1. Resistance to the Bacillus sphaericus Toxin in Cultured Mosquito Cells
Monique Gallegos, Brittany Ortega & Mercedes vigil
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2. 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

3. 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

4. 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

5. 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.

6. 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

7. 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

8. 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)

9. 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 

10. 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 

11. 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

12. 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?

13. 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

14. 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

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

16. 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: /bf
Su, Tianyun. “Resistance and Its Management to Microbial and Insect Growth Regulator Larvicides in Mosquitoes.” Insecticides Resistance, 2016, doi: /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: /b /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.