What’s the Problem?

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 was 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 achieved in increasing the potency of or decreased the environment’s effects on a fungus species, they did not take a step further to consider the evaluation the fungal insecticide must follow before commercialization, which includes the assessment of its toxicity towards humans and animals, dispersal, horizontal gene transfer, and 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, are designing light-induced kill switch aimed to reduce the dispersal and horizontal gene transfer of genetically engineered fungal insecticides. Using an entomopathogenic fungus that is applied as an insecticide around the world, Metarhizium anisopliae, as our chassis, we 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 environment tolerant fungal pesticides with reduced residual specimen in the area of application. M. anisopliae’s infection cycle starts with conidia 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, which will change gene expression alongside with any fungal cells that is in contact with the hemolymph. This is also when the production phase of our kill switch begins. Utilizing a hemolymph induced promoter from M. anisopliae, Pmcl 1, with fast activation and high production rate, original transcripts of the gene controlled by this promoter could be detected within 20 minutes of cellular contact with insect hemolymph and amounts for 5.6% of all ESTs [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 hemolymph and other interior organs of the insect, KillerRed molecules will remain inert due to the lack of yellow light. When the fungus depletes the nutrients in its host, M. anisopliae will emerge for conidiation. This put fungal cells in direct contact with sunlight, allowing KillerRed to create O2․- and disrupt the metabolism 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 an post-deployment clean up, lowering their threat to the surrounding environment.