveness of P. persimilis introductions. The objectives of this study were to; 1. Measure the effects of acaricides on the density and age structure of T. urticae populations. 2. Determine the compatibility of acaricides in an IPM program by measuring the toxicity of residues to P. persimilis and T. urticae adults. 3. Study the feeding behavior of P. persimilis on T. urticae. 4. Measure the effects of combinations of acaricides followed by release of P. persimilis on T. urticae populations using greenhouse trials. The effects of ten acaricides on T. urticae populations were measured on infested Buddleia x davidii 'White Profusion' cuttings. Acaricides did not alter age structure in predictable manner. Initial analysis of results demonstrates that cuttings treated with acaricides had age structures that were different from control treatments. However, these differences were not distinguishable from natural fluctuations in the age structure. Chlorfenapyr may have changed the age structure of T. urticae. Azadirachtin, pyridaben and spinosad did not suppress T. urticae populations at the rates and formulations tested in this trial. Abamectin, bifenthrin, chlorfenapyr, Gowan 1725,oil and neem oil suppressed T. urticae populations. Hexythiazox suppressed T. urticae populations but these results were not seen until two weeks after application. The effects of acaricide residues were tested on adult P. persimilis and T. urticae 1, 3, 7, and 14 days after application using a leaf disk system. Abamectin, Gowan 1725, hexythiazox, horticultural oil, neem oil, pyridaben and spionsad were not toxic to P. persimilis adults while bifenthrin and chlorfenapyr residues were toxic to P. persimilis. Tetranychus urticae mortality from chlorfenapyr residues was significantly greater than the control 1,3,7 and 14 days after application. Tetranychus urticae mortality from bifenthrin and abamectin residues was significantly greater than the control 3, 7, and 14 days after application. Tetranychus urticae mortality caused by Gowan 1725, horticultural oil, and neem oil residues was significantly greater than the control 1 day after application, while mortality from hexythiazox and spinosad residues was not significantly greater than the control at any of the times tested in this study. Phytoseiulus persimilis feeding behavior studies examining life stage preference tests and functional response studies were conducted on bean leaf disks. We found P. persimilis functional response to be a type II response for both eggs and adults with handling times of 0.079 hours for eggs and 3.399 hours for adults. The effects of a combination of acaricides followed by release of P. persimilis on T. urticae populations was tested using greenhouse studies conducted on infested Buddleia plants. In the first trial, severe plant damage occurred despite a reduction in the mean number of mites per leaf in treatments with oil+ predator treatments 7 days after release. Results from the second greenhouse trial produced plants with less visual damage compared to those in the first greenhouse trial. Treatments with predators alone and predators + acaricides produced similar results. However, treatments with predators had a mean numbers of mites per leaf that were significantly less than treatments with acaricides alone. The results demonstrate that the acaricides tested in the second greenhouse trial allowed the predators to provide suppression of T. urticae populations. A high release rate was used in the second greenhouse trial and lower release rates as well as different acaricide predator combinations need be tested to explore the possibility of new management techniques. Our results suggest that the number of pest mites present in the crop may be the most important factor affecting the success of biological control with predators. Combinations of oil applications followed by introduction of P. persimlis 3 days after release provided suppression of T. urticae populations in a meaningful time frame. I was not able to shape the age structure of T. urticae populations in a predictable manner with acaricide applications. Phytoseiulus persimilis does not have a prey-stage preference when feeding on T.urticae, but the shorter handling time for eggs may indicate that they are better able to suppress populations with higher proportions of eggs. However, we cannot determine if P. persimilis can keep T. urticae populations composed of predominately of eggs below threshold levels because our greenhouse trials did not test this hypothesis. Phytoseiulus persimilis feeding on adult T. urticae may suppress T. urticae populations below threshold levels because a reduction in the number of adults will lead to a reduction in the number of T. urticae eggs deposited on a plant. Our research suggests that abamectin and oil are two acaricides that would be less detrimental to the survival of P. persimilis. Additional greenhouse trials with compatible acaricides should be conducted as well as research on the threshold density of T. urticae that will allow P. persimilis to provide adequate control. / Master of Science

Master of Science / Department of Entomology / David C. Margolies / Yoonseong Park / Phytoseiulus persimilis (Acari: Phytoseiidae) is a specialist predator on tetranychid mites, especially on the twospotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae). The foraging environment of the predatory mites consists of prey colonies distributed in patches within and among plants. Quantitative genetic studies have shown genetic variation in, and phenotypic correlations among, several foraging behaviors within populations of the predatory mite, P. persimilis. The correlations between patch location, patch residence, consumption and oviposition imply possible fitness trade-offs. We used molecular techniques to investigate genetic variation underlying the foraging behaviors. However, these genetic studies require a sufficiently large amount of DNA which was a limiting factor in our studies. Therefore, we developed a method for obtaining DNA from a single mite by using a chelex extraction followed by whole genome amplification. Whole genome amplification from a single mite provided us with a large quantity of high-quality DNA. We obtained more than a ten thousand-fold amplified DNA from a single mite using 0.01ng as template DNA. Sequence polymorphisms of P. persimilis were analyzed for nuclear DNA Inter Transcribed Spacers (ITS1 & ITS2) and for a mitochondrial 12S rRNA. The sequence comparisons among individuals identified a number of polymorphisms in the 12S sequences. The foraging gene (for) associated with rover-sitter behavioral strategies of Drosophila is known to have role in feeding behaviors of honeybee and other arthropods. We surmised that the same or a similar gene may be present in P. persimilis. Among the foraging behavior(s) exhibited by this predatory mite, we were particularly interested in resource/prey-dependent dispersal behavior. We isolated a partial sequence that is presumed to be the orthologue of the foraging (for) gene. We named the putative foraging gene as Ppfor (for Phytoseiulus persimilis foraging gene). We used a fragment of Ppfor gene as a molecular marker between populations and among individuals and, further, to help understand behavioral phenotypes.

Master of Science / Department of Entomology / David C. Margolies / James R. Nechols / The study of how extrinsic factors affect the foraging efficiency and behavior of predaceous arthropods like Phytoseiulus persimilis is important to understand their various processes of acquiring prey, mates, refuges, oviposition sites, and overcoming obstacles posed by the environment. Many intrinsic and extrinsic factors affect predator foraging efficiency and behavior. One of the most influential extrinsic factors may be the host plant on which herbivorous prey are found. Recent studies suggest that plant architecture plays an important role in tritrophic interactions. In this work, I studied the effects of cucumber plant architecture and prey distribution on the foraging efficiency (prey-finding time and prey-consumption rate) and behavior (time allocated between moving, resting and feeding) of P. persimilis. Plant architecture represented differences in leaf number and size; however, all plants had the same total surface area. Plants with 6 small leaves (ca. 82.98 square cm each) were considered as complex architecture, whereas plants with only 2 large leaves (ca. 240.60 square cm each) were considered as simple. The prey distributions were: prey patch on a single basal leaf (closest leaf to the soil) and prey patch on all leaves. The foraging efficiency was assessed by measuring prey-finding time and prey-consumption rate, whereas the behavior was assessed by conducting observational studies on specific foraging activities. When placed either on the top or at the base of the plant P. persimilis encountered prey more rapidly (interval 0-30 minute) on complex and simple plants with prey patches distributed on all leaves. Differences in prey density (number of prey per leaf) had no effect on the prey-finding time of P. persimilis. The predator consumed more eggs on complex plants with prey patches distributed on all leaves. Phytoseiulus perismilis tended to find prey patches more quickly as well as consume more eggs on leaves close to its release point. Furthermore, the predator was observed to lay more eggs on leaves where it had consumed higher number of prey eggs. The dimensions of individual parts of the plant e.g., stem, petiole and leaf, affected the time allocated by P. persimilis between searching, resting and feeding. The predatory mite spent more time foraging on the stems and petioles of the simple plants whereas on complex plants it spent more time foraging on the leaves.

Las arañas rojas constituyen una de las plagas más importantes en la producción de hortalizas del litoral mediterráneo español, tanto en invernadero como al aire libre. Estos ácaros son controlados por medios químicos, lo cual representa costos significativos en términos económicos y ecológicos. Otra alternativa sería el control biológico usando fitoseidos que son depredadores naturales de los tetraníquidos. Sin embargo, se desconocen informaciones básicas sobre la dinámica y la óptima relación depredador-presa necesarias para que el control biológico sea efectivo. Este trabajo evaluó la relación depredador-presa de los fitoseidos nativos Phytoseiulus persimilis Athias- Henriot y Neoseiulus californicus (McGregor) con las arañas rojas T. urticae Koch, T. turkestani Ugarov y Nikolski y T. evansi Baker y Pritchard. El estudio incluyó ensayos de laboratorio y de invernadero, realizados en plantas de judía (Phaseolus vulgaris L.) y patata (Solanum tuberosum L.), al igual que simulaciones de la dinámica poblacional de los fitoseidos depredadores y los ácaros fitófagos. En el laboratorio se estudió el consumo de presa y los principales parámetros biológicos y demográficos de los dos depredadores cuando se alimentan de T. urticae. Los resultados del laboratorio indicaron que la duración del desarrollo, la fecundidad y los parámetros demográficos de poblaciones locales de N. californicus y P. persimilis eran similares a los de otras regiones geográficas y que cuando estos depredadores consumían T. urticae sus parámetros alcanzaban valores óptimos. En el invernadero se evaluó su eficacia en tres relaciones depredador-presa: 1:12, 1:8 y 1:4 para los ensayos con T. urticae, y de1:16, 1:8 y 1:4 para ensayos con T. turkestani y T. evansi. También se determinó el índice de daño y la distribución de los ácaros en las plantas y su comportamiento dispersivo en función del tiempo y de la densidad poblacional. / Gómez Moya, CA. (2007). Dinámica del sistema depredador-presa de las arañas rojas y los fitoseidos (Acari: Tetranychidae, Phytoseiidae) en cultivos hortícolas [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/15228 / Palancia