Team:NYMU-Taipei/Project-Background
What’s the Problem?
Chemical insecticides:
Although chemical insecticides have improved the lives of countless human beings by controlling the population of both agricultural and urban pests, since the second half of last century, the numbers of insecticide resistant pests have been rising at an alarming rate [1]. Some would argue that as researchers and chemical pesticide companies develop new insecticides, the resistance couldn’t possibly catch up. In reality, the amount of insecticides applied is actually proportional to the increase in resistance in the target pest population [2], [3]. This means if no action is taken to change the status quo, the ongoing population control of insect pest will become even more of a struggle.
Current Solution
Biopesticides, including insecticidal plant extract, bacteria, and fungi, are some of the more popular alternatives to chemical pesticides. Currently, the biopesticides that are the most widespread, in terms of usage, are the entomopathogenic-fungi insecticides[4]. Certain species of entomopathogenic fungi are capable of targeting a small range of hosts, making them the ideal solution to many regional insect pests. However, these biological control agents come with highly variable outcomes due to the variation in environmental (e.g. temperature and humidity) and host (e.g. nutrition and immune response) conditions [5].
Many researchers have tried to improve these fungal insecticides through biochemical or genetic means [6], [7]. Though they might have succeeded in increasing the potency or hardiness of a fungus specie, they did not take a step further to consider that the evaluation the fungal insecticide must follow before commercialization, which includes the assessment of its toxicity towards humans and animals, dispersal, horizontal gene transfer rate, and its effects on the resident microflora [5].
Our Biosafety Solution
The lack of biosafety development for genetically engineered fungal insecticides hinders its commercialization and public acceptance. To address this problem, we, 2016 NYMU_Taipei, have designed a light-induced kill switch aimed to reduce the dispersal and horizontal gene transfer of genetically engineered fungal insecticides. Using an entomopathogenic fungus, Metarhizium anisopliae, that is applied as an insecticide around the world as our chassis, we have constructed a genetically modified M. anisopliae with wildtype lethality and the additional ability to self-terminate after killing its host.
In-Out-Suicide
Through synchronizing the different stages of the kill switch activation with that of the M. ansiopliae’s infection cycle, our genetically modified M. anisopliae could infect and kill pest insects before self-termination. This allows for researchers to create highly lethal and environmental tolerant fungal insecticides with reduced residue in the area of insecticide deployment. The infection cycle of M. anisopliae starts with conidial adhesion to the cuticle of the host. The fungus will then attempt to penetrate the cuticle by secreting various proteases, chitinases, and lipases. When the mycelium reaches the hemolymph, it will start to produce yeast-like blastospores, The change in cell morphology is accompanied by changes of gene expression in cells in contact with the insect's hemolymph. We designed our circuit to utilize the gene expression change to activate the production phase of our kill switch. Utilizing Pmcl 1, a hemolymph induced promoter from M. anisopliae with fast activation and high production rate [8], high amounts of KillerRed protein will be produced by fungal cells within the hemolymph. KillerRed, BBa_K1184000, is a fluorescent protein that can be activated by yellow-orange light (540-585 nm) to produce highly reactive oxygen species (O2․-). During M. anisopliae’s stay within the hemocoel and other interior organs of the insect, KillerRed molecules will remain inert due to the lack of yellow light. When M. anisopliae depletes the nutrients in host's body, the fungus will emerge from the carcass of its host for conidiation. This put fungal cells in direct contact with sunlight, allowing KillerRed to create reactive oxygen species like O2․-, which will disrupt the metabolic functions of the cells and eventually killing the fungi. In conclusion, our In-Out-Suicide system allows for more lethal genetically modified fungal pesticides to be developed because it provides them with a ability to clean up after itself, lowering their threat to the surrounding environment.
Reference
1. Mallet J. The evolution of insecticide resistance: have the insects won? Trends Ecol Evol.1989;4(11):336–40. doi: 10.1016/0169-5347(89)90088-8.
2. Zhu, F., Lavine, L., O’Neal, S., Lavine, M., Foss, C., & Walsh, D. (2016). Insecticide Resistance and Management Strategies in Urban Ecosystems.Insects, 7(1), 2.
3. Reid, M. C., & McKenzie, F. E. (2016). The contribution of agricultural insecticide use to increasing insecticide resistance in African malaria vectors. Malaria Journal, 15, 107. http://doi.org/10.1186/s12936-016-1162-4
4. Sudakin D.L. Biopesticides. Toxicol. Rev. 2003;22:83–90.
5. Bonaterra, A., Badosa, E., Cabrefiga, J., Francés, J., & Montesinos, E. (2012). Prospects and limitations of microbial pesticides for control of bacterial and fungal pomefruit tree diseases. Trees (Berlin, Germany : West), 26(1), 215–226.
6. St Leger, R., Joshi, L., Bidochka, M. J., & Roberts, D. W. (1996). Construction of an improved mycoinsecticide overexpressing a toxic protease. Proceedings of the National Academy of Sciences of the United States of America, 93(13), 6349–6354.
7. Wang CS, St Leger RJ (2007) A scorpion neurotoxin increases the potency of a fungal insecticide. Nat Biotechnol 25: 1455–1456.
8. Wang, C., & St. Leger, R. J. (2006). A collagenous protective coat enablesMetarhizium anisopliae to evade insect immune responses. Proceedings of the National Academy of Sciences of the United States of America, 103(17), 6647–6652.
