Insect rearing
The creation started from larvae and pupae collected in an organic kale plantation in the municipality of Realeza, Paraná, Brazil. The stock rearing was kept at a temperature of 25 ± 2 ºC, relative humidity of 60 ± 2%, and photoperiod 12/12 in the laboratory 101 of the Universidade Federal da Fronteira Sul, campus Realeza, Paraná. The procedure for rearing of P. xylostella follows the methodology proposed by Barros et al. (2012).
Production of the Seaweed-biopolymer Solution
The production of the seaweed-biopolymer solution (SbS) consisted of three steps: the first refers to the production of the L. calcareum solution, the second consists of the production of the biopolymer solution, and finally, the third step corresponds to the interaction of the biopolymer solution with the L. calcareum solution, thus obtaining the SbS. For the production of the L. calcareum solution (Step 1), 5g of seaweed flour were diluted in 250 ml of acetic acid (P.A). The solution was heated to 40ºC, under constant agitation for 24 hours to the complete extraction of the compounds, filtered and reserved. Then (Step 2), 0.1g of guar gum (biopolymer) were added to a volumetric flask, which was calibrated with 100 ml of distilled water and kept under constant agitation for 24 hours at room temperature for the complete solubilization of the biopolymer. After, to miniaturize the particles in solution to the nanometric scale, the biopolymer solution was subjected to ultrasound treatment in an ultrasonic processor model MARCONI, UP 200S with a large gauge tip, for 3 minutes and amplitude of 100%, since results obtained in previous tests indicate this as the best time for miniaturization of guar gum. Finally (Step 3), the complexation of the solution of L. calcareum and guar gum was carried out, in which a solution at concentration of 10% was prepared, which consisted of the complexation of 1 ml of the solution of L. calcareum to 9 ml of the solution of nanoparticles of biopolymer forming the SbS. From this SbS, new solutions at concentrations of 1.25%, 2.5%, and 5.0% were prepared which were subjected to agitation for approximately 4 hours and then used in the tests with P. xylostella. The chemical description and analysis of the SbS can be found in Almeida et al. (in preparation).
In each test (see below) the SbS at the mentioned concentrations and their respective controls (also in the same concentrations 1.25%, 2.5%, and 5.0%), were used, being them, i) solution only of biopolymer, ii) solution only of flour seaweed, iii) only acetic acid solution and, iv) distilled water as a negative control, totaling 13 treatments.
Contact Test
For the contact test, 10 larvae of second instar of P. xylostella were placed in Petri dishes, along with previously sanitized kale leaf discs (8 cm² Ø). Sanitation was performed in a 5% sodium hypochlorite solution for 5 minutes, then the leaves were rinsed in running water and dried with filter paper. Subsequently, with the aid of a 100 ml spray bottle, approximately 0.324 ml of the SbS as well as the controls were sprayed onto the larvae in each plate, at a 30 cm distance. The larvae were evaluated under a stereoscopic microscope every hour after the contact with the solution until 16 hours of exposure to verify whether there was mortality. Mechanical stimuli were performed on the larvae with a brush, which was only considered dead when no movement was observed.
Ingestion Test
For the ingestion test, kale leaf discs (8 cm² Ø) previously sanitized as described above, were immersed in the SbS as well as in their respective controls, for 60 seconds and placed on filter paper to remove excess solution.
Then, 10 larvae of second instar of P. xylostella were kept in Petri dishes and fed daily with kale leaf discs, also treated daily with SbS, as described above, until pupae were obtained. After 24 hours of obtaining the pupae, 35 pupae from each treatment were weighed using a Shimadzu AUY2020 precision electronic scale with five decimal places. All pupae obtained were individually transferred to test tubes until the emergence of adults, which were then sexed through terminalia differentiation (Capinera 2008). Then, six couples were formed with adults of up to 24 hours of emergency for each treatment. The couples were transferred to laying cages containing cotton soaked in a 10 mg/ml honey solution, a filter paper disc, and one cabbage disc (8 cm² Ø). The cages were monitored daily, and the laying discs were replaced daily for four days (Ferrreira et al. 2021) to verify the fecundity of the females by counting the number of eggs. The couples were kept in cages and fed, following the rearing methodology, being followed up until their death to verify their longevity. In addition, the following parameters were evaluated: larval viability, which was determined by the percentage of larvae that reached the pupal stage; pupal viability, corresponding to the percentage of adults that emerged from the pupae and, pupal weight (Silva et al. 2019).
In each of the tests (contact and ingestion), for each of the 13 treatments, three repetitions were performed, each repetition being represented by three Petri dishes containing 10 larvae in each, totaling 30 larvae per repetition and a final N of 1,170 larvae of P. xylostella used in each experiment.
Data Analysis
The results obtained from the tests were submitted to statistical analysis performed in the Past4Project® statistical program. Data normality was assessed using the Shapiro-Wilk test. Confirming the non-normality of the data, the non-parametric Kruskal-Wallis test was performed, followed by the Mann-Whitney test.