Thesis (MSc (Biomedical Sciences))--Cape Peninsula University of Technology, 2019 / Background: Baculoviruses have great potential as alternatives to conventional chemical insecticides. The large scale adoption of such agents has however been hampered by the slow killing times exhibited by these bio-insecticides, limitation to single target insect and difficulty of large scale production of these preparations. Trichoplusia ni single nucleopolyhedrovirus (TnSNPV), initially identified in the Eastern Cape region of South Africa, has potential as a biocontrol agent as it possesses a higher speed of kill compared to other baculoviruses. Aims and methods: The main objective of this study was the identification, molecular characterisation and cloning of a structural core gene (polyhedrin) and three auxiliary genes, the inhibitor of apoptosis (iap2 and iap3) and the ecdysteroid UDP-glucosyltransferase (egt) genes, from TnSNPV in order to delineate its phylogenetic relationship to a Canadian isolate of the same virus and to other baculoviruses. In addition, the genes were expressed in an Escherichia coli (E. coli) based system as a prelude to genetic modification to increase the pesticidal property of the virus. Results: The genome size of the South African strain of TnSNPV was estimated at 160 kb and is significantly larger than the Canadian isolate of TnSNPV and may reflect genetic variation as the two strains have adapted to varying environmental conditions. Occlusion bodies of the South African strain of TnSNPV were visualised by Transmission Electron Microscopy and consisted of rod shaped single virions composed of a single enveloped nucleocapsid. Insect bioassays showed that the median lethal time (LT50) of the virus strain averaged 1.8 days which is significantly faster than other baculoviruses. The South African and Canadian strains of TnSNPV share nucleotide similarities greater than 95% for the genes analysed in this study, which indicates that they are closely related. From this analysis, the South African strain of TnSNPV identifies as a Group II NPV with the closest relatives being the Canadian strain of TnSNPV and ChchNPV. The topology of the tree for the polyhedrin protein was better resolved than that of the IAP2, IAP3 and EGT proteins and was comparable to the tree inferred from a concatenated data set consisting of complete polyhedrin/granulin, LEF8, and LEF9 proteins of 48 completely sequenced genomes. For the IAP2, IAP3 and EGT proteins, the separation of the lepidopteran and hymenopteran specific baculoviruses was not evident while the separation of Group I and II Alphabaculoviruses diverged from that observed from the baculovirus core gene polyhedrin as well as the tree inferred from complete polyhedrin/granulin, LEF8, and LEF9 proteins. Five distinct groups relating to IAP-1, 2, 3, 4 and 5 could be distinguished from the tree inferred from all IAP proteins from 48 fully sequenced baculoviruses. From this analysis, the IAP protein from the South African isolate of TnSNPV can be designated as an IAP3 due to sequence homology to other IAP3 proteins. Similarly, the IAP2 can be confirmed as an IAP2 protein as it clusters with other IAP2 proteins. RNA transcripts of the four genes were detected by RT-PCR at one hr after induction with Larabinose in BL21-A1 E. coli and persisted until four hrs post induction. Antisera directed against the C-terminal 6X His tag was able to detect the recombinant proteins at two hours after induction confirming the rapid rise in expression of the proteins which persisted at high levels until four hrs after induction. The discrepancy observed with the predicted molecular mass of the EGT protein and the migration on SDS-PAGE may be due to the absence of posttranslational modification in the E. coli expression system and the hydrophobic residues present in the N-terminal signal sequence. Conclusion: Sequence and phylogenetic analysis suggest that the two isolates of TnSNPV have been exposed to similar evolutionary pressures and evolved at similar rates and represent closely related but distinct variants of the same virus. The difference in genome size between the two strains is likely to reflect actual genetic differences as the strains have adapted to their local environments and hosts and the extent of the differences will only be apparent as more sequencing results become available. Phylogenetic analysis of the IAP and EGT proteins yields a tree that varies from the phylogenetic reconstruction observed for the polyhedrin gene as well as the concatenated data set consisting of complete polh/gran, LEF8, and LEF9 proteins and highlights the risks inherent in inferring phylogenetic relationships based on single gene sequences. The tree inferred from the concatenated data set of polh/gran, LEF8, and LEF9 proteins was a quick and reliable method of identification particularly, when whole genome data is unavailable and mirrors the accepted lineage of baculoviruses. Expression of the recombinant IAP2, IAP3, EGT and polyhedrin was confirmed by RT-PCR and immunoblot analysis and rose rapidly after induction and persisted at high levels. It is as yet unclear if the expressed proteins are functional particularly as post translation modifications are lacking in this system.

A behavioral model for acute responses in bivalves, was developed using time series analysis for use in a real-time biomonitoring unit. Stressed bivalves closed their shell and waited for the stressful conditions to pass. Baseline data showed that group behavior of fifteen bivalves was periodic, however, individuals behaved independently. Group behavior did not change over a period of 20 minutes more than 30 percent, however, following toxic exposures the group behavior changed by more than 30 percent within 20 minutes. Behavior was mathematically modeled using autoregression to compare current and past behavior. A logical alarm applied to the behavior model determined when organisms were stressed. The ability to disseminate data collected in real time via the Internet was demonstrated.

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Insecticide resistance in disease-transmitting arthropods has become a serious hindrance for successful vector control. Mosquitoes, in particular, are notorious vectors of potentially deadly diseases like malaria, dengue fever, and West Nile virus. Anopheles gambiae and Culex quinquefasciatus are just two examples of mosquito vectors that possess genetic mutations (denoted kdr and ace-1 ) and/or enhanced detoxifying enzymes (oxidases, esterases, and glutathione-s-transferases) that confer insecticide resistance. Culex tarsalis, a primary vector for West Nile virus among other arboviruses in Northern California, is a target for insecticide application and is under constant insecticide pressure, making it likely to adapt resistance mechanisms like kdr or ace-1 or increased detoxifying enzymes. Culex tarsalis adult females were collected from Yolo and Sutter counties. A bottle bioassay was completed to determine prevalence of resistance to Sumithrin (a pyrethroid; N=217) and Naled (an organophosphate; N=154). A susceptible lab-reared colony was used for comparison. Microplate assays were completed to investigate elevated levels of detoxification enzymes present as well as AChE. PCR was used to amplify the VGSC and ace-1 genes. Amplicons were sequenced and aligned to determine if mutations were present. No evidence of the ace-1 mutation was found in any mosquitoes, but the kdr mutation was seen in all semi-resistant and resistant individuals exposed to Sumithrin. Microplate data revealed significant differences between certain detoxifying enzymes within mosquitoes collected from Sutter and Yolo Counties exposed to both Sumithrin and Naled. The data obtained from this study suggests that resistance to Sumithrin in both populations is carried out by both metabolic and target site insensitivity, while resistance to Naled is caused by metabolic resistance only.

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A physiologically-based toxicokinetic and toxicodynamic (PBTK/TD) model was developed, from the open literature, to predict the toxicokinetic disposition and toxicodynamic response (acetylcholinesterase inhibition) of a ternary organophosphorus (OP) insecticide mixture: chlorpyrifos (CP), methyl parathion (MP) and parathion (P). In vivo studies were conducted in adult male Sprague-Dawley rats, orally administered one of two CP/MP/P mixtures (2.5, 0.5, 0.5 mg/kg or 5, 1, 1 mg/kg) with selected tissues (blood, brain, diaphragm, liver, lung and skeletal muscle) collected at 30min, 4, 12 and 24hr postdosing. Low dosages were studied so the mixture did not result in significant disruption of cardiovascular function nor invalidate the model’s underlying general physiological assumptions. The data were used to validate the model. CP and its metabolites (CP-oxon, 3,5,6-trichloro-2-pyridinol (TCP)), as well as MP, P and 4-nitrophenol, were quantified in the tissues of interest. Peak concentrations of CP were attained by 4hr in all tissues with the exception of the liver, whose peak occurred at 30min; MP, 30min in all tissues; P, 12hr in all tissues with the exception of the liver, 30min. This was supported by the model simulations. MP, P, and their respective oxons were below limits of quantitation for the lower dosage. No toxicokinetic interactions were observed in the present study. Cholinesterase inhibition in the tissues ranged from 11- 37% for the lower dosage, and 29-93% for the higher dosage group; with few exceptions, inhibition was generally additive and was also supported by the model simulations. This study demonstrates the utility of using previously developed individual PBTK/TD models and in vitro/in vivo data from the open literature to construct reliable mixture PBTK/TD models.

京都大学 / 新制・課程博士 / 博士(地球環境学) / 甲第24153号 / 地環博第231号 / 新制||地環||44(附属図書館) / 京都大学大学院地球環境学舎地球環境学専攻 / (主査)教授 梶井 克純, 教授 高野 裕久, 准教授 田中 周平 / 学位規則第4条第1項該当 / Doctor of Global Environmental Studies / Kyoto University / DFAM

Thrips are pests in a variety of crops and are responsible for millions of dollars in damage worldwide. In Virginia there are a few key thrips species that cause a large portion of damage to both vegetable and floricultural crops. Three prominent pests include Frankliniella tritici (Fitch), Frankliniella fusca (Hinds), and Frankliniella occidentalis (Pergande). Significant yield losses in row crops such as cotton, peanuts and vegetables have been attributed to feeding and oviposition of these insects in high densities. In addition, both F. fusca and F. occidentalis can transmit plant pathogenic tospoviruses, such as tomato spotted wilt virus (TSWV), in certain susceptible crops. While all of these thrips species are difficult to detect due to their cryptic lifestyles, F. occidentalis is a particularly challenging pest to manage due to its resistance to many insecticides commonly used for thrips treatment. Early spring weeds were sampled for the presence of F. occidentalis in 2008 and 2009 in eastern Virginia. Weed samples consisted of mustard, henbit and wild radish and were collected from several different sites on the Eastern Shore of Virginia. During the summer of 2008, 2009 and 2010 various agroecosystems were sampled for the relative incidence of F. occidentalis. Overall, thrips numbers were very low in weed samples. F. occidentalis was detected in early spring weed samples in 2009 at a few of the sites sampled. In nearly every habitat, the species composition was dominated by F. fusca and F. tritici, with F. occidentalis occurring in very low numbers. Two different lures were evaluated in their ability to attract Frankliniella spp. thrips. The lures included Chemtica P-178 floral kairomone (AgBio Inc., Westminster, CO), a floral iii kairomone lure composed of a proprietary floral compound mixture, and ThriplineAMS (Syngenta Bioline Ltd., Oxnard, CA) pheromone lure, containing the aggregation pheromone of F. occidentalis. In spring 2009 and 2010 lure experiments were conducted in several different agroecosystems including: a tomato and potato field in Painter, VA, a cotton and peanut field in Suffolk, VA, and grass fields near a greenhouse in Virginia Beach, VA, and a high tunnel in Chesapeake, VA, as well as within these structures. Baited and non-baited sticky cards were arranged in a completely randomized design, with a pan trap located in the center of each plot. Traps were collected approximately twice weekly. F. fusca numbers were low and catches on sticky cards were not significantly affected by either lure. Sticky cards baited with the kairomone caught more flower thrips than traps baited with the pheromone, or the non-baited traps, especially when thrips numbers were high. Several biologically derived insecticides including: essential oils, spinetoram, spinosad, pyrethrins, and azadirachtin, were evaluated in their efficacy against thrips in several different crops. Randomized complete block design experiments were carried out in: tomatoes, snap beans, collards, soybeans, cotton and peanuts grown in several locations in southeastern Virginia in 2009 and 2010. Both spinetoram and spinosad reduced thrips numbers the most effectively compared with the untreated control. Peanut and cotton treated with spinosad, and treatments containing spinetoram suffered less thrips injury compared with the control, and yield was higher in cotton plots treated with spinetoram. / Master of Science in Life Sciences