Resistance to the Bacillus sphaericus Toxin in Cultured Mosquito Cells Monique Gallegos, Brittany Ortega & Mercedes vigil Image: https://www.bing.com/images/search?view=detailV2&ccid=%2bEhrVyLC&id=011AE9A6A2D78411313B315884791C20EE4E0C00&thid=OIP.-EhrVyLClVJVknNv7_303gHaEL&mediaurl=https%3a%2f%2fwww.ecolab.com%2f-%2fmedia%2fEcolab%2fEcolab-Home%2fImages%2fCategory123%2fPest-Elimination%2fMosquito-Culex-quinquefasciatus.jpg%3fh%3d310%26la%3den%26w%3d550%26hash%3dC833D52AAA3256BFC3FD69079E5023289617A9F8&exph=310&expw=550&q=c.+quinquefasciatus&simid=608024263839449340&selectedIndex=20&ajaxhist=0

Insecticidal toxins play a detrimental role in our environment These toxins can harm targeted insects and non-targets by contaminating water, soil, vegetation and bioaccumulate in organisms which result in health issues or even death This study focuses on the bacterium Bacillus; which is the most common toxin used in insecticides Introduction

Why This Topic? Human interactions? Mosquitos encounter blood-born pathogens of different species without consequences, unlike humans--> but HOW? We wanted to understand how constant exposure to fatal toxins within the environment can build resistance

Bacillus The genus, Bacillus, produces inclusion bodies which are toxic to mosquito larvae Inclusion bodies aggregate nuclear and cytoplasmic substances such as proteins which causes them to denature Representing sites of viral replication Members of the genus Bacillus have proven to be pathogenic to organisms with little evidence of resistance

What is Bacillus sphaericus? A rod- shaped bacterium Aerobic, spore-forming Tolerant to high temperatures Pathogenic for larvae Photo taken using a scanning electron microscope (SEM)  Photo by: Krishnakumar, Keerthana.

Why Should You Care? How would your life expectancy differ if you were resistant to toxins? By introducing the Bacillus sphaericus toxin to cultured cells, we can have a better understanding of resistance By conducting a toxic challenge, we can see how cultured mosquito cell react to increased increments of the toxin

Mechanism of Action The main toxicity factor is a binary toxin (BIN) The ligand binds to the receptor and activates the toxic component This causes the protein to denature in larvae https://www.researchgate.net/publication/300379890_Mosquitocidal_Bacillus_sphaericus_Toxins_Genetics_Mode_of_Action_Use_and_Resistance_Mechanisms

Methods The Culex quinquefasciatus (Southern House Mosquito) was used as the subject Cloned cells were given nutrients and cultured for 3 days on a soy- based medium The B. sphaericus toxin was crudely introduced at a low dosage After 1 week, cells were transferred to a medium without toxin any toxin until they reached confluency—threshold of cells present—then were reintroduced with toxin of varying concentration Parameters for resistance selection- G7 cells were selected by survival of a titer assay (compares health of cells to their distribution over the plate)

Results  These results show that over environmental strain (caused by constant presence and increased concentration) of the B. sphaericus toxin resulted in the mosquito cells increased resistance that plateaued after week 7 Overall tolerance of the cells increased over time and lasted 4 months after the final dosage was completed 

How Does It Work? A tagged toxin (FITC) was found bonded to the periphery  of the cells and internalized in spherical vesicles in both G-7 and resistant cells This indicated that the toxin is membrane bound and most likely alters the function of the cell ultimately resulting in lysing of the cell Resistant cells maintained their spindle shape, while the G7 cells that were not selected became more rounded  Possibly because of the disruption of concentration gradient which affects water flow into the cell 

Results This graph, formatted from a table found in the article and summarizes the resistance percentage of mosquito to the B. sphaericus toxin  The highest percentage of resistance was found following week 6  Similar to the Ballmer's peak

Discussion Cells/mechanisms are very receptive to their environment and will alter their structure in order to benefit and survive in a situation  Other studies show that both "predator" and "prey" will adapt in paradox of evolution to compete for evolutionary fitness  Is there a limit to the limit of adaptation? Will we be able to predict the evolution of toxins in order to benefit us, through crop production or medical advantages?

Summary A toxin challenge was conducted by increasing the concentration of the toxin into the cultured cells The cultured mosquito cells                  (C. quinquefasciatus) became resistant to the B. sphaericus toxin This study shows us that toxin sensitivity can be lost by culturing cells in the presence of a toxin

A high level of resistance to the B A high level of resistance to the B. sphaericus toxin is due to the adaptation of the toxin in the mambrane of the cell that affects the every-day survival of the cell Evidence demonstrates tolerance of a toxin increases over time This is important because we can use this method and apply it to different cultured cells and toxins for future research Conclusion

Future Directions This information can be adapted for: Medical research Agricultural benefits  Further understanding cellular adaptation

Citations  Schroeder, Jamie M., et al. “Resistance to TheBacillus Sphaericus Toxin in Cultured Mosquito Cells.” In Vitro Cellular & Developmental Biology, vol. 25, no. 10, 1989, pp. 887–891., doi:10.1007/bf02624000. Su, Tianyun. “Resistance and Its Management to Microbial and Insect Growth Regulator Larvicides in Mosquitoes.” Insecticides Resistance, 2016, doi:10.5772/61658. Charles, J.-F., et al. “Mosquitocidal Bacillus Sphaericus: Toxins, Genetics, Mode of Action, Use, and Resistance Mechanisms.” Comprehensive Molecular Insect Science, 2005, pp. 207–231., doi:10.1016/b0-44-451924-6/00082-x. Webster, Daniel, and Bliss Perry. “Webster.” Webster, Pub. for Nelson Doubleday, Inc., by Doubleday, Page & Co., 1924. SEM picture for Bacillus sphaericus Krishnakumar, Keerthana. SEM-Image-for-Bacillus- Sphaericus_fig4.