Digitized by Kansas Correctional Industries

Thesis (Ph. D.)--Ohio state University, 1924. / Autobiography. "Reprinted from the Ohio journal of science, vol. XXVIII, no. 1, January, 1928." Bibliography: p. 38.

Thesis (Ph. D.)--Cornell University, 1930. / Cover title. Published also as Bulletin 356 of the Maine Agricultural experiment station. "Literature cited": p. 219-221.

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Antifeedants and growth regulators have potential as alternatives to conventional methods of insect pest control. As with conventional chemicals it is important to consider their effects on natural enemies of the pests. The aim of this project was to determine whether the bitter-tasting synthetic chemical denatonium benzoate as an antifeedant and compare with neem derivatives. Other aims were to investigate their effect on parasitoids and the field infestation of cabbage pests. Denatonium benzoate is systemic and had no contact effect but acted as an antifeedant on a generalist sap feeder <I>Myzus persicae</I> and reduced the survival, fecundity, and lifespan. Neem derivatives, Azatin EC and Pestistat R were more effective of which Azatin EC was the most effective. Antifeedants further reduced the fecundity and survival when parasitized with its parasitoid, <I>Aphidius matricariae</I> and neem derivatives had adverse effects on both aphid and the parasitoid. Denatonium benzoate and Pestistat R with a specialist leaf feeder, <I>Pieris brassicae</I> and all antifeedants with <I>Plutella xylostella</I> (generalist leaf feeder) and <I>Chrysodeixis eriosoma</I> (specialist brassica leaf feeder) reduced the leaf area consumed and it was inversely proportional to the concentration. The highest leaf area consumption was in denatonium benzoate and the lowest in Pestistat R. Higher azadirachtin concentrations were detrimental but denatonium benzoate was safer to the larval parasitoid, <I>Cotesia plutellae</I>. Denatonium benzoate and Azatin EC reduced field infestation of <I>M. persicae, P. xylostella</I> and <I>C. eriosoma</I> in Peradeniya, Sri Lanka and Azatin EC was the most effective. Denatonium benzoate acts as an antifeedant against cabbage pests and safer towards parasitoids hence can be used in integrated pest management programmes. Neem derivatives are comparatively more effective on pests but harmful to parasitoids. Denatonium benzoate and Azatin EC effectively reduced the pest infestation under field conditions.

Bacillus thuringiensis subsp. isrealensis (B.t.i.) produces a crystalline endotoxin specific for some larvae of mosquitoes that are vectors of the malaria parasite and other infectious diseases. Fragments were obtained from the 108 kb plasmid from B.t.i. strain 4Q2 which encodes several proteins comprising the delta-endotoxin. These DNA fragments were inserted into the hybrid cyanobacterial cloning vector, pTNTV, downstream from its powerful lambda promoter, and the chimaeras were transformed into Escherichia coli. Ampicillin resistant transformants were screened with radioactively labelled oligonucleotides whose sequences were determined from the published sequences of the B.t.i. 130 kDa polypeptide. Clones showing hybridization were used in bioassays to determine their level of toxicity to the fourth instar larvae of the Aedes aegypti mosquito. Twelve clones were found that demonstrated toxicity which was statistically significantly greater than that observed in controls. Plasmid DNA from some of these clones was isolated, cut with restriction endonucleases, and viewed through agarose gel electrophoresis to confirm that B.t.i. fragments had been inserted into the vector. Future work will investigate the expression of these cloned toxin genes in transformable cyanobacteria and will determine their subsequent activity against the fourth instar larvae of Aedes aegypti and Anopheles quadrimaculatus. / Department of Biology

The use of the larvacidal delta-endotoxin of the sporeforming bacterium Bacillus thuringiensis var. israelensis has been examined as a promising means to control insects that carry diseases such as malaria. An ultimate goal of this project was to genetically engineer both E. coli and the cyanobacterium Synechococcus PCC 7942 to express high levels of this delta-endotoxin and to construct the recombinant to carry a gene which would allow for monitoring of recombinants in the field. Previous research performed by a member of our laboratory involved cloning the gene fragment encoding the delta-endotoxin into a hybrid plasmid yielding recombinant E. coli clones which were toxic to mosquito larvae. Unfortunately, upon further examination of these recombinants using agarose gel electrophoresis and mosquitocidal assays, the clones were found to be unstable and lost their toxin encoding genes readily. Furthermore, cloning of the stabilizing parB locus into one of the recombinant plasmids did not enhance segregational stability as had been shown with some plasmids in E. coli. In another approach oligonucleotide primers were constructed which flanked the 130 kDa toxin gene but excluded a transposon-likesequence postulated to contribute to instability. These primers were used in the polymerase chain reaction in order to amplify this smaller DNA fragment for cloning experiments. Only a small quantity of primers were made and amplification of the DNA was not achieved prior to depletion of the primers. Future work will involve synthesizing new primers to be used for amplification and cloning of the B.t.i. toxin gene.In order to construct a traceable recombinant, the luciferase reporter gene (Luc) had been previously cloned into a hybrid plasmid that was capable of transforming both E. coli and the cyanobacterium Synechococcus PCC 7942. The new construction was then transformed into E. coli, to yield a pool of uncharacterized recombinants. In the present work, I determined that the luciferase enzyme was being expressed in the E. coli recombinants in the presence of the substrate luciferin. Initially, bioluminescence of these E. coli clones was detected by using OG-1 film which fogs in the presence of light. In order to quantify expression of the clones, lysates of the E. coli recombinants were also examined using a luminometer. Comparisons of bioluminescence were made between lysates with the parent E. coli plasmid harboring the luciferase gene and recombinants in which the Luc gene was placed downstream of the powerful rightward lambda promoter. Luminometer readings indicated that luciferase expression was enhanced six fold (from 2.0 X 10-6 to 3.0 X 10-5 by units/cell) in the recombinant plasmid. Plasmid DNA was isolated from the two luciferase expressing E. coli clones. Recombinants were obtained as determined by agarose gel electrophoresis examination of the plasmid DNA. This recombinant DNA was used to transform Synechococcus PCC 7942. However, because enzyme releasing methods were unsuccessful for the more rigid Synechococcus PCC 7942, the level of expression of the Luc gene could not be determined by either method mentioned above. Apparently, the methods used either failed to lyse the cells or they were too harsh and inactivated the enzyme. Future endeavors will involve the use of a French press to more gently lyse the cells so that the level of expression can be determined. / Department of Biology

The larvae of Tenebrio molitor Linne and Alphitobius diaperinus (Panzer) both aggregated upon substrates treated with aqueous extracts of conspecific larval frass. Lactic acid is a pheromone in the frass of both species. Alphitobius larvae were attracted to lactic acid, while lactic acid caused Tenebrio larvae to arrest. / Propionic acid is a repellent pheromone present in Tenebrio frass, but the lactic acid-induced response is dominant. The role of these chemical factors in population orientation of the larvae of these mealworm species is discussed. / The cuticular lipids of the larvae of both species contained close-range attractants that had a role in aggregate formation. The Tenebrio cuticular lipid pheromone is predominately 8,9-pentacosanediol. The Alphitobius cuticular pheromone is a mixture of at least two compounds. / The ecological preferences of these larvae suggested that these aggregation pheromones increased the density of individuals per unit volume. This increased density had varying effects on the physiological development of Tenebrio individuals.