Investigation of Mosquitocidal Activity of Paraclostridium bifermentans serovar malaysia and Bacillus thuringiensis subspecies israelensis

Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus serve as vectors for some of the worlds most debilitating diseases; these diseases include Dengue Fever, Malaria, and West Nile Virus respectively. One method currently employed to combat these diseases, and the deaths they cause, is to control the mosquito populations that allow them to persist. Multiple methods have been used to control mosquito populations, one method is through chemical control through pesticides, another is biological control through bacterial toxins. Bacillus thuringiensis israelensis and Lysinibacillus sphaericus have been used to control Aedes and Culex populations respectively, but no current biological control methods primarily target Anopheles mosquitoes. Additionally, some mosquito populations have started to develop resistance against Lysinibacillus sphaericus toxins, while resistance has also been seen against single toxins produced by Bacillus thuringiensis israelensis. A new bacteria called Paraclostridium bifermentans malaysia was discovered in an Anopheline environment and produces toxins that target Anopheles mosquitoes. A large 109Kb plasmid was found in Pbm, this plasmid contains two loci that are toxic, the Cry operon and the Ptox locus. The Cry operon is toxic to Aedes mosquitoes while the Ptox locus is toxic to Anopheles mosquitoes. The general mechanism of Cry toxins includes binding to receptors in the midgut of their target organism followed by pore formation and subsequent osmotic imbalance. We report steps forward in elucidating the mechanism of action of the Cry operon in that mutations in loop regions of Domain II in Cry17 caused a loss of toxicity of the full Cry operon. This loss of toxicity was not seen when Domain II loop regions were mutated in Cry16. We also report a lack of binding between purified Cry16 proteins and Aedes brush border membrane fractions. Competition assays between purified Cry16 and Cry11 showed Cry16 did not bind but suggested Cry16 may facilitate binding of Cry11 to Aedes brush border membrane fractions. The Ptox locus, also found in Pbm, is toxic to Anopheles mosquitoes. The Ptox locus is similar to botulinum neurotoxin in that there is a neurotoxin, NTNH, and three OrfXs. We report an estimated size of the Ptox complex which is consistent with reports of Botulinum neurotoxin progenitor complexes. We also report different methods that aid in the purification of the Ptox complex, as well as methods that were not advantageous. Bacillus thuringiensis israelensis is currently used as a biological control method that targets Aedes and Anopheles mosquitoes. We report the use of Crispr-Cas9 to attempt to edit the genome of this bacteria to target Anopheles mosquitoes as well. Here all Crispr elements were functional, but more screening was required to find a positively edited colony