Efficacy evaluation of spinosad bioinsecticide capsules suspension for the control of Helicoverpa armigera

for control of H. armigera. The experiment was conducted in three different groups treated separately with emulsifiable concentrate (EC) with 48% of spinosad; suspension capsules (SC) with 48% of spinosad; and control group treatment with deionized water. Caterpillars were used in the early stage (1 and 2 days old larvae), and maintained under laboratory conditions (24 ± 1°C, 65 ± 5% RH and a 12:12 h light: dark photoperiod). The mortality was recorded and the affected behavior of treated larvae was checked every 30 min for a period of 8 h. The results show the efficacy of spinosad for control of H. armigera in the emulsifiable concentrate group treatment at 92.6 and 88.9%, and in the suspension capsule group treatment at 88.9 and 87.0%, respectively, in the first and second bioassay. It was concluded that the spinosad 48 g i.a. ha -1 is efficient in the control of H. armigera in micro encapsulated formulations, promoting less environmental damage by reducing the level of pesticides in the environment by the smaller concentration of the active ingredient through capsule suspension formulation and revealed economic viability and promotion of extended release of active ingredients.


INTRODUCTION
Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) belongs to the subfamily, Heliothinae.It has broad occurrence throughout the world, being found in various countries from Asia-China and India, Africa-Benin, Cameroon and Nigeria, Europe and Oceania-Australia (Brévault et al., 2009;Perini et al., 2016).Female H. armigera generally lays around 1,000 to 1,500 eggs and the hatching takes place between five and seven days after the laying (Czepak et al., 2013).H. armigera larvae eats leaves, stems, buds, fruits and pods, it causes damage in the vegetative and reproductive plant stages and serious economic crop damage in Brazil, mainly in the corn, cotton and soybean that are important crops for Brazilian agriculture (Pomari-Fernandes et al., 2015).Bueno and Sosa (2014) has reported that in Brazil, the damage caused by H. armigera in 2012/2013, reached approximately $0.8 billion.
Population growth of the Helicoverpa as well as losses of the production systems were caused by following a cumulative process of inadequate cultivation practices and successive planting of host vegetable species (corn, soybean, etc.) in very extensive management contiguous areas with non-judiciars use of high toxicity pathology category agrochemicals has contributed to resistance development in H. armigera, which resultand in heavy crop losses each year (Vijayabharathi et al., 2014;Specht et al., 2013;Suzana et al., 2015).
The success of the control in pest management depends on the choice, stability and acceptance of after years of use, in which chemical control was used the most (Samri et al., 2015).It was considered that abusive use and lack of technical criteria can result in problems, jeopardizing sustainability by the development of plague resistance to defensive products (Obopile, 2006).
Due to their effectiveness and availability, the products mostly used in farming for biological control of plagues in crops are strains of pathogenic bacteria like Bacillus thuringiensis, whose pathogenicity has already been well established against demonstrated insect pest of lepidopteron, coleopteran, dipteran, arthropod and nematode orders (Faten et al., 2011).Among other biopesticides which have been exploited in predators crop management is spinosad, an aerobic fermentation of Saccharopolyspora spinosa products spinosyns, which is found to show a broad spectrum activity as an insecticide for the control of insects of the lepidopteran and dipteran species (Amiri-Besheli, 2009).
Pesticides are conventionally applied to crops by periodic broadcasting or spraying.Very high and possibly toxic concentrations are applied initially, and decrease rapidly in the field to concentrations that fall below the minimum effective level, for example, the emulsifiable formular.As a result, repeated level applications are needed to maintain pest control.Knowles (2008) mentioned that the insecticide formulations used are normally in the form of emulsifiable concentrate, in which the active ingredient is solubilized in hydrocarbons and other synthetic solvents, and associated with surfactants and emulsifiers to stabilize the emulsion on the aspersion tanks.
According to Liu et al. (2016), the encapsulated or release-control formulations present better benefits when compared with emulsifiable concentrates, due to reduced levels of hydrocarbons on this formule, longer action time of the active ingredient, reduction of the evaporation rate of the insecticides.All these specifications are important for the reduction of the level of pesticides in the environment by the smaller concentration of the active ingredient and, consequently, less damage to the plants, non-target insects and humans, lower photodegradation, hydrolysis, bacterial decomposition and leaching.
The aim of this study was to evaluate the efficiency of the insecticide, spinosad in the form of suspension capsules in comparison with the bioinsecticide, in the form of emulsifiable concentrate, in the concentration of 48% active ingredients in the dosage of 48 g i.a.ha -1 for control of H. armigera.

MATERIALS AND METHODS
The current research was conducted in the bioterium of Universidade Positivo, Curitiba, PR, Brazil.Caterpillar eggs of H. armigera were used; acquired from the Company Promip Manejo Integrado de Pragas.After the eggs have hatched, the larvae were placed in plastic cups (diameter of 15 cm, and depth of 0.94 cm).The plastics cups were kept in glass terrariums with an approximate area of 10 x 10 x 20 cm and adapted to the environment for 12 h.The glass terrariums were maintained in acclimatized room, with a temperature of 24 ± 1°C and relative humidity of 70 ± 10% and a 12L:12D photoperiod, absence of ventilation and artificial feeding.These conditions were maintained during all the periods of the experiment.
In this study, the concentration of spinosad for the control of Helicoverpa sp. in soybean crop was used; Tracer®, that is, a commercial product used in Brazil for the control of Helicoverpa sp.(Perini et al., 2016).It is used in the concentration of 48 g active ingredient per hectare (equivalent to 100 mL.ha -1 ).
In the form of emulsifiable concentrate, the bioinsecticide was formulated through a mixture of active ingredients, emulsifiers and solvents (Knowles, 2008); in the form of suspension capsules, it was made through the interfacial polymerization method by coacervation in liquid phase (Benita et al., 1984); and deionized water was used for aspersion in the negative control group.
Three experimental groups were formed, each group comprised three replications, each treatment contained 18 larvae of first instar stage (1 day).The groups were treated with spinosad in the form of emulsifiable concentrate (EC) with 48% of spinosad, 46% of C9 solvent (AB9 ) and 6% of calcium dodecylbenzene sulfonate; spinosad in the form of suspension capsules (SC) with 48% of spinosad, 41.8% of deionized water, 7% of amine, 3% of ethoxylated fatty alcohol and 0.2% of xanthan gum; and control group (CG) was treated with deionized water.For the aspersion of the insecticides formulations (SC, EC and water), a glass sprinklers was used.
The formulations were observed in a digital microscope with an increase of 1600 times.A micrometric slide for biological microscope of 0.10 mm scaled in 0.01 mm was used in order to confirm the formation of mycelium in the bioinsecticide formulation of the emulsifiable concentrate, as well as the formation of microcapsules in the formulation of suspension capsules.Spherical capsules that are characteristic in encapsulation and micelle formation in the emulsifiable concentrate are shown in Figure 1.
Data on mortality and mobility were collected by means of observations carried out every 30 min for a period of 8 h, aiming to measure control results within a time interval similar to the interval that can be observed in the field, using natural luminosity; initially through the performance of a first bioassay and then by a second bioassay (24 h later).The number of dead larvae was noted down and the mobility of the larvae was observed from the record of photographs.Efficacy in each treatment was calculated according to Abbott (1925): (1)  Where, E (%) is the efficacy of the treatment, n1 is the average number of living moths after treatment (EC and SC) and n2 is the number of living moths in control (CG).
The statistical analysis was carried out by the piece of software ACTION, as supplement to Excel software.The treatments were submitted to distribution and normality analyses by the Anderson-Darling statistical method.As the distribution presented linearity, Tstudent test was also applied.The groups treated with spinosad in the form of EC, SC and CG were compared and considered as statistically different when they presented value of p≤0.05.

RESULTS AND DISCUSSION
To prevent problems with the pulverization of the formulated substances (density must be lower than 1.0 g mL -1 ), the bioinsecticides were subjected to density analysis, with a result of 0.93 g mL -1 at 25°C for the product in the form of emulsifiable concentrate and 1.03 g L -1 at 25°C for the microencapsulated product.
The increase of the mortality of caterpillars can be observed in Figure 2, which refers to the control group (A), the group treated with spinosad in the form of emulsifiable concentrate (B), and the group treated with spinosad in the form of encapsulated suspension (C).
In relation to mortality, after the data analyses of the groups, EC and SC presented a significant difference in relation to the control group, where no death of caterpillars was observed.The treatments made in groups EC and SC, when compared, also showed significant differences between them in the bioassay.Significant differences were observed between the efficiency indexes in the mortality of the H. armigera moths in the early stage when treated with spinosad in the form of emulsifiable concentrate and suspension capsules.
After 8 h from applying the bioinsecticide, the efficiency in mortality of the H. armigera caterpillars was obtained as follows: in the form of EC, it was 92.6 and 88.9%, respectively, for the first and second bioassays; and for the moths treated with the bioinsecticide in the form of SC, it was 88.9 and 87.0%, respectively, for the first and   second bioassays.
According to Tsunoda and Nishimoto (1986), the value of efficiency must be above 80% so that a chemical formulation can be considered efficient.In this manner, the satisfactory efficiencies in the group EC were obtained from the intervals of 150 and 210 min, respectively, after applying the bioinsecticide for bioassays 01 and 02 with the values of the average and standard deviations of 14.0 ± 1.1 and 16.0 ± 0.6.For the group SC, satisfactory efficiencies were obtained after applying the bioinsecticide at intervals of 210 and 420 min after the treatment with results of the averages and standard deviations, respectively, 15.0 ± 1.0 and 16.0 ± 1.0 (Table 1); this suggests that efficiency in the treatment above 80% took place faster in the control of moths treated with the bioinsecticide in the form of emulsifiable concentrate.
The efficiency index of the SC formulation, when compared with the groups treated with EC, showed a greater toxicity effect found in the components of the EC formulation, that is, lipophilic action of the organic solvents on the moths larvae as greater amount of the active ingredient available in the syrup in the form of encapsulation as most part of the active ingredients at the time of application found within the capsules.Wang et al. (2012) reported that, traditionally, evaluation of the effects of the spinosad against the target-pest is based on the average lethal dose (DL 50 ) or on the average lethal concentration (CL 50 ) of the active ingredients and, in addition to direct mortality, the survived larval after treatment of insecticide can be found to malfunction, resulting in retarded development, fertility and fecundity.These consequences can lead to negative impacts and must be taken into account in the management of integrated handling of the pest.The greater time of bioavailability of the active ingredient in the form of suspension capsules for the length of the time of permanence in the environment can also favor and establish the lethal concentration as necessary for the desired control.Harter et al. (2015) found efficiency of above 80% in up to 7 days after applying spinosad (Tracer®) 0.096 g i.a.ha -1 in the control of Anastrepha fraterculus.Wang et al. (2012) demonstrated the high toxicity of the spinosad in the control of S. exigua in the second instar stage and Wang et al. (2009) reported on the high toxicity of the spinosad for lepidopteron, in corroboration with Sayyed et al. (2012), who showed that the toxicity of the spinosad chlorfenapyr is significantly greater for S. exigua than compounds of the groups of pyrethroids and organophosphates.Tello et al. (2013) presented a significant result of mortality for adults E. paulistus in the concentration of 0.48 mg of i.a.L -1 of spinosad after 12 and 24 h.Spinosad also has the capacity of reduction in weight gain of the moth neonates, possibly associated with the toxicity of the bioinsecticide.
Moths have defense mechanisms such as a short development period, polyphagia, mimicry, thanatopsis and also genetic and biochemical adaptations that favor the maintenance of the species (Mello and Silva-Filho, 2002).In relation to mobility, a low mobility rate of moths was ascertained in the group treated with bioinsecticide spinosad in the form of EC, when compared with the control group and the group treated with the bioinsecticide spinosad in the form of SC; this is shown in Figure 2A and C through the demarcation of the moths on the walls of the containers.Mobility of the moths was ascertained in 100% of the observation period.Figure 2B shows that, with low mobility, the moths held themselves at the base of the container during the entire period of the bioassay.
The reduction of mobility observed in the moths of group EC (Figure 2B) can be associated either with the stress caused by primary dermal irritability of a few formulation components, mainly solvents and a few tensioactives, or with the presence of a greater amount of insecticide in its free form in this kind of formulation (Benita et al., 1984).In the present study, the behavior of thanatopsis was observed, so, the insect was considered dead when it did not move for a period of three minutes of observation after being stimulated.According to Couturier et al. (1996), thanatopsis is the behavioral reaction in the insects that simulate death when threatened.This is a defense strategy presented by insects, and it simulates death by remaining immobile or static, or with the intention of avoiding an attack, predation and death itself.
Thanatopsis can reduce the efficiency of the insecticide, which would have its action lowered by the consumption of leaves and fruits; the feeding of the moths can be retaken after the concentration of the insecticide is reduced through evaporation and/or leaching.
The absence of thanatopsis in the moths treated with the bioinsecticide in the form of suspension capsules can allow for continuity in the ingestion of treated plants, thus favoring the action by ingestion of the bioinsecticide and extending its efficiency, since capsules are more resistant to the evaporation and leaching processes.

Conclusion
The bioinsecticide, spinosad in the form of concentrate emulsion, showed better efficacy with 80% higher efficiency in a shorter time interval after application of the biopesticide for the control of caterpillars, H. armigera when compared with capsules suspension.Microencapsulated formulations of bioinsecticides are a feasible and innovative alternative to control caterpillars, but in this study, the formulation used for capsules suspension did not induce the caterpillars to apparent death (thanatopsis) from environmental and toxicological point of view; the use of spinosad bioinsecticide in encapsulated form showed increased protection when compared with formulations using aliphatic hydrocarbons.

Figure 1 .
Figure 1.Photomicrography of the spinosad bioinsecticide formulation in digital microscopy with an increase of 1600 times, scale 14 µm (a) microencapsulated formulation and (b) emulsifiable concentrated formulation.

Figure 2 .
Figure 2. Activity of H. armigera in the presence of the bioinsecticide spinosad.A) control group, B) group treated with spinosad in the form of emulsifiable concentrate (EC) and C) group treated with spinosad in the form of suspension capsules (SC).The circled areas show the caterpillars on the walls of the plastic containers indicating their constant mobility during the study.
b and c express the significant difference (p<0.05) through the T-student test among the control group (CG), EC and SC.

Table 1 .
Average mortality of H. armigera after eight hours of applying bioinsecticide spinosad in the form of emulsifiable concentrate (EC) and suspension capsules (SC).