alexa Impact of Loop Residues of the Receptor Binding Domain of Bacillus thuringiensis Cry2ac11 Toxin on Insecticidal Activity

Clinical Infectious Diseases: Open Access

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  • Research Article   
  • Clin Infect Dis 2018, Vol 2(2): 109

Impact of Loop Residues of the Receptor Binding Domain of Bacillus thuringiensis Cry2ac11 Toxin on Insecticidal Activity

Quratulain Amjad*
1School of Biological Sciences, University of the Punjab, Lahore, Punjab, Pakistan
2Abeedha, Tu-Allah Khan, Pakistan
*Corresponding Author: Quratulain Amjad, School of Biological Sciences, University of the Punjab, Lahore, Punjab, Pakistan, Email: [email protected]

Received Date: May 23, 2018 / Accepted Date: May 31, 2018 / Published Date: Jun 07, 2018

Abstract

Bacillus thuringiensis is an excellent candidate to be used as bio-pesticide. Various techniques have been used to exploit Bt genes for our own benefits. In the present work, detailed study on the receptor binding epitopes of Bacillus thuringiensis Cry2Ac11 toxin is done by inducing different mutations in loop 2 of Domain II. The mutants are characterized to estimate the effect of mutations on the toxicity and insecticidal activities by performing bioassays against Pakistani populations of Culex quinquefasciatus, Aedes aegypti, Helicoverpa armigera and Spodoptera litura. The results revealed that Cry2Ac11 is not toxic to local populations of Aedes aegypti but two of its mutants with G384D and A386N (30% mortality) T387D (6% mortality) showed some toxicity. Against Culex quinquefasciatus and Helicoverpa armigera, Cry2Ac11 showed 46% and 8.35% mortality respectively with all mutants showing variable percentage mortalities. Cry2Ac11 and its mutants showed only growth retardation in local populations of Spodoptera litura.

Keywords: Cry2Ac11; Bt genes; Culex quinquefasciatus; Aedes aegypti; Helicoverpa armigera; Spodoptera litura; Bacillus thuringiensis; Biopesticide

Introduction

A major cause of the loss of agricultural crops is the destruction caused by insect pests. In a country like Pakistan, where crops are major source of food, such loss is not bearable. Chemical pesticides may however play an important role in eradicating these insect pests and boosting agricultural production, but their haphazard use can also lead to harmful consequences eventually affecting the human health. These chemical pesticides also cause severe environment pollution. During the past few years, the use of chemically synthetic pesticides has widely increased. The usage of chemical pesticides to this extend has created a great concern. Chemical residues from insecticides and pesticides remain in vegetables and fruits. These harmful residues were also reported to be found in milk samples, in drinking water of cattle and animals feed in various reports. This situation is quite alarming and can lead to severe human diseases.

Bio-pesticides provide quite safer alternative to their chemically synthetic counterparts. For increasing percentage yield and pest control Bacillus thuringiensis has been used as substitute or supplement to chemical pesticides [1,2]. The insecticidal properties of Bt toxins were discovered in the early 20th century [1] and since then it has been successfully used to control various insect pests [3]. Cloning of the cry gene was reported by Schnepf & Whiteley [4,5] and now it is widely used either in form of bio-pesticides or transgenic plants.

Bacillus thuringiensis is a rod shaped gram-positive bacteria found in many different habitats including soil, stored products, insects, insect cadavers and the phylloplane (plant surfaces) [3]. It produces several types of toxins including heat labile α-exotoxin possibly lecithinase; heat stable beta exotoxin including an adenine containing compound; and δ-endotoxin (crystal proteins). These designations are according to the type of activity of toxin. The biochemical nature of each toxin varies from strain to strain [6].

There are hundreds of Bt strains distributed worldwide in multiple habitats and most of them produce one or more parasporal inclusions comprising of either one or several correlated insecticidal protoxins called delta-endotoxins [1].

During stationary phase of BT at the end of sporulation these insecticidal proteins are produced and accumulate as inclusion bodies in their mother cell compartment. There they constitute up to 25% of total dry weight of the sporulated cells and with spores they are also released to the environment [7]. These proteinecious crystals are produced only during sporulation but crystal protein is accumulated in large amounts as inclusions as well as a part of the spores coat making them different from many other sporulation-dependent proteins [7]. When these inclusion bodies are ingested by the insects; the proteins solubilize and cause the formation of pores in the apical membrane of midgut cells. As a result of this the insect dies [4].

The best part about using BT toxins is that they are highly specific in their action. These toxins are present in form of a protoxin which converts to biologically active toxin only when exposed to an alkaline pH that is present in pest’s gut. This indicates that these proteins are non toxic to non-targetted insects. This protein exhibits specific insecticidal activity against different insect orders including Lepidoptera, Coleoptera, Diptera, Hymenoptera, Homoptera, Mallophaga some invertebrates such as nematodes, mites and protozoans [8]. The insecticidal activity of Bt is not reported in vertebrates till now.

There are hundreds of Cry proteins reported until now and have been classified on several different basis. Cry1 type proteins have been used commercially since long but the problem with their use is their large sizes; unsuitable for transformation into transgenic plants and the increasing insect resistance development against them. These problems are believed to be overcome by Cry 2 type proteins that have relatively smaller sizes ranging from mostly 60 to 70 kDa, making them quite suitable candidate for transgenic plants. Among the Cry2-type proteins; Cry2Ab; Cry2Ac; Cry2Ad; Cry2Ae; and Cry2Af are toxic only to Lepidopteran pests [8]. Cry2Aa exhibit broader toxicity spectrum against Diptera (mosquitoes and flies) and Lepidoptera (butterflies and moths). Where Cry2Aa proteins are different from all other proteins (showing only 17-20% similarity) [9]. Cry2Ac is a closest relative of Cry2Aa showing almost 100% sequence similarity so like Cry2Aa, Cry2Ac is also present in form of an operon and can be assumed to show dual insecticidal activity due to high similarity with dual toxic Cry2Aa protein.

Although there are various Cry2 proteins already reported and a number of studies have already been conducted on Cry proteins but due to the increasing insects resistance emergence against a number of Cry proteins the isolation of noble cry genes and characterization and genetic manipulation of reported Cry proteins to improve their toxic efficacy always remains the need of the day. In the present study also different cry gene mutants are produced and are characterized for toxicity and insecticidal activity. The toxic efficacies of the mutants are also compared with wild types.

Materials and Methods

Cloning and expression

Mutants of loop 2 of receptor binding Domain II were formed by site-directed mutagenesis. Each construct contained 1 or 2 residues replaced by designing primers (Table 1). Cry2Ac11 gene was cloned in PHT3101 shuttle vector under the control of cyt1A-p and STAB-SD. The mutated genes were sub-cloned to acrystalliferous BT strain 4Q7.

Sr. No Constructs Primers AA sequence Mutation Nucleotide sequence 5’→3’
1. pPFS-2Ac11L2-1 2AcL2-1 R reGKATst V386K Tgt aga ggt ggc ctt gcc ctc ccg
2. pPFS-2Ac11L2-2 2AcL2-2 R reGVKTst A386K Tgt aga ggt ctt aac gcc ctc ccg
3. pPFS-2Ac11L2-3 2AcL2-3 R reGVETst A386E Tgt aga ggt ttc aac gcc ctc ccg
4. pPFS-2Ac11L2-4 2AcL2-4 R reDVNTst G384D
A386N
Tgt aga ggt gtt aac gtc ctc ccg
5. pPFS-2Ac11L2-5 2AcL2-5 R reGSATst V385S Tgt aga ggt ggc aga gcc ctc ccg
6. pPFS-2Ac11L2-6 2AcL2-6 R reGVADst T387D Tgt aga gtc ggc aac gcc ctc ccg
7. pPFS-2Ac11L2-7 2AcL2-7 R reGVAAst T387A Tgt aga ggt ggc agc gcc ctc ccg
8. pPFS-2Ac11L2-8 2AcL2-8 R reGAAAst V385A
T387A
Tgt aga ggc ggc agc gcc ctc ccg
9. pPFS-2Ac11L2-9 2AcL2-9 R reGVPTst A386P Tgt aga ggt ggg aac gcc ctc ccg

Table 1: Primers sequences for site-directed mutagenesis by PCR.

Toxin preparation

Bt strains harbouring pHT3101 plasmid with different mutants of Cry2Ac11 gene were cultured at 30°C and 100 rpm for approximately 72 hours until complete sporulation in GYS medium supplemented with 10 mg/ml erythromycim [9]. To observe sporulation; cultures were microscopically analyzed after 24, 48 and 72 hours of inoculation. A sterilized inoculating loop was used to transfer an aliquot of the sporulating media on to a microscopic slide. The slide was then air dried and heat fixed. The slides were stained with crystal violet and rinsed with distilled water. After air drying the slides were observed under light microscope using 100x oil immersion objective.

When about 90% of cells were sporulated and lysed the crystal spores mixtures were harvested by centrifugation at 7000 rpm for 25 minutes.

Washing of the cell pellets was done with 1 M NaCl for removal of proteases and 10 mM EDTA 3-4 times. The cell pellets were lyophilized and stored at -20°C for further use.

Protein quantification

Protein quantification was done by taking O.D280 (1O.D unit=1 mg) of protein and by Bradford assay. The total protein profile of spore–crystal mixtures was determined by SDS–PAGE analysis 12% polyacrylamide separating gels.

Insect toxicity assays

To evaluate the insect specificity and toxicity of all proteins bioassays were performed with Helicoverpa armigera, Spodoptera litura, Culex quinquefasciatus and Aedes aegypti. Freeze dried powder of crystal-spore mixtures were used for performing bioassays and both surface contamination and diet incorporation methods were used for toxicity assays of Lepidopterans.

Insect rearing

The larvae were fed on semi solid diet in the laboratory at 20-25°C and 60-65% humidity with 14:10 light:dark ratios. Diet was placed in the chambers of 3 x 12 trays with one larva per chamber. The pupae were collected after turning into dark brown color. At moth emergence; the adults were placed for egg laying in glass chambers having two baby nappy strips suspended in each for egg laying. The adults were fed on sugar solution. The male to female ratio was kept 4:6 per chamber. The diet was prepared by method [10].

Toxicity Assays with Helicoverpa armigera and Spodoptera litura Surface contamination and diet incorporation bioassays were done with first instar larvae of Helicoverpa armigera using a 24 well plate. Diet overlay bioassay was done with first instar larvae of Helicoverpa armigera using a 24 well plate. A single concentration of 2 ug/cm2 was used for surface contamination method and 25 ug/ml for diet incorporation method. Diet without protein was used as negative control. The weight and growth instars of larvae were critically noted before experiment. Mortality and growth retardation was observed till 7 days. At 7th day the weight of survived larvae was also noted.

With Spodoptera litura the bioassay was done employing diet incorporation method using the same procedure as for Helicoverpa armigera. A single concentration of spore crystal mixture was used that was 25 ug/ml. First instar larvae of Spodoptera litura was used per well and mortality, growth retardation and weight loss was observed for 7 days.

Toxicity Assays with Culex quinquefasciatus and Aedes aegypti

Bioassays were performed using single concentration in triplicate at 25°C. Ten second instar mosquito larvae were placed in 50 ml plastic cups with 20 ml of autoclaved water with known concentrations of spore crystal mixture. A single dose containing high concentration of 200 ug/ml was used. Autoclaved water was used as negative control.

The percentage larval mortality was recorded after 24 hours by counting the number of dead larvae in each cup.

Results

Toxin preparation and spores harvesting

All mutants were cultured at 30°C and 100 rpm for approximately 72 hours in nutrient broth sporulation medium (GYS medium) supplemented with 10 mg/ml erythromycin until complete sporulation.

The status of sporulation in all constructs was microscopically analysed using Gram staining. When the sporulation was complete in more than 90% of cells the spores crystal mixtures were harvested by centrifugation at 4°C. The samples were freeze dried and stored at -20°C untill further use. Protein quantification was done by Bradford assay and by taking O.D280 (Tables 2 and 3). Total protein profile was analyzed by SDS-PAGE.

Sr.No Mutant Proteins O.D280
(O.D Units)
1 pPFS-2Ac11 1.261
2. pPFS-2Ac11L2-1 0.873
3. pPFS-2Ac11L2-2 1.172
4. pPFS-2Ac11L2-3 0.962
5. pPFS-2Ac11L2-4 1.034
6. pPFS-2Ac11L2-5 0.902
7. pPFS-2Ac11L2-6 1.006
8. pPFS-2Ac11L2-7 0.940
9. pPFS-2Ac11L2-8 1.504
10 pPFS-2Ac11L2-9 1.118

Table 2: O.D280 Loop 2 Mutants(10 mg protein dissolved in 1 ml SDS).

Sr.No Mutant Proteins O.D595 Final protein concentration (mg/ml) Percentage of protein
1. pPFS-2Ac11L2-1 0.412 38 73
2. pPFS-2Ac11L2-2 0.426 39 81
3. pPFS-2Ac11L2-3 0.512 48 98
4. pPFS-2Ac11L2-4 0.592 59 99
5. pPFS-2Ac11L2-5 0.527 51 86
6. pPFS-2Ac11L2-6 0.596 59 99
7. pPFS-2Ac11L2-7 0.600 59 99
8. pPFS-2Ac11L2-8 0.696 51 98

Table 3: Protein concentrations of Loop 2 mutants by Bradford assay.

Bioassay with Culex quinquefasciatus

Second-third instar mosquito larvae were used for bioassays and single protein concentration of 200 ug/ml was used. Bioassays were performed in triplets. The larval mortality was recorded after 12 hours by counting the number of dead mosquitoes in each cup. The bioassays were done three times on different temperatures and environmental conditions. All proteins showed different mortality rates in a period of 24 hours. Loop 2 mutant pPFS-2Ac11L2-3 showed 56% mortality which is higher than Cry2Ac11 that showed 46% mortality. The least mortality was shown by pPFS-2Ac11L2-1 and pPFS-2Ac11L2-8 which was only 6% (Table 4 and Figure 1).

Proteins Dead Mosquitoes after 24 hrs
200ug/ml
Percentage Mortality
1 2 3 Average
pPFS-2Ac11 7 7 0 4.6 46%
pPFS-2Ac11L2-1 1 1 0 0.6 6%
pPFS-2Ac11L2-2 2 3 4 3.0 30%
pPFS-2Ac11L2-3 6 5 6 5.6 56%
pPFS-2Ac11L2-4 6 3 3 4.0 40%
pPFS-2Ac11L2-5 1 2 5 2.6 26%
pPFS-2Ac11L2-6 3 3 2 2.6 26%
pPFS-2Ac11L2-7 3 0 3 2.0 20%
pPFS-2Ac11L2-8 0 2 0 0.6 6%
pPFS-2Ac11L2-9 4 2 6 4.0 40%

Table 4: Insecticidal activity of Loop2 mutants against Culex quinquefasciatus 2ndinstar.

clinical-infectious-diseases-mutants

Figure 1: Toxicity of Cry2Ac11 mutants against Culex quinquefasciatus and Aedes aegypti.

Bioassays with Aedes aegypti

Ten second-third instars Aedes larvae were used per cup with 20 ml of autoclaved distilled water. A single protein concentration of 200 ug/ml was used in each cup and bioassays were performed in triplets. The larval mortality was recorded after 12 hours, 48 hours and 72 hours period by counting the number of dead mosquitoes in each cup. Temperature was maintained at 25°C. Loop 2 mutants showed no mortality against Aedes aegypti even after 72 hours. Only pPFS- 2Ac11L2-4 showed little toxicity that was 30% but the toxicity didn’t increased that remarkably with a passage of time. Loop 2 mutant pPFS- 2Ac11L2-6 showed 6% mortality. There was no growth retardation as well. The molting of cuticle (ecdysis) was also visible indicating the change in growth instar (Table 5).

Proteins Time Duration Dead Mosquitoes
(200ug/ml)
Percentage Mortality
1 2 3 Average
pPFS-2Ac11 After 24 hrs 0 0 0 0 0%
  After 48 hrs 0 0 0 0 0%
After 72 hrs 0 0 0 0 0%
pPFS-2Ac11L2-1 After 24 hrs 0 0 0 0 0%
  After 48 hrs 0 0 0 0 0%
After 72 hrs 0 0 0 0 0%
pPFS-2Ac11L2-2 After 24 hrs 0 0 0 0 0%
  After 48 hrs 0 0 0 0 0%
After 72 hrs 0 0 0 0 0%
pPFS-2Ac11L2-3 After 24 hrs 0 0 0 0 0%
  After 48 hrs 0 0 0 0 0%
After 72 hrs 0 0 0 0 0%
pPFS-2Ac11L2-4 After 24 hrs 5 1 2 2.6 26%
  After 48 hrs 6 1 2 3 30%
After 72 hrs 7 1 2 3.3 33%
pPFS-2Ac11L2-5 After 24 hrs 0 0 0 0 0%
  After 48 hrs 0 0 0 0 0%
After 72 hrs 0 0 0 0 0%
pPFS-2Ac11L2-6 After 24 hrs 0 0 2 0.6 6%
  After 48 hrs 0 0 2 0.6 6%
After 72 hrs 0 0 2 0.6 6%
pPFS-2Ac11L2-7 After 24 hrs 0 0 0 0 0%
  After 48 hrs 0 0 0 0 0%
After 72 hrs 0 0 0 0 0%
pPFS-2Ac11L2-8 After 24 hrs 0 0 0 0 0%
  After 48 hrs 0 0 0 0 0%
After 72 hrs 0 0 0 0 0%
pPFS-2Ac11L2-9 After 24 hrs 0 0 0 0 0%
  After 48 hrs 0 0 0 0 0%
  After 72 hrs 0 0 0 0 0%

Table 5: Insecticidal activity of Loop2 mutants against second- third instar Aedes aegypti larvae.

Bioassays with Helicoverpa armigera

Toxicity assays with Helicoverpa armigera were done using spore-crystal mixtures with both surface contamination and diet overlay method.

Using surface contamination method and dose concentration of 2 ug/cm2. The insects did not show significant percentage mortality using this concentration (Tables 6-8). Cy2Ac11 showed no mortality till the seventh day. Among all loop 2 mutants maximum mortality was shown by pPFS2Ac11L2-7 that was11.1% with 2 larvae killed out of 18. No mortality was shown by pPFS2Ac11L-4and pPFS2Ac11L-6 and all remaining mutants showed only 5.6% mortality. As far as weight loss and growth retardation is concerned growth was not retarded with any of protein but there was a slight weight loss caused by all proteins except pPFS2Ac11L-4. Cry2Ac11 also showed no weight loss but there was little growth retardation.

Mutants Weight of Control
g/larvae
Growth instars of control Weight of treated
g/larvae
Growth instar of treated Mortality (out of 18) Weight loss %age Mortality
pPFS-2Ac11 0.191 5th 0.147 4th 0 0.044 0.0
pPFS2Ac11L2-1 0.216 5th 0.197 4-5th 0 0.019 0.0
pPFS-2Ac11L2-2 0.133 5th 0.174 4th 0 -0.041 0.0
pPFS-2Ac11L2-3 0.262 4th 0.252 4th 0 0.011 0.0
pPFS-2Ac11L2-4 0.176 5th 0.225 4th 0 -0.048 0.0
pPFS-2Ac11L2-5 0.108 5th 0.129 5th 0 -0.021 0.0
pPFS-2Ac11L2-6 0.138 5th 0.235 5th 0 -0.097 0.0
pPFS-2Ac11L2-7 0.218 4th 0.111 4th 0 0.108 0.0
pPFS-2Ac11L2-8 0.225 5th 0.187 5th 0 0.038 0.0
pPFS-2Ac11L2-9 0.193 5th 0.158 5th 0 0.035 0.0

Table 6: Dose response of Cry2Ac11 mutants against Spodoptera litura with single dose of 25 ug/ml using diet incorporation method.

Mutants Weight of Control
g/larvae
Growth instars of control Weight of treated
g/larvae
Growth instars of treated Mortality (out of 18) Weight loss %age Mortality
pPFS-2Ac11 0.085 4-5th 0.136 3-5th 0 -0.057 0
pPFS2Ac11L2-1 0.247 4th 0.205 4th 1 0.042 5.6
pPFS-2Ac11L2-2 0.162 4th 0.179 4th 1 -0;018 5.6
pPFS-2Ac11L2-3 0.282 4-5th 0.261 4th 1 0.020 5.6
pPFS-2Ac11L2-4 0.185 3-4th 0.227 4th 0 -0.042 0.0
pPFS-2Ac11L2-5 0.086 4th 0.133 3-4th 1 -0.047 5.6
pPFS-2Ac11L2-6 0.141 4th 0.238 4th 0 0.122 0.0
pPFS-2Ac11L2-7 0.227 4th 0.104 3-4th 2 0.072 11.1
pPFS-2Ac11L2-8 0.270 4-5th 0.198 3-4th 1 0.085 5.6
pPFS-2Ac11L2-9 0.238 4-5th 0.153 3-4th 1 0.008 5.6

Table 7: Dose response of Cry2Ac11 loop 2 mutants against Helicoverpa armigera with single dose of 2 ug/cm2 using surface contamination method.

Mutants Weight of Control
g/larvae
Growth instars of control Weight of treated
g/larvae
Growth instar of treated Mortality (out of 18) Weight loss %age Mortality
pPFS-2Ac11 0.227 4-5th 0.180 4th 1 0.047 8.3
pPFS2Ac11L2-1 0.206 4-5th 0.174 3-4th 1 0.033 8.3
pPFS-2Ac11L2-2 0.123 4th 0.158 3-4th 1 -0.034 8.3
pPFS-2Ac11L2-3 0.256 4-5th 0.223 4th 2 0.033 16.7
pPFS-2Ac11L2-4 0.173 4th 0.217 4th 0 -0.044 0.0
pPFS-2Ac11L2-5 0.115 4th 0.115 3-4th 1 0.000 8.3
pPFS-2Ac11L2-6 0.137 4th 0.224 3-4th 0 -0.087 0.0
pPFS-2Ac11L2-7 0.216 4th 0.129 4th 2 0.087 16.7
pPFS-2Ac11L2-8 0.210 4th 0.156 3-4th 1 0.054 8.3
pPFS-2Ac11L2-9 0.178 4th 0.174 4th 2 0.003 16.7

Table 8: Dose response of Cry2Ac11 mutants against helicoverpa armigera with single dose of 25 ug/ml using diet incorporation method.

Using diet incorporation method the bioassays were done with a concentration of 25 ug/ml. Cry2Ac11 showed 8.3% mortality (Figure 2). Among Cry2Ac11 mutants the maximum percentage mortality was shown by pPFS2Ac11L2-3 and pPFS2Ac11L2-7 that was 16.7% with killing 2 mosquitoes each out of 12.

clinical-infectious-diseases-Protein-marker

Figure 2: SDS-PAGE analysis.(1) Protein marker, (2) pPFS2AcL2-1, (3) pPFS2AcL2-2, (4) pPFS2AcL2-3, (5) pPFS2AcL2-4, (6) pPFS2AcL2-5, (7) pPFS2AcL2-6, (8) pPFS2AcL2-7, (9) pPFS2AcL2-8.

Bioassays with Spodoptera litura

Bioassays against Spodoptera litura were done with a concentration of 25 ug/ml of freeze dried spore-crystal mixture. None of them showed any mortality. However some weight loss and growth retardation was observed. Cry2Ac11 also showed weight loss of 0.044 g as well as growth retardation from fourth to fifth instar (Table 6).

pPFS2Ac11L2-2 and pPFS2Ac11L2-4 showed no weight loss but showed growth retardation from fourth to fifth instar. pPFS2Ac11L2-6 did not show growth retardation as well as weight loss.

Discussion

Bacillus thuringiensis is a potential candidate for pest control due to its ability to produce insecticidal proteins but very few data has been generated about the receptor ligand interactions of Cry proteins.

In present work a detailed study on binding interactions of Cry2Ac11 is done with its putative receptors. Also the binding epitope of Cry2Ac11 is analyzed by generating different mutations and observing their role in receptor binding and toxicity.

Considering the findings of Liang [6] the peptide stretch including residues 307-412 is the specificity determining region. As the mutations done were present in this area so there was little possibility that the insect specificity and host range of Cry2Ac11 may also get affected due to the induced mutation. To check the effects of mutations on insect toxicity, the mutants were characterized by bioassays.

A number of studies regarding the mutational analysis of the binding epitope have been done previously and the effect of these mutations has been analyzed on receptor binding and toxicity. For example in case of Cry3Aa the replacement of N353 and D354 of loop 1 with alanine resulted in loss of the receptor binding and toxicity. The replacement of W357 of loop 1 in binding domain of Cry19Aa with alanine resulted in the loss of toxicity against mosquitoes. The mutations Y410A, W416A, and D418A of the loop 2 in binding domain of Cry19Aa resulted in reduced toxicity against Culex, Aedes aegypti. The alanine scanning of Cry4Aa mutants showed comparable toxicity to wildtypes [11]. Unlike these reports the mutational analysis in present study indicated no marked difference in insect specificity and toxicity as compare to wild type and the toxicity profiles were quite similar to wild type.

As far as the toxicity of Cry2A toxins is concerned for dipterans. It is reported that unlike Cry2Aa, Cry2Ac is not toxic to dipterans [12]. Cry2Aa has been repored to be toxic to dipterans but in this case also various dissimilarities are also present. Park et al. performed bioassays with different mosquito species. Against Culex quinquefasciatus, using Cry2Aa with a concentration of 200 ug/ml for 48 hours but Cry2Aa showed no mortality indicating that Cry2Aa is not effective against this specie. With Aedes aegypti and Anopheles gambiae, using same concentration Cry2Aa showed very low toxicity [13]. In another study, using different concentrations of Cry2Aa, no toxicity was reported against second instar Culex quinquefasciatus and Aedes agepti larvae [7]. Although the results of present study cannot be compared with previous reports directly due to different environmental conditions like temperature, humidity, type of water used etc but still in present study also Cry2Ac11 which shares various characteristics with Cry2Aa [12] showing more than 98% sequence similarity with Cry2Aa, along with its mutants showed no toxicity against Aedes agepti even at a high concentration of 200 ug/ml. Only the mutant with G384D and A386N showed 30% mortality against Aedes agepti and mutant withT387D showed only 6% mortality.

With Culex quinquefasciatus Cry2Ac11 and its mutants showed toxicity at concentration of 200 ug/ml. Cry2Ac11 showed 46% mortality in 24 hours. The mutant with A386E showed maximum mortality among all mutants and that was 56%.

As for Lepidopteran insects, various reports confirm the toxicity of Cry2A type proteins for lepidopteran insects especially for Helicoverpa armigera, but there are others reports as well that indicate less toxicity of Cry2A type proteins for lepidopteran insects. A Cry2A type protein named Cry2(SKW) showing high homology with Cry2Aa, was also reported to be less toxic to a lepidopteran specie Bombyx mori [12]. In another study it was reported that due to insolubility in alkaline conditions Cry2Ab is also not toxic to Helicoverpa armigera [14]. In present study as well, no mortality of Cry2Ac11 and its mutants against lepidopteran species is reported. At a concentration of 25 ug/ml, using diet incorporation method, Cry2Ac11 showed only 8.3% mortality against Helicoverpa armigera and only little growth retardation against Spodoptera litura. Among loop2 mutants and operon variants maximum 16.7% mortality was observed against Helicoverpa armigera.

Hence in conclusion, the results indicate that Cry2Ac11 is not toxic to Aedes agepti and Spodoptera litura (little growth retardation was present) and show low toxicity against Helicoverpa armigera and Culex quinquefasciatus at least in the conditions that we used. Also it can be concluded that mutations of one or two amino acids in loop 2 of Domain II cannot impart any remarkable difference in insect specificity and toxicity spectrum for both lepidopteran and dipteran insect species as compare to wildtype. This study can help in future for estimating the role of individual amino acid residues in insecticidal activity, making it easy to induce mutations in only those particular residues that may have significant role in increasing specificity spectra and insect toxicity.

References

Citation: Amjad Q, Khan ATA (2018) Impact of Loop Residues of the Receptor Binding Domain of Bacillus thuringiensis Cry2ac11 Toxin on Insecticidal Activity. Clin Infect Dis 2: 109.

Copyright: © 2018 Amjad Q, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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