The bioassays, as well as the rearing of T. pretiosum and C. cephalonica, were conducted in the Laboratório de Biologia e Criação de Insetos (LBCI) of the Departamento de Ciências da Produção Agrícola (Fitossanidade), Faculdade de Ciências Agrárias e Veterinárias, UNESP, Jaboticabal, SP, in controlled conditions of temperature (25 ± 2°C), relative humidity (70 ± 10%) and daily photoperiod (12 h light and 12 h dark).
Rearing of the host Corcyra cephalonica
The eggs of C. cephalonica were obtained from the LBCI stock rearing, and a parallel rearing was conducted to supply the eggs needed for bioassays using a diet consisting of wheat germ (94%), sterilized at 150°C for two hours, and beer yest (6%) (Bernardi et al. 2000). The diet was arranged in 47.0 cm x 29.5 cm x 10.5 cm plastic containers, distributed in four parallel grooves and in direction of the length of the container 0.15 g of eggs, with subsequent closure with plastic lids containing central holes covered with voile-type fabric, measuring 6.0 cm x 8.0 cm. The newly emerged adults were collected daily using a vacuum cleaner adapted with a capture chamber built with a PET bottle and PVC pipe. The collected adults were transferred to cylindrical glass containers (11.0 cm in diameter x 17.0 cm in height) containing a piece of sombrite type-screen folded in “Z” (substrate for oviposition), the container being closed with the same type of screen. The eggs were collected daily by inverting the adults’ container on a white plastic tray, tapping the bottom of the container with the palm of the hand, so that the eggs adhered to the laying substrate would detach and fall into the tray. The eggs were sieved to remove impurities, part of which was used to maintain the rearing itself and another part to carry out the bioassays. The eggs produced and momentarily not used were stored in a refrigerator (temperature close to 10 ºC) (Jacob and Cox 1977).
Rearing of the parasitoid Trichogramma pretiosum
The parasitoids used in the bioassays came from the LBCI stock rearing, and their maintenance was carried out according to the methodology described by Parra (1997). Before making the cards with C. cephalonica eggs, they underwent a non-viability process with exposure to the germicidal lamp for 45 minutes. Then, the unviable eggs adhered to cards of sky-blue paperboard (3.5 cm x 1.5 cm), with fixation on double-sided adhesive tape (2.5 cm x 1.2 cm), were exposed to parasitism, with the cards positioned inside flat-bottom test tubes (2.0 cm in diameter x 8.0 cm in high), containing adults of T. pretiosum newly emerged. On the surface of the inner side of the tube, a droplet of honey was added to feed the parasitoids. The card was replaced every 24 hours, for five days, and those with parasitized eggs were transferred to another similar tube, with the tubes always being sealed with PVC film, with the emergence of adult insects in these tubes.
Assessment of parasitism capacity
Females of T. pretiosum were individualized in 30 Duran glass tubes (3.0 cm in length x 0.90 mm in diameter) and sealed with PVC film. For each female, 30 eggs of C. cephalonica were offered, aged between 0 and 24 h, adhered to cards of sky-blue paperboard. The cards were replaced every 24 hours for the entire longevity of the females, being identified and maintained in the same conditions. Daily it was determined: the number of parasitized eggs, accumulated percentage of parasitism, total number of parasitized eggs per female, female longevity and percentage of emergence of adults. The design used was completely randomized design (CRD), and the data analysis was conducted with the SAS® Software. For the analysis, the data were submitted to Bartlett’s tests to verify homoscedasticity (PROC GLM), and Cramer von Mises test for normality (PROC UNIVARIATE). As the data did not present such requirements, the transformation at x + 0.5 was used for the Analysis of Variance (PROC ANOVA). The means, when significant, were compared using the Student Newman Kells test (p > 0.05).
Functional response of Trichogramma pretiosum with Corcyra cephalonica eggs
Females of T. pretiosum were individualized in 140 flat-bottom test tubes (2.0 cm in diameter x 8.0 cm in high) and sealed with PVC film. For each female, eggs were offered between 0 and 24 h of laying and in densities of 1, 2, 4, 8, 16, 32 and 64, adhered to cards of sky-blue paperboard. The cards were removed after 24 h of exposure to parasitism, with 20 repetitions per egg density. The assessments were carried out after 10 days, determining the number parasitized eggs and the percentage of emergence of adults.
Subsequently, the random equation of functional response proposed by Rogers (1972) was used, calculating the handling time (Th) and attack rate (a), with the construction of the functional response equation. The parameters were determined by the graphical linearization of the values of the number of parasitized eggs (x-axis) and the natural logarithm (Ln) of the proportion of remaining eggs (y-axis). Using the slope of the straight line divided by 24 h, the attack rate (a) (eggs/h) was obtained in hours. Dividing this value by the slope of the line gave the handling time (Th), also measured in hours. The estimated parameters of attack rate (h-1) and handling time (h) were compared between densities using the confidence interval (CI) at 95% probability, differing the means in which the CI did not overlap.
The predation data were submitted to Student’s test to determine the significance among treatments, using the SAS® Software, with the functional response determined by means of the logistic regression of the proportion of eggs consumed according to the original densities, using different models, using PROC CATMOD, with the random parasitism equation adjusted to the results by PROC NLIN (Juliano 2001).