The research aimed to understand how to better control mosquito populations using Bacillus thuringiensis Cry19Aa toxin. Experiments were conducted from 1/11/16 to 5/1/16 under a PhD student to determine the toxicity of Cry19Aa against Anopheles gambiae cell lines (Ag55). Preliminary results showed trypsin-activated Cry19Aa was cytotoxic to Ag55 cells in a time- and concentration-dependent manner, reducing cell viability to zero after 24 hours. This suggests Cry19Aa could be an effective toxin for mosquito control.

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Determining the toxicity of Cry19Aa against Ag55(anopheles gambiae) cell line
Dr Adang’s Lab
Research done from 1/11/16 to 5/1/16
Supervison under Ruchir Mishra P.H.D student
Mihir Panchal

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Determining the toxicity of Cry19Aa against Ag55(anopheles gambiae) cell lines
Summary:
The overall purpose of this laboratory research conducted from 1/11/16 to
5/1/16 in Dr. Adang’s molecular lab was to understand how to better control
mosquito populations in regions susceptible to diseases in which mosquitoes are a
vector. The use of Bacillus thuringiensis Cry19Aa toxin was central to our research.
Working with a doctoral student (Ruchir Mishra), our research project focused on
the affects of the BT Cry19Aa toxin on the cell membrane proteins of different
species of mosquitoes (Culex, and Anopheles), Cry19Aa is also especially effective
against Culex strains resistant to Cry4Aa, Cry4Ba, Cry11Aa toxins, investigating this
resistance and its effectively to mosquito cells was key to our research.
In order to conduct our research we used Ag55 cell lines. Ag55 cell lines have
been generally considered relatively easy to study Cry interaction and cellular
responses to those interactions. Thus we used Ag55 cells to carry out assays to
figure out the toxicity of Cry19Aa to Ag55 cell and larval cell lines. Preliminary
results showed that trypsin present in activated Cry19Aa was cytotoxic to Ag55 cells
and these results can give insights to other more efficient methods for mosquito
control.
Introduction:
Mosquito control has been central to human civilization for over millennia,
many civilizations have tried to control mosquitoes and the various disease they are
vectors for such as malaria, Chickungunya, Dengue, West Nile virus and various
other emerging diseases such as the Zika Virus. The most common form of mosquito
control is sanitation and insecticides (Teixeira, 2012).

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The ability of diseases vectored by mosquitoes to disrupt life and increase
morbidity, especially in the developing world, where Malaria kills nearly 660,000
people every year (Nadjm,2012) led us to research more effective toxins against
different types of mosquitoes. We decided to use Bacillus thuringiensis, a toxin of the
bacterial origin that targets the larval stages of certain mosquitoes. A key advantage
to using B. thuringiensis toxins is they affect few non target species, which makes
them much more environmentally friendly (Roh,2009). Bt(Bacillus thuringiensis)
toxins incorporate various different Cry and Cyt toxins, the crystal aggregation
which these toxins form contains at least four major toxic compounds. Cry and Cyt
toxins both are pore-forming toxins that lyse midgut epithelial cells by inserting into
a target cell membrane and forming pores.
Our research focused on Cry19Aa, a toxin found in the pBtoxis plasmid which
is isolated from B.thuringiensis subsp. Jegathesan . Our research targeted Cry19Aa
because it is known to be toxic to Anopheles and Culex but not to Aedes and it is also
toxic to Culex strains resistant to Cry4Aa, Cry4ba, and Cry11Aa, showing no cross-
resistance. The lack of resistance suggests a mechanism of action distinct from other
Bti toxins and Cry11Ba. This lack of resistance makes studying and investigating
Cry19Aa and its affectivity compared to other toxins of importance for developing
better ways of controlling mosquito-vectored diseases.
Understanding the efficiency and rate at which the Cry19Aa protein kills the
Ag55 cells, a cell line of Anopheles gambiae, can lead to further research to
genetically develop even more effective toxins against mosquito cells.

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Experimental Methods:
In order to perform our experiment, we first grew a cell culture model conducive
for investigating Cry toxin action. The cell cultures we used and found effective were
the Ag55 cell lines. Ag55 cell lines were previously effective for investigating
malarial plasmodium research. After growing 1x10^6 Ag55 cell lines, we prepared
activated Cry19Aa using trypsin. Additional details and order of the experiment is
provided below:
Culturing Cry19Aa toxin:
A single colony was seed cultured in 5ml LB supplemented with 10ul erythromycin
(10mg/ml in ethanol filtered and stored at -20 degrees Celsius) to make a final
concentration of 20ug/ml, shake at 250-350rpm at 30 degrees Celsius overnight.
First we prepared a medium to grow the toxin, we added 2g/L Peptone, 5g/L Yeast
extract, 12.54g/L K2HPO4(Dibasic), 2.31g/L KH2PO4 (Monobasic), we also filter
sterilized and added 5 ml 20% glucose + 1.23% MgSO4 , 1 ml 7.5% Cacl-2H2O
,1 ml 0.15 MnSO4- 7H2O and 1ml 0.014% FeSO4- 7H2O to the Erlenmeyer flask.
and then add 2ml Erythromycin stock (10mg/ml), Shake at 250-300 rpm at 30
degrees Celsius. After one day, we added 1L of sodium phosphate solution
[(Na2HPO4 8g/L (Na2HPO4-12H2O 20.18g) + NaH2PO4 5g/L (NaH2PO4-H2O 5.75g)]
into the growing medium. After this, we cultured the toxin for another two days
until complete sporulation. Then we centrifuged the solution having sporulated
Cry19Aa at 5000rpm for 20 minutes. Then we discarded the supernatant. Next, we
purified the Cry19Aa.

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Purification of Cry19Aa:
Resuspended the pellet in 80ml of CWI(0.1M NaCl, 2%Triton X-100, 20mM Bis-Tris
pH 6.5) and sonicated it for 3 miniutes twice. Then Centrifuged and re-suspended
the pellet in 80ml of CWI. Then we repeated the above step with CWI twice and with
ultra pure H2O once. Then once again repeated the above step with CWII (1M Nacl)
twice and with ultra pure water once. After that we re-suspended the pellet in 5ml
of ultra pure H2O.
Separation of spores and crystals:
To further separate spores from crystals, we used a discontinues NaBr gradient. We
prepared a 30-60% NaBr gradient, after the gradient formed in the tubes we
overlayed the pellet on the gradient. Then we ultra centrifuged the gradient at
20,000 rpm for 1 hours and 15 minutes. This caused a layer of crystals to float up to
the top; we then extracted the crystals and washed them twice with ultra pure
water.
Solubilization and trypsin activation of Cry19Aa crystals:
To solubilize the cry19aa crystals, we solubilized the crystals in carbonate
buffer(Na2CO3/NaHCO3,buffer, pH 11.5) and 10mM DTT. We solubilized the crystals
at 37OC overnight. After solubilization, we added trypsin in 1:10(weight by weight
ratio).
Anion exchange chromatography was performed using Buffer A= 50 mM Carbonate
buffer + 100mM NaCl(pH 11.50) and Buffer B= 50mM Carbonate buffer + 1 M
Nacl(pH 11.50) and this separated the activated cry19Aa from other protein

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degrading toxin proteins. Then we carried out a concentration course and time
course assay.
To carry out the Cry19Aa concentration course and time course quantitate assay on
Ag55 cells to measure toxicity:
 We seeded the 1x10^6 cells in the 7 wells of the 12 wells in the cell culture
plate.
 The cells were treated with different concentrations of trypsin activated
Cry19Aa.
 Live and dead cells were counted after 16 hours of incubation for
concentration course assay @ 2,4,6,8,24 hours for time course assay with
trypsin activated Cry19Aa using Trypan blue and Hemocytometer(an
instrument for visual counting of number of cells). Cells were counted from 4
corners. Each corner square having 16 small squares.
 We then calculated the percentage of cells viable. (% cells viable/total
number of cells)x100.
 We then repeated the experiment 3 and 2 times for time course and
concentration course respectively.
This part of the experiment helped us measure the toxicity and affectivity of
Cry19Aa to Ag55 cells.
Results:
The size of the activated Cry19Aa toxin was approximately 42 kDa. This is
confirmed below in the gel-electrophoresis. Lane 1 is the marker while lane 2 is the
solubilized Cry19Aa. While lane 3 is trypsin activated Cry19Aa.

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The approximate band length of trypsin activated Cry19Aa was 42 kDa. This was
obtained using SDS PAGE electrophoresis. The results of the toxicity of trypsin
activated Cry19Aa on the Ag55 cell lines is shown below. We obtained these results
using an inverted phase contrast microscope.
Lane 1 2 3
97.4
66.2
45
31
21.5
kDA

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The results show rapid toxicity to Ag55 cell lines by 8 minutes. Using these pictures,
we were able to carry out a concentration and time course assay. The results of the
time and concentration course are also shown below.

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Viability of cells was checked at 16 hrs. after incubation with activated Cry19Aa
using trypan blue.
Trypan Blue Ag55 cell viability time course assay. Blue: Control (0.8 nM Carbonate
buffer pH 9.6).
Red: 0.8 nM Cry19Aa. Readings were taken at 2 hrs , 4 hrs , 6 hrs , 8 hrs & 24 hrs.

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Discussion:
Our results were consistent in showing that the trypsin activated Cry19Aa
toxin was indeed cytotoxic to the Anopheles gambiae cell lines. The time course and
concentration course assay showed that the percentage of viable cells with
increasing Cry19Aa concentration decreased significantly with larger Cry19Aa
concentrations. The time course assay also showed that after 24 hours of exposure
to Cry19Aa, the cell viability decreased to zero. These results show that Cry19Aa is
an efficient toxin that could be used to effectively control major mosquito vectored
diseases around the world by eliminating mosquitoes rapidly from the area. Further
research on the Cry19Aa toxin and its mechanism on cell membrane proteins of the
Ag 55 cell lines could give insights into developing or discovering even more
efficient toxins for mosquito control.

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References
Nadjm, B. (2012). Malaria: An update for physicians. PubMed. Retrieved April 27,
2016, from http://www.ncbi.nlm.nih.gov/pubmed/22632637
Roh et al.2009. Mutagenic analysis of putative domain II and surface residues in
mosquitocidal Bacillus thuringiensis Cry19Aa toxin. FEMS Microbiol
Lett. 295(2):156-63.
Teixeira Correa et al. 2012.Cytotoxicity Analysis of three Bacillus thuringiensis
subsp. Israelensis δ-Endotoxins towards Insect and Mammalian Cells. PLoS ONE
7(9): e46121