Toxicity Evaluation and Genetic Improvement of Bacillus thuringiensis Isolated from Different Regions in Assiut, Egypt against Mosquito Larvae

This study aims to evaluate the toxicity and genetic improvement of Bacillus thuringiensis isolates. Isolates were obtained from soil, insect and water samples from different regions of Assiut, Egypt for biological control of mosquito larvae. B. thuringiensis colonies were identified based on morphological and then by PCR which detect the Cry toxic genes in the isolates. Bioassays were performed to evaluate the toxicity of different strains of B. thuringiensis against mosquito larvae such as ( Culex spp). In general, 36 B. thuringiensis isolates were obtained (31 from soil, 4 from insects, and 1 from water). And they were all toxic to mosquito larvae with different mortality percentages from 7 to 97% after 48 hours. Isolate Am2 recorded the highest mortality percentage 97% and Mn3 lowest mortality percentage 7%. PCR revealed that Am2 isolate which caused the highest mortality encodes three different types of Cry toxins, Cyt1AA, Cry1Ac and Cry2Aa . This isolate Am2 was examined by scanning electron microscopy to observe the shape of the Cry proteins. The results showed that the Am2 isolate contained of spherical and cuboidal toxic proteins. Then UV-mutagenesis was performed on the Am2 isolate to improve its toxicity. Out of 30 obtained UV-mutants, only one mutant showed improvement in the mortality of mosquito larvae since it caused a mortality rate of 100%. The results of the present study revealed the larvicidal efficacy of B. thuringiensis (Am2) isolate found in the soil of Assiut, could be used in biological control program of mosquito larvae.


INTRODUCTION
Mosquitoes are among the most important insects that transmit diseases to humans, as they infect them with diseases due to their transmission of viruses and parasites. 1,2The mosquito transmits the virus that causes several diseases, including dengue fever, yellow fever, and chikungunya. 3Where they are found in places where humans and animals live and breed in places that contain ponds with stagnant water and swamps, as they multiply rapidly and the resulting eggs are resistant to drought and withstand harsh conditions until the appropriate conditions are available to complete the life cycle of the insect, so there is difficulty in controlling them. 4he current strategy for controlling insect vectors of different infectious diseases is based on the use of programs of chemical insecticides, the development of resistance is a threat to the program's efficacy or by eliminating breeding sites. 5,68][9] Therefore, it is necessary to search for alternative and more harmless control methods including biological methods such as the use of entomopathogenic bacteria, in which the bacterium B. thuringiensis represents a very promising alternative. 10. thuringiensisis is a Gram-positive bacteria as well as facultative in that they are aerobic or anaerobic and produce spores in the form of protein crystals that are toxic against many orders of insects including B. thuringiensis subsp.israelensis (Diptera), B. thuringiensis subsp.tenebrionis (Coleoptera), B. thuringiensis subsp.kurstaki, B. thuringiensis subsp.aizawai (Lepidoptera) are examples of subspecies with specific bacterium against at variance groups of insects.11 These crystals are δ-endotoxins proteins, including Cry and Cyt proteins.12 These toxic proteins are encoded by the Cry and Cyt genes during the sporulation.1,13More than 700 Cry genes have been sequenced and classified into at least 70 groups named Cry1, Cry2, Cry3 ... Cry70, whose corresponding insect toxicity.13,14 The severity of toxicity to the insect also depends on the combinations of different proteins expressed by the Cry genes.15,16 Some strains can encode for more than one toxic protein, a well-known example is B. thuringiensis var.israelensis, which contains six proteins that are toxic to Diptera, this strain contains a plasmid called megaplasmid pBtoxis that encodes six different toxic proteins to mosquito larvae.17,18 It was observed in some strains that were isolated from different places that they contain different combinations of genes and have specialized toxicity to different insects order.19,20 These toxic proteins bind to receptors located in the lining membrane of the intestine of the mosquito larvae, where they feed on crystalline proteins, which leads to an imbalance of ions that leads to cell rupture and death of the larva.[21][22][23] The main objective of this study was to collect different isolates of B. thuringiensis from different locations in Assiut governorate, Egypt that cause mortality to mosquito larvae, molecularly characterize its cry genes, and enhance the toxicity by UV-mutagenesis.

Isolation of B. thuringiensis
Samples were collected from different areas and different sources (soil, water and insects) of Assiut governorate, Egypt.Samples were collected at a depth of 10 cm below soil surface using cylindrical sampler and kept in polyethylene bags.All samples were immediately transported to the laboratory and stored in a refrigerator at 4°C until used for the isolation of B. thuringiensis.B. thuringiensis was isolated according to previous studies with some modifications. 19,24One gram of soil sample was taken and mixed in 10 ml of sterilized distilled water.100 μl of each sample was plated to a petri dish containing the nutrient agar supplemented with penicillin G (100 mg/L).Insect samples were cut into small pieces and mixed with 2 ml of sterilized distilled water then 100 μl were transferred to petri plates containing the selective media.And then, 100 μl of the water samples were transferred directly to petri plates.Then all the plates were placed in the incubation for 48 hours and incubated at a temperature of 28°C.The colonies showed round shapes with wavy edges and no pigment morphology were inoculated in a nutrient broth and selected for further studies.

B. thuringiensis Bioassay Activity
To test the ability of different B. thuringiensis isolates to kill the mosquito larvae, all the B. thuringiensis isolates were incubated in nutrient broth for five days at 28°C and 180 rpm until the sporulation was observed.The concentration of each isolate was measured and adjusted to be equal at a concentration of OD 600 = 0.2.The sporulated isolate were added to 10 ml of sterile water that contains 10 mosquito larvae and incubated at of 28 ± 2°C.The number of deaths mosquito larvae was recorded after 48 hours of the treatment. 25The treatment was carried out in three replicates for each isolate.For comparison, an additional replicate containing the commercial bio-insecticide named Dacron 54% WP (SAFA TARIM. A. S. Turky) was used as a positive control.

Detection of the Presence of Toxic Genes within the Genome of the Isolates by PCR
7][28] PCR technique was used to screen the presence of some toxic genes in collected B. thuringiensis isolates.Five primer pairs were newly designed to target 5 different Cry and Cyt genes (Cry11AA, Cyt1AA, Cry1Ac, Cry2Aa and Cry4AA) from B. thuringiensis (Table 1).All gene sequences were obtained from NCBI (www.ncbi.nlm.nih.gov).Sequences of DNA primers were manually selected by considering, sequence location, GC content, number of nucleotides and simulated with In-Silico PCR amplification website (http://insilico.ehu.es/PCR/).There is no probability of complementarity between one primer with other or bases in the same primer. 29NA isolation was performed by the boiling method. 24,30PCR was performed in a final volume of 20 μl containing 10 μl of GoTaq blue master mix (Promega, Madison, WI, USA), 1 μl DNA sample, 0.2 μl of each primer and 8.5 μl of water, nuclease-free.The amplification reactions were carried out in a thermocycler (Sensoquest, Biomedical Electronics, Germany) under the following initial denaturation at 93°C for 5 min, followed by 30 cycles of, 30 sec of denaturation at 95°C, 1 min for annealing at 55 -58°C and 2 min for the extension at 72°C, then followed by 5 min final extension at 72°C and a final hold at 4°C.Then 1% agarose gel and 1X TAE were used to perform the electrophoresis.The gel was stained with ethidium bromide, visualized under UV rays and photographed.The molecular sizes of DNA fragments were measured by comparison with a 100-bp molecular marker (Hyperladder 100 bp, Bioline, Meridian Biosience, UK).

Scanning Electron Microscope
To observe the crystalline proteins under the electron microscope, isolate Am2 was grown on nutrient agar medium for 5 days at 28°C until sporulation.The spores were washed three times by cacodylate buffer for 13 minutes and post fixed

Induction of Mutagenesis with Ultraviolet Rays (UV)
The most mortal B. thuringiensis isolate to mosquito larvae was selected to improve its efficiency by UV-mutagenesis. 1 ml of an overnight culture was poured and spread on the petri plates that contain nutrient agar medium.The plates were exposed to UV source at different time intervals (0 sec as control), 20, 40, 80, 120, 160, 200, 240 and 280sec).The plates were covered in a black polyethylene bag and incubated at 28°C for 48h to get first generation mutants.The plates that were exposed to the time causing the death of half the colonies (LC 50 ) were used to isolate the mutants. 33And all the selected mutants were screened for their mortality by the bioassay test which was carried out in three replicates.

Isolation of B. thuringiensis isolates from different Locations and Sources
Around 100 bacterial isolates with similar morphological characteristics to B. thuringiensis (Table 2) were obtained from 9 different locations (Assiut university farm, Menqabad, Al-Ghanaim, Posra, Arab almadabigh, Sedfa, Sahel Salim, Al-Fath, West country) and different sources (water, soil and insects).The 100 bacterial isolates were morphologically screened, and colonies showed round shapes with wavy edges and no pigment morphology were furthermore checked by inoculation on a selection media containing penicillin G (100 mg/L).

Isolates Toxicity Evaluation
After the morphological screening, all the isolates were examined for their mortality to mosquito larvae.Out of the 100 isolates, only 36 isolates showed a mortality effect on mosquito larvae (Table 2), so these isolates were selected for further investigations.The rest of the isolates (64 isolates) did not give a mortality rate (results are not shown), so they were excluded from further experiments.
Am2 isolate showed the highest mortality rate (97%) among all the isolates, while Mn3 isolate showed the lowest one (7%) after 48h of incubation.
The results illustrated in (Figure 1) showed that, the used bio-pesticide recorded a very low mortality rate (27%) compared to bacterial isolates especially the Am2 isolate which showed the highest mortality rate.Previous studies indicated that it is possible to distinguish between different isolates with the mortality rate of mosquito larvae, which can be used to develop biological control tools to help control diseases caused by mosquitoes. 14,43,44he results showed that B. thuringiensis isolated from soil samples were high toxicity and more abundant than those from other sources (insects and water), these result were also found by Asokan and Puttaswamy 45 who also isolated different B. thuringiensis isolates from different sources.

Detection of the Presence of B. thuringiensis Toxin Genes by PCR
Polymerase chain reaction (PCR) was performed to determine the presence of different Cry genes (Cry11AA, Cyt1AA, Cry1Ac, Cry2Aa and Cry4AA) in the 36 selected isolates using specific primers illustrated in (Table 1).The results of the PCR screening were illustrated in (Figure 2) and (Table 3).
The Cyt1AA gene was more frequent among all the 5 tested genes and was present in six different isolates.Cyt1AA was found to cause high mortality since Ar7 and Gh5.2 isolates showed only the presence of Cyt1AA gene and caused a mortality percent of 87% and 80%, respectively.On the other hand, lepidopteran-specific Cry1Ac gene causes the least mortality rate, since it was detected individually in three isolates (Mn9, Am4 and Am4.2) and the maximum mortality rate was 30%, thus it can be concluded that the Cry1Ac gene has no major effect on the mortality.The results showed the lowest frequent amplified gene was Cry4AA since it was detected in only one isolate (Ps6) which showed a mortality rate with 30%.
In this study, 23 isolates did not show any presence of any tested toxic genes, but these isolates still have toxicity on mosquito larvae.This indicates the presence of other toxic genes that were not included in this study.Among those isolates, Ps1 isolate showed the highest mortality percentage 90%.On the other hand, Mn3 isolate showed the least mortality percentage 7%.
The absence of the PCR product in some isolates was detected was also obtained by 46,47 that found 89 out of 215 field-collected strains of B. thuringiensis did not produce PCR products with universal primers.
PCR was used as a basic parameter to detect the different types of toxic proteins of the tested isolates and their activity against mosquito +: amplified the gene; -: did not amplify the gene larvae. 13,487][28] The Cry1Ac gene is associated with toxicity against Lepidoptera larvae. 49Results in Figure 2 showed that, most of the isolates did not produce a positive single band with the specific primers, and an extra band/bands were amplified.Similar findings were reported by 50,51 who suggested that, isolates containing novel Cry genes may give PCR products different in size relative to the standard or may completely lack PCR products.This could be because two or three Cry genes might be positioned next to each other, forming an operon. 52

Scanning Electronic Microscopy (SEM)
Am2 isolate which caused the highest mortality 97% were preliminarily screened with phase contrast microscopy 100X magnification to observe the presence of shape of the Cry crystals.Scanning electron microscopy showed crystal architecture of cuboidal shapes for the toxic proteins and confirmed the presence of rod-shaped bacterial cells, the morphological characteristics of B. thuringiensis (Figure 3).
A previous study, [53][54][55] reported different shapes of crystals protein cuboidal, spherical, irregular, bipyramidal, balloon shaped and flat square all these crystal structures were found in different isolates of B. thuringiensis and isolated from different regions.In the present study, Am2 isolate amplified three different types of toxic genes Cyt1AA, Cry1Ac and Cry2Aa and the scanning electron microscopy showed crystal structure of cuboidal shape for the toxic proteins.Cry proteins related to some crystal structures. 46,56,57In this study, Am2 isolate demonstrated cuboidal crystals related to amplify Cry2Aa protein. 56,58

Genetic Improvement with Mutagenesis by Ultraviolet rays (UV)
0][61] Previous studies were obtained in B. thuringiensis mutants that were produced by successive rounds of ultraviolet rays, the mutants were more UV resistant and some of them were lost encoding their toxin Cry genes due to mutations. 33,62,63V-mutagenesis was performed to improve the toxicity of the best killing BT isolate (Am2).Am2 isolate was exposed to UV irradiation for different time intervals (0 sec (as control), 20, 40, 80, 120, 160, 200, 240 and 280sec).The mutants were selected from plates that showed a survivability rate of 50% after exposure (280 sec of UV irradiation).
Thirty mutants were obtained from the mutagenesis of the wild Am2 isolate.All the mutants were screened for their mortality.Out of 30 mutants, only one mutant Mu25 showed better mortality than the wild type Am2 isolate.The improved Mu25 mutant showed toxicity to mosquito larvae with a mortality rate of 100% after 48 hours (Figure 4).
Results in Figure (4) showed that, the toxicity of the isolates was decreased in all the isolates after the UV-mutagenesis.These results were also found by 64,65 since they found that, insecticidal activity of B. thuringiensis were negatively affected by UV rays, which leads to a decrease in toxicity and a decrease in stability.Only one mutant Mu25 was improved and caused a high mortality for mosquito larvae after UVmutagenesis.

CONCLUSION
In this study, the genetic variability was obvious among different isolates and caused variation in their toxicity against mosquito larvae.The isolate Am2 amplified the highest number of different types of genes (Cyt1AA, Cry1Ac and Cry2Aa) and caused high mortality rates against mosquito larvae.The Cyt1AA gene toxin has predominant efficiency against dipterous according to. 19,66The Cry2Aa protein demonstrated form cuboidal crystal in Am2 isolate under scanning electron microscopy according to. 58,67,68V-mutagenesis showed high toxicity and stability in mutant Mu25 against mosquito larvae.Further studies will be performed to investigate the effect of UV mutagenesis on different cry gene sequences.Moreover, the gene expression levels of different cry gene will be determined by qRT-PCR and SDS-PAGE.

Figure 1 .
Figure 1.Mortality rate caused by B. thuringiensis isolates on mosquito larvae

Figure 2 .
Figure 2. PCR amplification of the different Cry genes on the different isolates

Table 1 .
Primers used in the PCR to amplify Cry and Cyt genes of B. thuringiensis that cause toxic activity against mosquito, showing the primer sequences, the size of the target fragment, and the annealing temperature

Table 2 .
Different isolates code, location, type of sample, number of bacterial isolates, and number of B. thuringiensis isolates