Selective inactivation of spores maintaining larvicidal activity in Bt serovar israelensis irradiated with gamma rays

Bacillus thuringiensis is used to produce biopesticides against target-insects of importance in agroindustry and human health. However, B. thuringiensis can carry the same enterotoxin-encoding genes as Bacillus cereus, suggesting a potential risk of food poisoning. The objective of this work was to study the application of gamma radiation to eliminate spores in biopesticides based on B. thuringiensis serovar israelensis in order to avoid the liberation of these spores in the environment. The application of 20 kGy of radiation ensures total inactivation of the spores in the insecticide without changing the larvicidal activity. Results showed that it is possible to develop formulations containing B. thuringiensis serovar israelensis (IPS-82) eliminating viable spores with gamma radiation without affecting the larvicidal activity. Irradiated formulations would be safer for the environment and humans since the elimination of viable spores abolishes the pathogenic potential of B. thuringiensis strains.


Introduction
The use of chemical pesticides to control insects has revealed several different risks for man and the environment. The occurrence of chemical pesticides resistance in target insects and human health problems related to their use have been reported (Aranda et al., 2000;van Rie and Ferrè, 2000;Cadavid-Restrepo et al., 2012). These facts prompted the emergence of industrialized biopesticides based on strains of Bacillus thuringiensis, a member of Bacillus cereus senso lato group, that are capable of producing different crystal δ-endotoxins encoded by cry genes. These toxins are the active ingredients of a large number of preparations used against larvae of many insect orders, especially species of Coleoptera, Diptera and Lepidoptera (Aranda et al., 2000;Libman and MacIntosh, 2000;Hansen and Salamitou, 2000). In 1978, a previously described B. thuringiensis strain (Goldberg and Maragalit, 1977) was designated as the 14 th B. thuringiensis serovar (Bacillus thuringiensis var israelensis, H-14) (de Barjac, 1978). This bacterium is highly toxic for mosquitos and blackflies (LC 50 ranging from 10-13mg/ml against fourth instar larvae of many species) and presents three inclusions containing four main proteins, Cyt1A, Cry4A, Cry4B and Cry11A. These proteins have different molecular weights and act in synergy with other Cyt toxins (Federici et al. 2000;Crickmore et al. 2006;Cadavid-Restrepo et al., 2012). This serovar became the active compound of industrialized biopesticides used all over the world in polycentric programs to control Aedes spp. larvae, especially those of Aedes aegypti, the yellow fever and dengue vector (Mittal, 2003). It is noteworthy that in Germany, B. thuringiensis serovar israelensis has been used since 1981 in a program that is conducted by the German Association for Mosquito Control (Kommunale Aktionsgemeinschaft zur Bekämpfung der Stechmückenplage -KABS) (Becker, 2002). Despite the advantages of using biopesticides instead of chemical pesticides, there are some considerations to be made: B. thuringiensis shares a high degree of genetic similarity with B. cereus, and it can be distinguished from the last mainly by the production of parasporal bodies containing δ-endotoxins (Yuan et al., 2002). It has been described that B. thuringiensis strains can carry the same enterotoxins as B. cereus, suggesting that this species may also pose potential risk of food poisoning (McIntyre et al., 2008;Bartoszewicz et al., 2008). The aim of the present work was to evaluate the application of gamma radiation to reduce the presence of viable spores in biopesticides based on B. thuringiensis serovar israelensis, in order to decrease or prevent the release of these spores in the environments where the breeding sites of mosquito larvae and black flies are located. This inactivation methodology can be used as an alternative to create an effective, safer and more environment-friendly biopesticide.

Bacterial strains
The toxigeni c strain B. thuringiensis serovar israelensis (H-14) IPS-82 (LFB-FIOCRUZ 584, deposited in the Coleção de Culturas do Gênero Bacillus e Gêneros Correlatos -CCGB since 1983) was used. This strain was preserved as lyophilized spores produced in AES medium according to Rabinovitch et al. (Rabinovitch et al., 1975) and kept in refrigerator. Cytomorphological, biochemical and physiological characteristics were confirmed as previously described (Gordon et al., 1973;Claus and Berkeley, 1986). The spores obtained from AES medium were conserved in distillated water under refrigeration as suspensions containing 8x10 6 colony forming unit (CFU)/mL -1 .

Production of B. thuringiensis IPS-82 formulation
In order to prepare the primary inoculum, the spores contained into two tubes with AES medium (at least 90% of viable spores) were harvested and transferred to 2.0 mL of sterile distillated water and homogenized. One milliliter of this suspension (10 9 -10 10 CFU) was transferred to 100 mL of liquid medium and incubated at 35 o C under 120 rpm for 4h. After that, 30 mL of this growth were inoculated in 6L of fermentation medium (Soy meal 60-70% of protein 20 g/L; glu- Total fermentation time was 22h-24h. The end-point of the process was determined by observation under an optical microscope as the point when the sporogenesis reached at least 90% of free of spores and, in addition, by the pH measure (values between 8.5 and 9.0). After the fermentation, the pH of the fermented medium was adjusted to 4.5 with laboratory grade propionic acid. This medium was centrifuged in a continuous flow Alfa-Laval Gyro Tester centrifuge at 3450 rpm. The final formulation was obtained by the addition of thickener, preservatives, emulsifying, disintegrants and water to the centrifuged, not exceeding 2% (w/w). These additives were mechanically mixed at room temperature (25 o C to 30 o C) until reaching a uniform creamy texture. The biomass was kept in the refrigerator. The fermentation process generated around 170g of biomass with 70% water content.

Irradiation
Aliquots of formulated biomass were placed in neutral glass tubes with screw caps (75mm x 15mm) until filling them, and sent to IPEN-CNEN/SP, São Paulo to be exposed to an ionizing radiation source of Co-60 (Gammacell 220 Atomic Energy of Canada Ltd.) at an average dose of 4.86 h -1 kGy and dose uniformity factor of 1.13. Doses ranged from 0 to 20 kGy.

Quantification of spores in the irradiated biomass
In order to evaluate the effects of different doses of irradiation on the viability of B. thuringiensis serovar israelensis IPS-82 spores, suspensions containing 25 mg or 50 mg of each irradiated and non-irradiated samples in 25 mL or 50 mL of sterile distillated water were prepared. Two milliliters of each suspension were heated in 100°C water bath for 15 min. After that, heat-treated suspensions were diluted in sterile distilled water at a ratio of 1:10 and 3 aliquots of 0.1 mL of the most diluted suspensions were spread in three Petri dishes containing nutrient agar culture medium (meat peptone 10 g/L; meat extract 3 g/L; NaCl 5g/L; agar-agar 20 g/L; pH 7.5). Colony counting was performed after incubation at 30°C for 24h-30h in order to determine the average number of colony forming units per mg (CFU mg -1 ).

Biological assays and potency determination
The effect of the irradiation on the insecticidal activity was evaluated by assays with sensitive Aedes aegypti larvae. These assays were conducted according to the methodology recommended by the World Health Organization (WHO, 1981), with minor modifications, in order to determine the potency of the material expressed by International Toxic Units per mg (ITU mg -1 ). For this purpose, 25 mg of each of the irradiated and non-irradiated samples were diluted in 50ml of sterile distilled water and, after homogenization, different aliquots were transferred into plastic cups containing 25 young L4 larvae of A. aegypti (Rockefeller strain) in 150 ml of dechlorinated water, comprising different concentrations, at least six. Four cups for each concentration and four cups for control samples, where no insecticide was added, were used. Mortality of the larvae was evaluated 24 hours after incubation at 27 o C -28 o C, and the lethal concentration to 50% of the larvae (LC 50 ) was determined by the analysis of the probits. The tests were repeated for each sample at least three times on different days and the mean value and the standard deviation were calculated. In addition to each test sample, the LC 50 of the standard powder IPS-82 (batch #91519) was determined. This powder was provided by the Institut Pasteur (Paris, France) and preserved in refrigerator under vacuum. Its potency was arbitrated at 15,000 ITU per mg, and it allowed determining the average potency of the bioinsecticide samples exposed to different doses of radiation and also of those not irradiated.

Data analysis
The analysis was performed to evaluate the effect of irradiation on the larvicidal activity of formulations based on B. thuringiensis serovar israelensis IPS-82. The value of decimal reduction (D 10 ) for B. thuringiensis spores, representing the radioresistance of the spores, was calculated through linear regression. Larvicidal activity (expressed as potency) of samples of the insecticide that were exposed to different doses of gamma radiation was determined in A. aegypti and compared with that of the non-irradiated samples. The ANOVA test was applied in order to compare the potency of irradiated and non-irradiated samples.

Effect of irradiation on the viability of B. thuringiensis serovar israelensis IPS-82 spores
Samples of formulated insecticides biomass containing an average of 3.5 x 10 6 viable spores per mg were subjected to different doses of irradiation. Table 1 shows the average number of viable spores (N) per mg for each dose of radiation (D) used for inactivation. A linear response between N and D logarithms was obtained (Figure 1). The value of decimal reduction (D 10 ) for B. thuringiensis spores was 2.89. According to the equation of the curve obtained by the correlation of the data showed in Table 1, only one spore, from 3.5x10 6 that were irradiated, survive after a dose of 19.83 kGy. The application of a dose of 20 kGy ensured total inactivation of the spores in the insecticide. Effect of irradiation on the larvicidal activity of emulsifiable concentrate based on B. thuringiensis serovar israelensis IPS-82 The variance analysis of the larvicidal activity of samples of the insecticide (expressed as potency) revealed that there is no statistical significance between the mean values of different treatments (Table 1). These results indicate that there was no change in the effectiveness of insecticide after irradiation with Co-60 up to a maximum dose of 20 kGy (p=0.05, confidence interval 95%).

Discussion
This study showed that it is possible to develop formulations containing B. thuringiensis serovar israelensis (IPS-82) eliminating viable spores with gamma radiation without affecting the larvicidal activity. There are a few works in the literature reporting irradiation of B. thuringiensis serovar israelensis with Co-60. One of these works reported that gamma radiation doses of 20-25kGy produced a 20-30% reduction in the effectiveness of B. thuringiensis serovar israelensis powder against L4 Culicidae larvae (Becker, 2002). Melo-Santos et al. (2009) reported that two Bti IPS-82 preparations (tablet and technical powder) lost up to 83% of potency after irradiation. Asano et al. (2000) found that gamma irradiation of B. thuringiensis serovar kurstaki spores results in a dramatic reduction of their effect against the diamondback moth larvae, Plutella xylostella, indicating that live spores are necessary to achieve full insecticidal activity against these Lepidoptera larvae. Our results showed that irradiation with Co-60 (ranging from 5 to 20 kGy) gradually decreases the amount of viable spores of B. thuringiensis serovar israelensis in the formulated biomass. Moreover, it was shown that the larvicidal activity of the preparation remains unchanged. Thus, the results suggest that the mosquitocidal activity of the B. thuringiensis serovar israelensis strain IPS-82 (LFB-FI-OCRUZ 584) used in the formulation tested does not depend on the germination of the spore inside the A. aegypti larvae and thus it is not affected by radiation. Our results showed that irradiation with Co-60 (ranging from 5 to 20 kGy) gradually decreases the amount of viable spores of B. thuringiensis serovar israelensis in the formulated biomass, but the larvicidal activity of the preparation remains unchanged. The discrepancy between the results presented in this work and those described by Asano et al. (2000) and Becker (2002) regarding the insecticidal efficiency of irradiated spores may be, in part, due to the different B. thuringiensis strains and serovars that were used in each study. This is particularly more relevant when comparing our results with those of Asano et al. (2000) where the germination of the spores seems to play an important role on the pathogenicity of B. thuringiensis serovar kurstaki on P. xylostella. In this work, an "in house" emulsifiable formulation was used instead of the commercial wettable powder used by Becker (2002) or the table and the technical powder used by Melo- Santos et al. (2009). In contrast with results reported in both studies, we did not observe any significant loss of potency after the irradiation. Moreover, it seems that the addition of proprionic acid to the fermentation product, creating an acidic environment, protects the crystalline structure of the protoxin. Our results suggest that the type of formulation may influence the conservation of the potency in irradiated products and this should be considered when developing these products. The original, non-irradiated formulation described in the present study was once tested in the field in three streams with different flows located in Rio Grande do Sul, Brazil (Mardini et al., 1999). The formulation tested against larvae of Simulium spp., in doses ranging from 20 to 40 ppm per point of application, was able to totally eliminate the larvae of this insect in a distance of 380m from the point of application (Mardini et al., 1999). Nevertheless, further studies are necessary to evaluate the efficacy of the irradiated formulation in the field. The present work demonstrates that it is possible to develop industrially formulated products containing B. thuringiensis serovar israelensis (IPS-82) eliminating viable spores with gamma radiation without affecting the larvicidal activity. Irradiated formulations would be safer for the environment and humans since the elimination of viable spores abolishes the pathogenic potential of insecticidal B. thuringiensis strains. Further studies should evaluate the application of the methodology described here on different formulations using other B. thuringiensis serovars active against agricultural pests, in order to assess how the insecticidal activity of these strains is affected by radiation.