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Cyt2Ba

Description

Bacillus thuringiensis toxins, or Bt toxins, are toxins that derive from Bt bacteria and are commonly used as insecticides as they can target specific insects without causing harm to other species. There are a wide variety of strains derived from a selection of Bt bacteria and each has similar effects but targets different species of insects. There are three main categories of the Bt toxin: Cry, Cyt, and Vip. There are also the Xpp strains which we renamed from Cry strains.[1]

Usage and Biology

Cyt2Ba is a potent larvicide that works in the digestive tract of Aedes mosquitos. The Cyt protein’s reactivity to alkaline conditions in Aedes mosquito larvae’s midgut causes it to solubilize by increasing membrane permeability, and undergo proteolytic cleavage. This activates the protein, leading to its binding to unsaturated phospholipids present in the cell membrane of the larvae’s epithelial cells lining the alimentary canal. Such a disruption in an epithelial cell’s membranes changes its membrane permeability, hindering normal cell processes, and ultimately, cell lysis. Once this takes place on a large scale, larvae midgut tissues lose their function and cause symptoms in the Aedes mosquito larvae including starvation, electrolyte imbalance and eventually death.[2][3][4]

The results from lanes 3 and 4 show that the IPTG has successfully inducted Cyt2Ba. Lane 3 has shown a faint band, indicating only a low protein concentration compared to the band on lane 4, which is vibrant and clear. Moreover, the band shows the correct size, 30KDa.

Analysis of Aedes mosquito larvae exposed to Cyt2Ba synthesised by our engineered bacteria

The larvicidal efficacy of Cyt2Ba against Aedes albopictus larvae was tested by exposing 10 larvae in a cup to Cyt2Ba by dissolving its serially diluted wet pellets. (Pellets containing recombinant biolarvicidal toxins were collected from BL21(DE3) bacterial cultures.) The larvicidal efficacy of toxins was compared with ddH2O as a control. (Note: control larvae were still alive when collected, whereas larvae exposed to larvicides were collected when they died.) The LC50 (lethal concentration required to kill 50% of the population) and morphology of larvae were examined and analyzed.

LC₅₀

Morphology

Fig 3. Microscope amplification of control larvae

In Figure 3, features can be seen as annotated. An unbroken and food-filled midgut and continuous epithelium.

Fig 4. Microscope amplification of midgut breakage in larvae exposed to Cyt2Ba

In Figure 4, there is a breakage in the larvae midgut. A clean spit in its epithelium is present, unlike in the control, where it is continuous. Despite the breakage, there is no observable damage to its surrounding body, meaning that the damage was unlikely caused by errors during the making of microscope slides and likely caused by the Cyt2Ba toxin.

Fig 5. Second microscope amplification of Midgut damage in larvae exposed to Cyt2Ba.

Figure 5 is an example of a larva that shows both external damage and internal damage. The area circled in white shows breakage similar to that in Figure 4, where it is purely internal and likely the action of the Cyt2Ba toxin. The area bracketed in black is external damage, likely from errors during the moving of the larva from its cups to its microscope slide. Additionally, both Figure 4 and 5 larvae have an absence of food in their midgut.

Deviating from the effects of Cyt2Ba on larvae which had been predicted by theory, some larvae showed different characteristics.

Fig 6. Third microscope amplification of Midgut damage in larvae exposed to Cyt2Ba.

The larva in Figure 6 shows characteristics not predicted by theory: darkening in the midgut. Deformities in the midgut are also a characteristic of midgut damage, but it is less certain that darkening is also a characteristic of midgut damage. The reasons for darkening will require additional research.

Larvae Scale

Larvae scale is a continuance of morphology; it's hypothesized that the potent recombinant larvicidal toxin Cyt2Ba would result in early mortality, and hence, the length of each larva was measured. The length of larvae exposed to Cyt2Ba and control groups were analyzed with ImageJ.

Fig 7. Control Larva length

Fig 8. Cyt2Ba Larva length

These larvicide assays were all carried out within the same time frame, but the larvae were mounted at different times. The control larvae were in the late stage three of their development during mounting, while most of the larvae from the treatment group were mounted at various time frames when they died. Therefore, the length of the larvae reflected the true length of their size in real life. The larvae were washed and preserved in absolute alcohol and mounted with the mounting solution. The difference observed in length, therefore suggests the relative efficacy of the toxins.

Fig 9. The relative length of larvae treated by Cyt2Ba and Xpp81Aa1 Compared to the control group. (For this registry, only Cyt2Ba is relevant)

The deviations of the results from Cyt2Ba larvicidal assays were taken relative to the control group. The values above and below the 0 point represent the extent to which a value deviates from the average control measurement (0.5338 cm). Cyt2Ba is represented by the pink values.

This graph shows that there is no significant difference between the bodies of the larvae exposed to Cyt2Ba and Xpp81Aa1, but relatively, the treated group was shorter in length compared to the control groups. It was deduced that the shorter length was due to the early mortality experienced by the treatment groups compared to the control groups.

The biolarvicidal assay started with larvae of the same stage, stage 3, and the control group was mounted 2 days after the test. Due to the larvae’s early mortality, they were unable to grow past the stage at which they were upon death. This allows us to see that the effects of Cyt2Ba and Xpp81Aa1 manifested in the form of early death, showing that the larvicides indeed impact larvae mortality.

Sequence and Features

Reference

[1] Shilling, P. J., Mirzadeh, K., Cumming, A. J., Widesheim, M., Köck, Z., & Daley, D. O. (2020). Improved designs for pET expression plasmids increase protein production yield in Escherichia coli. Communications Biology, 3(1). https://doi.org/10.1038/s42003-020-0939-8

[2] Bravo, A., Likitvivatanavong, S., Gill, S. S., & Soberón, M. (2011). Bacillus thuringiensis: A story of a successful bioinsecticide. Insect Biochemistry and Molecular Biology, 41(7), 423–431. https://doi.org/10.1016/j.ibmb.2011.02.006

[3] Wu, J., Wei, L., He, J., Fu, K., Li, X., Jia, L., Wang, R., & Zhang, W. (2021). Characterization of a novel Bacillus thuringiensis toxin active against Aedes aegypti larvae. Acta Tropica, 223, 106088. https://doi.org/10.1016/j.actatropica.2021.106088

[4] Gu, J.-B., Dong, Y.-Q., Peng, H.-J., & Chen, X.-G. (2010). A Recombinant AeDNA Containing the Insect-Specific Toxin, BmK IT1, Displayed an Increasing Pathogenicity on Aedes albopictus. American Journal of Tropical Medicine and Hygiene, 83(3), 614–623. https://doi.org/10.4269/ajtmh.2010.10-0074