Mortality of apple leafroller exposed to different Bacillus thuringiensis Subspecies in artificial diet , in three assessment periods

Bioinsecticides based on Bacillus thuringiensis are the entomopathogen most marketed worldwide. The aim of this study was to evaluate the bioactivity of kurstaki, aizawai subspecies and kurstaki x aizawai hybrid on B. salubricola larvae fed on artificial diet in different assessment periods. Mortality was assessed on the seventh, tenth and fifteenth days after treatment. Microplates containing 150 μl of diet in each cell were used. Suspensions containing Bt subspecies was deposited on the surface of the diet, then neonate larvae were released. The mortality assessment performed after fifteen days of treatment was significant for all three subspecies. Bt kurstaki was more efficient in all assessment periods, except for fifteen days, which was similar to Bt aizawai. Assessments performed up to seven days were more efficient to determine mortality. The three subspecies have influenced the ability for instar change, regardless of concentration. The LC50 of Bt kurstaki on the seventh and tenth days of assessment was from three to six times smaller than that observed in Bt aizawai x kurstaki, and Bt aizawai, respectively.

The evolution of fermentation techniques and isolation of new subspecies allowed the emergence of new efficient formulations for the control of lepidoptera containing kurstaki (Btk) and aizawai (Bta) subspecies.Larval mesentery has ideal pH to promote the action of proteases that activate the toxin, causing the infection of larvae (BULUSHOVA et al., 2011).
The effect caused by Bt differs according to subspecies, each producing specific pro-toxins that act differently on insects.Formulations containing two Bt subspecies, called azaiwai x kurstaki (Btak) transconjugate, were elaborated to increase control efficiency.Another important factor in the use of Bt is the exposure time of larvae to the pathogen, which influences mortality.Schesser et. al (1977) showed that larval mortality may vary according to the assessment period, influencing the result.The assessment period differs among authors, ranging from three (JYOTI and BREWER, 1999), four days (AMARAL et al., 2013), five days (BONCHEVA et al., 2006), seven days (OSTREM et al. 2016) and ten days (SCHESSER and BULLA, 1978).
The aim of this study was to compare the efficiency of three Bt subspecies for the control of B. salubricola in artificial diet and in different periods of mortality assessment.

Material and Methods
Experiments were carried out at the Laboratory of Integrated Pest Management (LAMIP), located in the Sector of Agrarian Sciences of the Federal University of Paraná (SCA-UFPR).
Bonagota salubricola.The population was collected in a commercial apple orchard in Porto Amazonas-PR in 2009.Larvae were grown in tubes (80 mm x 25 mm) containing artificial bean-based diet (PARRA et al., 1995).Pupae were kept in greenhouse with photophase of 14 hours, temperature 24 ± 2 ° C, RH of 60 ± 10% until emergence of adults, which were released in cages (35 cm x 35 cm x 35 cm) containing polyethylene strips for oviposition.
Bioassays.Commercial products based on Bacillus thuringiensis were from kurstaki (Dipel WG, 32 gl -1 , Abbott Laboratories, North Chicago, USA), aizawai (Xentari WG, 540 g.kg -1 , Abbott Laboratories, North Chicago, USA) and azaiwai x kurstaki (Bt) transconjugate (Agree WP, 500 g.kg -1 , Certis, Columbia, USA) subspecies.96-well microplates received 150μl of artificial diet for lepidopterans (Soybean-Wheat, Stonefly Industries, TX).Each well was added of 6 μL of Bt suspension on the diet.Each well received a neonate aged up to three hours after hatching, about 20 minutes after addition of Bt.Three replicates were tested with 24 subjects at each concentration and control.Wells were closed with paraffin waxed paper (Parafilm, Chicago, IL) to prevent larval escape and diet dehydration.Microplates were maintained in BOD (Eletrolab, mod.112FC, São Paulo), RH 60 ± 10%, temperature of 24 ± 2°C, photophase of 14 hours.Seven concentrations measured in part per million (ppm) of the commercial product (p.c.) were tested and defined from preliminary bioassays and adjusted for each Bt subspecies as follows: 118.8,74.3,46 ,4.29,18.1,11.3 ppm;118.3,56.4,26.8,12.8,6.1,2.9 ppm;155.6,86.4,48.0,26.7,14.8,8.2 ppm.Assessment.Mortality was considered in three assessment periods (AP): seven (7), ten (10) and fifteen (15) days after application of insecticides.The stages of surviving larvae were monitored to verify the influence of Bt on their development, identifying the cephalic capsules.Larvae that did not react to the touch of a stylet were considered dead.
Mortality data were submitted to Probit analysis to determine LC 50 (PoloPlus 1.0, Software LeOra, 2003).Mortalities corrected by the Abbott formula (ABBOTT, 1925) in treatments were normalized using the sine-arc function and submitted to factorial analysis, and means were compared by the Tukey test considering significance of P <0.05 (Assistat Software Version Beta 7.6).

Results and discussion
Assessment period.The assessment period of the average mortality of B. salubricola larvae influenced the efficiency of the three B. thuringiensis subspecies (Table 1) (Btak F = 41.75, p <0.001, df = 2, Btk F = 28.30,0.001, df = 2; Bta F = 25.31,p <0.001, df = 2).The corrected mortality of B. salubricola at the highest concentration tested was directly proportional to AP (Btak r 2 = 0.97, Btk r 2 = 0.70, Bta r 2 = 0.80), ranging from 58.3 to 98.5% respectively at seven and fifteen days (Table 2).The results in B. salubricola suggest that assessments performed before seven days may underestimate the efficiency of Bt probably due to the food habit, since larvae of the first three instars consumed little diet, observing galleries of approximately three millimeters in the surface.For largecapacity larvae, evaluations occur earlier (BLANCO et al., 2009) and after seven days (LIAO et al., 2002;YEE et al., 2008); however, Schesser and Bulla (1978) showed differences in mortality rates obtained in different APs.
Development.Monitoring of surviving larvae from the seventh day showed that Bt influences development as a function of subspecies (Figure 1).Analyzing the highest concentration, it was observed that in the seventh day of assessment, most larvae were in the first instar, whereas control larvae were mostly between second and third instar.On average, 70% of larvae in Btak and Bta treatments were found in the second instar on the 10th day, while in Btk, all remained in the first instar, while in the control treatment, larvae occurred in the first four instars, with approximately 60% of them in the third instars.At the lowest concentration, the three Bt subspecies influenced the development of the larvae in relation to the control, although with low mortality rate at seven and ten days.In assessment performed at the fifteenth day, larval escape was observed, reducing the number of samples per treatment (Table 3).Individuals who perforated the parafilm were in the fourth to fifth instars and this generally occurred at concentrations below LC 50 .This behavior occurred in Btak (11.1%),Btk (8.7%) and Bta (7.8%) in the total of treated individuals.In literature, there are reports of mortality assessments up to ten days after treatment (ER et al., 2007;FRANKENHUYZEN et al., 2008) and there are no references for larval escape.  of concentrations: 190, 118.8, 74.3, 46.4, 29.0, 18.1, 11.3 parts per million (ppm) of the commercial product (p.c.); 2 Average of concentrations: 248.5, 118.3, 56.4, 26.8, 12.8, 6.1, 2.9 ppm of p.c. 3 Average of concentrations: 280.0, 155.6, 86.4, 48.0, 26.7, 14.8, 8.2 ppm of p.c. * Means followed by the same lowercase letter in the subspecies and upper case among species do not differ statistically by the Tukey test at 5% probability; 1 concentration expressed as part per million (ppm) of the commercial product (p.c.). 2 number of assessed individuals. 3Mortality corrected according to Abbott (1925).
Interference in larval development is evident through the stages they were in; however, it was not possible to confirm the death of Bt-affected larvae before 15 days.The results indicate a decrease in larval development with the use of sub-lethal concentrations, and this may be related to the behavior of larvae that after Bt ingestion present food reduction or stopping (CÍCERO et al., 2009), not obtaining the necessary energy for instar change.Other authors mention that there is no instar change in larvae due to starvation observed after Bt infection (Monner et al., 2000;Cicero, 2009).However, the concentrations used were high and caused the rapid larval death.Bioassay.The results showed that the death caused by kurstaki subspecies was statistically significant for the three assessment periods (PA7 F = 109.06,p <0.001, df = 2; PA10 F = 21.25,p <0.001df = 2, PA15 F = 77.05,p <0.001, df = 2) (Table 1), except when compared to Bta at 15 days.In PA7, the LC 50 of Btak and Bta were respectively 3.0 and 5.9 times greater than Btk (Table 4).This trend was also observed in the other two assessment periods.For B. salubricola, Btk was more efficient in the control, which may be related to the origin of the population, resistant or susceptible insects exposed to Bt present different mortality rates (Diaz-Gomez et al., 2000).In assessment of leaf injuries caused by Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), Btk was five times more efficient than Bta (GONZÁLEZ-CABRERA et al., 2011), corroborating results obtained in the present work.

Table 1 .
Average mortality (%) of Bonagota salubricola larvae fed on artificial diet with different concentrations of three B. thuringiensis subspecies in different assessment periods.
* Means followed by the same lowercase letter in vertical and upper case in horizontal, do not differ statistically by the Tukey test at 5% of probability.1Average

Table 2 .
Corrected mortality (%) of Bonagota salubricola larvae fed on artificial diet and exposed to different Bacillus thuringiensis subspecies at different assessment periods.

Table 4 .
Lethal concentration that kills 50% of the Bonagota salubricola population (LC 50 ) and angular coefficient in comparison with Bacillus thuringiensis aizawai x kurstaki, B. thuringiensis aizawai in relation to B. thuringiensis kurstaki in different assessment periods.Days after application in the diet; 2 Ratio calculating the LC 50 between Bt aizawai x kurstaki -Bt aizawai and Bt kurstaki Conclusion Bacillus thuringiensis subsp.kurstaki caused higher mortality of B. salubricola larvae when compared to the other subspecies.Post-treatment assessment time influenced the percentage of larval mortality and B. thuringiensis toxins influenced larval development at non-lethal conc