Evaluation of a new formulation of Bacillus thuringiensis israelensis

The aim of this study was to determine the potency (ITU) and efficacy of a liquid formulation of Bacillus thuringiensis israelensis developed by the State University of Londrina named BioUel, against early fourth instar larvae of Aedes aegypti and Culex quinquefasciatus. The ITU/mg of BioUel was 960, the LC 50 was of 0.271 (± 0.39) ppm, and the LC 95 was 0.634 (± 0.099) ppm, in larvae of C. quinquefasciatus. In A. aegypti larvae, LC 50 was 0.332 (± 0.042) ppm and LC 95 was 0.694 (± 0.073) ppm. The ITU level of BioUel and its control results were similar to most commercial products tested. Stability was of approximately 90 days, which allows for local production.


Introduction
According to Forattini (2002), mosquito control is carried out mainly with chemical products, such as organophosphates, carbamates and pyrethroids.The utilisation of these products, however, has been limited because they are non-specific, pollute the environment, and their target insects have high rates of resistance.This situation has led to the development of biological insecticides using bacteria, fungi, viruses, and other alternative organisms (Consoli et al., 1997;Regis et al., 2001;Arantes et al., 2002;Tauil, 2002;Zequi and Lopes, 2007;Russell and Kay, 2008).The emphasis of this study is on the use of entomopathogenic bacteria of the genus Bacillus because of its high degree of specificity and environmental safety, thus particularly suitable and environmentally friendly for use in the control of insect pests.
In 1975, a new line of Bacillus thuringiensis (Bt) known as the variety israelensis (Bti) was identified and
BioUel stability was determined by placing 500 mL of the product in a white opaque bottle kept in a cabinet in the laboratory, in local conditions.The bioassays were performed following the same method used in test the efficacy of BioUel, at concentrations of 0.5, 0.29, 0.22, 0.18, 0.13, and 0.10 ppm immediately after the product was produced, and also after 30, 60, 90, 120, and 150 days.Larval population densities were assessed 24 hours after treatment.The results were submitted to Probit Analysis (Micro Probit program) to determine BioUel LC 50 and LC 95 .The Tukey test was applied to the results obtained in the stability study.

Results and Discussion
The ITU level obtained for the new Bti formulation identified as BioUel, with A. aegypti larvae, was 960 (960 ITU/mg).BioUel has an ITU level comparable to commercial products and to products cited in the literature (Sharma et al., 2008) Bioassays evaluating BioUel efficacy to control C. quinquefasciatus larvae had LC 50 of 0.271 (± 0.39) ppm, and LC 95 of 0.634 (± 0.099) ppm (Table 2).For A. aegypti larvae, LC 50 was 0.332 (± 0.042) ppm and LC 95 was 0.694 (± 0.073) ppm (Table 3).These results suggest higher sensitivity in C. quinquefasciatus larvae.It is known that the control efficiency of insect disease vectors depends on formulation type and feeding behaviour of the mosquito larvae, in addition to the species susceptibility to the Bacillus toxin.As we applied the same formulation on species C. quinquelasciatus and A. aegypti kept in a container with less feeding space, it is supposed the LC50 and LC95 results were different for each species, due to differences in each species susceptibility.
The fermentation product was evaluated in bioassays, and the mean LC 50 for A. aegypti larvae was 4.81 ppm and 0.14 ppm, respectively, before and after being filtered.The mean of 0.33 ppm obtained for the BioUel (Table 3) shows a loss of potency of about 2.4 times, probably due to the interaction between formulation ingredients and protein crystals.A loss of 1.5 times was actually expected.
Studies to understand the lethal action of commercial or new formulations of bio-insecticides with Bti-based active principles are conducted regularly (Ibarra et al., 2003;Praça et al., 2004;Lima et al., 2005;Osborn et al., 2007).However, the target-species in experiments vary according to the mosquito species of major health concern in each region.The type of formulation used also depends on the species.This diversity makes comparison of results difficult.Rabinovitch et al. (1999) tested a new isolate of Bti, called LFB-Fiocruz 710, with an LC 50 of 0.03 mg/L for Aedes albopictus (Skuse, 1894), whereas for Anopheles darlingi, Root, 1926 the LC 50 was 4.8 mg/L.Nayar et al. (1999) tested Vectobac ® against larvae of Culex nigripalpus (Theobald, 1901) and found 0.131 and 0.208 ppm for LC 50 must be conducted to determine the formulation potency in international toxic units (ITU), as well as its efficacy and shelf life.
The ITU for the liquid formulation of BioUel was calculated by bioassays using larvae of Aedes (Stegomyia) aegypti (Linnaeus, 1762) and its efficiency and stability was compared with commercial products in the laboratory, with bioassays using larvae of A. aegypti and Culex quinquefasciatus Say, 1823.
The liquid formulation as concentrated suspension was produced by mixing Bti primary powder with detergent, emulsifier, sun block and dispersal agents for easy scattering and slow sedimentation.

Determination of potency
The experiments were conducted in the Laboratory of Entomology, Department of Animal and Plant Biology, UEL, using early fourth instar larvae of A. aegypti and C. quinquefasciatus obtained from eggs kept at the lab insectarium.
The BioUel potency (ITU/mg) was evaluated following procedures described by WHO (1999) comparing with the International Standard IPS-82, for titration, against laboratory-reared A. aegypti larvae.A battery of bioassays was performed with five repetitions for each of six different concentrations tested and one untreated control, performed at 25 ± 2 °C.For IPS-82 the dose consisted of 0.04, 0.03, 0.02, 0.01, 0.008, and 0.005 mg/L.Distilled water was added to reach 150 mL, where 25 A. aegypti larvae were added.For the BioUel suspension, doses were 0.5, 0.3, 0.23, 0.17, 0.10 and 0.06 mg/L.Initial bioassays were also conducted with the fermented solution for the determination of the adequate concentration of crystals.
Larval population densities were assessed 24 hours after treatment and data were submitted to Probit analysis (Micro Probit program) to determine the LC 50 of BioUel.The potency in ITU of BioUel was calculated using the following Equation 1: ) 15.000 ITU / mg standard x LC mg / L of standard ITU / m g LC mg / L of test product test product = (1) of storage.Five months later, the loss reached 35.4% and 26.3%, respectively (Table 4).Significant mean difference was observed for LC 50 in the following storage months, both for A. aegypti (ANOVA, F = 285.6,p < 0.01) and C. quinquefasciatus (ANOVA, F = 125.4,p < 0.02).There was no significant difference in LC 50 means between the second and the third months for A. aegypti larvae, and between the fourth and the fifth storage months for C. quinquefasciatus larvae (Tukey, p < 0,05) (Table 4).Product stability was maintained for three months.In a large country like Brazil, a product with three months of stability can be used for regional but not large scale production, for economic reasons.
So, for three months of storage, the effectiveness of BioUel is comparable to products available on the market.and LC 90 , respectively; for larvae of Aedes taenorhynchus (Wiedemann, 1821), LC 50 and LC 90 were respectively 0.208 and 0.374 ppm.According to these results, both species, C. nigripalpus and A. taenorhynchus, were more susceptible to the bio-insecticide than the two species used in our study, C. quinquefasciatus and A. aegypti.As they are classified in the same genera, are genetically close, and share the ability to colonise receptacles in urban areas, this comparative analysis can be allowed.
All the formulated commercial products should have a shelf life with no more than 20% potency loss (FAO and WHO, 2002).According to Thiery and Hamon (1998), the stability of IPS-82 has been regularly tested to evaluate its larvicidal activity and potency, and the yearly overall mean had a coefficient of variation lower than 20%.In the present study, BioUel lost potency against C. quinquefasciatus and A. aegypti by 8.7 and 17.6%, respectively, in the first month

Table 1 .
Products containing Bacillus thuringiensis israelensis and respective potencies.

Table 2 .
Lethal concentrations (LC 50 and LC 95 ) of BioUel using early fourth instar larvae of Culex quinquefasciatus at mean room temperature of 25 ± 2ºC and room photoperiod (n = 25 larvae).

Table 3 .
Lethal concentrations (LC 50 and LC 95 ) of BioUel using early fourth instar larvae of Aedes aegypti, at a mean room temperature of 25 ± 2 °C and room photoperiod (n = 25 larvae).

Table 4 .
Susceptibility of early fourth instar larvae of Culex quinquefasciatus and Aedes aegypti to BioUel stored for up to 150 days (LC 50 in ppm).