Part:BBa_K5066001
Xpp81Aa1
Description
Xpp81Aa is one of the Bacillus thuringiensis toxins, or Bt 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, and the Xpp strains were renamed from Cry strains. It has also shown synergetic effects with Bt toxins, heightening the toxic effects.[1]
Usage and Biology
Currently, there is a lack of information regarding Xpp81Aa1. Previous studies have demonstrated its role in assisting the mortality rate of mosquito larvae is its symbiotic relationship with Bt toxins (such as Cry2Aa and Cry4Aa). Chosen for its coactive properties, Xpp81Aa heightens the effectiveness of the other toxins.[2]
Characterizaation
Western Blot
Lane 1: pET-28a-Xpp81Aa1 incubated without IPTG; Lane 2: pET-28a-Xpp81Aa1 with 1 mM IPTG; Lane 3: pET-28a-Cyt2Ba without IPTG; Lane 4: pET-28a-Cyt2Ba incubated with 1mM IPTG
The Xpp81Aa1 was expressed using E. coli BL21 (DE3) cells. Results on lanes 1 and 2 indicate the success of the induction of Xpp81Aa1 via IPTG. The band on Lane 1 is significantly lighter compared to the band on Lane 2, hence we can deduce that IPTG has an impact on the expression of our protein and we have successfully produced a protein. The Protein was further confirmed to be Xpp81Aa1 as the band size is correct, showing 45KDa.
Larvicidal Assay
Larvicidal mortality of Aedes albopictus larvae exposed to various concentrations of the biolarvicidal toxins collected under different conditions including Xpp81Aa1 37°C overnight, Xpp81Aa1 37°C for 4 hours and Xpp81Aa1 20°C overnight. The LC50 was calculated to be 3.16x107 CFU/mL, 9.24x107 CFU/mL and 1.98x107 CFU/mL, respectively.
The larvae from our larvicidal assay were inspected under a light microscope. In the larvae treated by Xpp81Aa1, we observed a total of 6 larvae with visibly darkened areas, specifically from the anus through to the rectum, making the surrounding area darker. Darkening can also be observed through the rest of the larvae, generally in the head, thorax, and lower half of the abdomen. Some larvae were found to retain food in the midgut until death. To date, the structure and mechanism of how Xpp81Aa1 is not known. Further research studies could be done on Xpp81Aa1 to elucidate the function, mechanism and structure of the protein.
The larvae from our larvicidal assay were inspected under a light microscope. In the larvae treated by Xpp81Aa1, we observed a total of 6 larvae with visibly darkened areas, specifically from the anus through to the rectum, making the surrounding area darker. Darkening can also be observed through the rest of the larvae, generally in the head, thorax, and lower half of the abdomen. Some larvae were found to retain food in the midgut until death. To date, the structure and mechanism of how Xpp81Aa1 is not known. Further research studies could be done on Xpp81Aa1 to elucidate the function, mechanism and structure of the protein.
Immune responses of Aedes albopictus larvae by qPCR
Furthermore, to determine whether the mortality that we observed from the biolarvicidal assay is solely due to the mechanisms exerted by each of the toxins, such as the cytolytic effects of Xpp81Aa1, it is necessary to rule out the death of mosquito larvae due to E. coli.
The experiment was designed to observe the effect of Xpp81Aa1 toxins on Ae. albopictus larvae in comparison to control (negative control), BL21(DE3)-empty vector (control), and treatment groups. BL21(DE3)-empty vector is used to investigate whether the mortality observed was due to the cytotoxicity effect or bacterial infection of the E.coli or solely due to the potency of the Xpp81Aa1.
Hence, Real-time PCR, also known as quantitative PCR (qPCR), was used to confirm our hypothesis. This is a method used to determine the concentration of a target DNA (Dymond, 2013). PCR was performed to examine the expression levels of the target immune genes, including Gambicin, Rel1, Rel2, and Defensin of the larvae.
Gambicin
Figure 4 shows that after the 24-hour period for both 20°C and 37°C, the fold change of Gambicin in larvae left in Xpp81Aa1 solutions showed a significantly lower value than the controlled condition. However, only the results of the 37°C showed that the Xpp81Aa1 is effective after a 48-hour period. These results indicate that our Xpp81Aa1 is effective at targeting the larvae.
Rel1
Figure 5. Expression levels of Rel1 relative to the housekeeping gene S7 (A & B) Larvae exposed to biolarvicidal toxins with IPTG induction at 20°C overnight, and (C & D) IPTG induction at 37°C for 4 hours, respectively, compared to the control group treated with ddH2O and BL21(DE3)-empty vector at 24 hours and 48 hours
Figure 5 shows that after both the 24 and 48-hour periods, the fold change of Rel1 in larvae left in Xpp81Aa1 solutions showed a significantly lower value than the controlled condition in the two temperature conditions. These results indicate that our Xpp81Aa1 is effective at targeting the larvae. This result is different to the results of Gambincin where only the 24 hours condition at 37°C was effective.
Rel2
Figure 6. Expression levels of Rel2 relative to the housekeeping gene S7 (A & B) Larvae exposed to biolarvicidal toxins with IPTG induction at 20°C overnight, and (C & D) IPTG induction at 37°C for 4 hours, respectively, compared to the control group treated with ddH2O and BL21(DE3)-empty vector at 24 hours and 48 hours
Figure 6 shows that after both the 24 and 48-hour periods, only the protein inducted at 37°C showed fold changes lower than the control. These results indicate that only when incubated at 37°C is Xpp81Aa1 effective at inducing changes in the expression of Rel2.
Defensin
Figure 7. Expression levels of Defensin relative to the housekeeping gene S7 (A & B) Larvae exposed to biolarvicidal toxins with IPTG induction at 20°C overnight, and (C & D) IPTG induction at 37°C for 4 hours, respectively, compared to the control group treated with ddH2O and BL21(DE3)-empty vector at 24 hours and 48 hours
Figure 7 shows that after both the 24 and 48-hour periods, the protein inducted at 20°C showed fold changes lower than the control, moreover, the results from the 37°C condition also showed positive results after the 24 period. These results indicate that when incubated at 20°C and 37°C is Xpp81Aa1 effective at inducing changes in the expression of Defensin, but only at 24 and 48 hours or 24 hours periods, respectively.
Morphology of Larvae
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. We washed and preserved the larvae in absolute alcohol and mounted them with the mounting solution. The difference observed in length, therefore suggests the relative efficacy of the toxins.
This graph shows that the treated group was shorter in length compared to the control group. We deduced that the shorter length was due to the early mortality experienced by the treatment groups compared to the control groups.
We started the biolarvicidal assay with the larvae of the same stage, stage 3, and mounted the control group 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 Xpp81Aa1 manifested in the form of early death, showing that the larvicides indeed impact larvae mortality.
Conclusion from results
In conclusion, we gained insight from the qPCR study that the potency observed from the biolarvicidal toxins was due to the binding of crystal to the epithelium of midgut by Xpp81Aa1. The morphology of the larvae also offers a different perspective on the effects of our toxin, showing that the toxin could cause the early death of larvae.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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]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
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