Dicksonia sellowiana Hook. and Nepholepis cordifolia (L.) Lellinger extracts as potential green pesticides: insecticidal activity Extractos de Dicksonia sellowiana y Nepholepis cordifolia (L.) Lellinger como posibles

Known as green insecticides, natural plant-based products have become a promising alternative to conventional insecticides. The primary objective of the present study was to analyze, under laboratory conditions, the insecticidal activity of crude ethanol extracts of the fern species Dicksonia sellowiana and Nephrolepis cordifolia against Oncopeltus fasciatus (Hemiptera). Fern leaves were collected from Itatiaia National Park (Brazil), dried and ground using 96% ethanol, with the extract concentrated in a rotary evaporator. The crude extract was used to prepare a 50 mg/mL solution, with acetone as solvent. Qualitative analysis of the terpenoid and phenolic substance profile in the extracts was performed by thin-layer chromatography (TLC). Fourth-instar insects were used, in groups of 10 animals per treatment, with three repetitions. Four treatments were used: D. sellowiana and N. cordifolia extracts, acetone control and water control. Then, 1 μL either of the respective solution was topically applied to the insect abdominal cuticle. After 21 days, all the insects in the water control group had reached adult. The N. cordifolia extract was responsible for 63% (p < 0.0001) of insect mortality around 16 days after treatment, whereas the D. sellowiana exhibited 50% (p < 0.0001) on the 21 day post-treatment. The extracts also caused delays in insect molting and metamorphosis. The D. sellowiana and N. cordifolia extracts exhibited 18% similarity in the terpenoid profile and 0% for Research, Society and Development, v. 9, n. 8, e120985182, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i8.5182 3 phenolic substances. N. cordifolia and D. sellowiana show potential for research on selective biodegradable substances for use as green insecticides.


Introduction
There are an estimated 5 to 10 million different species of insect (Lewinsohn et al., 2012). Despite their economic and ecological importance, approximately 10% of insect species are considered pests worldwide, causing significant damage to major crops and heavy production losses (Zucchi et al., 1992), in addition to being harmful to domestic animals, humans and other plants (Gallo et al., 2002). Development, v. 9, n. 8, e120985182, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i8.5182 4 Most chemical insecticides available are harmful to non-target organisms, such as humans and wild animals (Rizzati et al., 2016). Given the need for new substances that act efficiently and specifically, with non-neurotoxic mechanisms of action and low persistence, green insecticides have become a viable alternative (Koul et al., 2008). One of the most prominent is insect growth regulators (IGRs), which can be hormonal or chitin synthesis inhibitors. Hormonal IGRs typically function by inhibiting or mimicking the juvenile hormone (JH), the latter causing abnormal reactions such as extranumerary stages in the life cycle, for example, the juvenoids. The aim is to prolong the larval and nymph stages, disrupting development and resulting in sterile adults (Ferreira, 1999). Anti-juvenile hormone agents inhibit JH production, blocking the insect molting (Quistad et al., 1982). Chitin synthesis inhibitors interfere in the formation of the exoskeleton (ecdysis) and chitin production, inhibiting insect development.
Plants produce a variety of substances that can act as IGRs, including essential oils, terpenoids and phenolic substances (Feder et al., 2019). As such, due to their efficacy/dose, environmental safety and lack of human toxicity, plants have become a promising source of green insecticides and an alternative to synthetic pesticides (Tietbohl et al., 2014).
Over the years, Oncopeltus fasciatus (Dallas, 1852) (Hemiptera: Lygaeoidea) has become a popular laboratory species because it is easy to grow and tolerates a wide range of conditions. It has also become one of the most important species worldwide in terms of entomology and physiology-related issues (Chipman, 2017).
The collections were authorized for scientific purposes, under license number 53534-2, issued on 11/28/2016 by the Chico Mendes Institute for Biodiversity Conservation (ICMBio). Part of the botanical material was collected in line with the techniques described by Fidalgo & Bononi (1989) and all the exsiccates were deposited in the Faculdade de Formação de Professores Herbarium (RFFP) at Universidade do Estado do Rio de Janeiro (UERJ), Brazil.

Extract preparation
The leaves of the fern species were dried in an oven at 50°C, then ground and weighed. The dry plant material was macerated in 96% (v/v) ethanol at a proportion of 1L of solvent to 100g of macerated plant for approximately 45 days to obtain the concentrated extract. The crude extract was used to prepare a 50 mg/mL solution, with acetone as solvent.
Each extract was submitted to ultrasound for three minutes to ensure total solubilization in the solvent.

Insect bioassays
The O. fasciatus colonies were established in the Laboratory of Insect Biology of the Universidade Federal Fluminense, which followed the methodology already in place in the lab (Duprat et al., 2017).
To evaluate the toxicity of the extracts, randomly chosen fourth instars of O. fasciatus were used, in groups of 10 insects per treatment, with three repetitions. Four treatments were used: D. sellowiana extract (DS), N. cordifolia extract (NP), acetone control (AC) and water control (WC). Then, 1 μL either of the respective solution was topically applied to the insect abdominal cuticle. Detailed evaluations of the various treatments were made from the day after treatments (1st day) and throughout the time for development from the fourth instars to the adults. Evaluations recorded were the toxicity (mortality over the 24 h following Research, Society and Development, v. 9, n. 8, e120985182, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i8.5182 6 treatment), lethality, range of intermolt period, molting and metamorphosis (Fernandes et al., 2013).

Qualitative analysis of the plant extracts
Qualitative analysis of the extracts was performed by thin-layer chromatography, using chemical markers for terpenoids (beta-sitosterol and fridelinol) and phenolic compounds (rutin and kaempferol). Samples of extract (4 μl) diluted in methanol (50 mg/ml) were applied to silica gel (F254) chromatography plates (SILICYCLE inc.). For the terpenoid plate, a hexane:acetate (7:3) mobile phase and vanillin-sulfuric reagent were used, with reading performed in a darkroom at a UV wavelength of 365nm. An acetate:formic acid:acetic acid:H2O (100:11:11:26) mobile phase and NP/PEG reagent (methanol solution with 1% 2-aminoethyl diphenylborinate (p/v) + ethanol solution with 5% polyethylene glycol (p/v)) were used for the phenolic compound plate, with reading carried out in a darkroom at a 254nm UV wavelength.

Statistical analysis
Data normality was analyzed using the Shapiro-Wilk test. The Kruskal-Wallis and Dunn's post hoc tests were applied in accordance with the PAST program (Paleontological Statistics software package for education and data analysis), version 3.10. Analyses were performed in triplicate, with 3 tubes for each treatment, containing 10 insects each.
Differences between the control groups and treated insects were considered statistically significant when p < 0.05. The p-values were specified throughout the results.

Molting time
After 21 days, all the insects in the water control group (NC) had reached adulthood, ending the experimental model (Table1). No significant differences were observed between the control and treatment groups in the intermolt period (X 2 = 3.993; p = 0.2584). However, molting in controls began two days after treatment, while the experimental groups took an average of 6 days to start molting. Six days after treatment, all the controls had reached 5 th Research, Society and Development, v. 9, n. 8, e120985182, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i8.5182 7 instar nymph, whereas the Dicksonia sellowiana (DS) and Nephrolepis cordifolia (NP) groups took 14 and 9 days, respectively. Metamorphosis onset was at 9 days after treatment in controls and approximately 12 days in the experimental group, but not all the insects reached this stage. These findings indicate a delay in molting.

Development
Analysis of development from 4 th and 5 th instar nymphs showed no significant intergroup differences (X 2 = 3.79; p= 0.2704). However, as shown in Table 1

Qualitative analysis of the plant extracts
The retention factors observed in TLC suggest the presence of 9 terpenoids in Dicksonia sellowiana and Nephrolepis cordifolia; however, similarity of the terpenoid profile between the species was only 18% (Table 2). With respect to phenolic substances (Table 2)

Terpenoids
Phenolic substances insect mortality. Toxicity of a chemical substance to insects does not qualify it as an insecticide; its efficacy even at low concentrations and lack of toxicity to mammals and humans must also be taken into account (Viegas Júnior, 2003).

Rf
According to the retention factors (Rfs) obtained in thin-layer chromatography (TLC), the Nephrolepis cordifolia and Dicksonia sellowiana extracts exhibited different terpenoid and phenolic substance profiles. These substances are described in the literature as having insecticidal action (Corrêa & Salgado 2011). Terpenoids act in plant defense against herbivores and as repellents, toxins or modifiers of insect development (Fürstenberg-Hägg et al., 2013), while phenolic substances repel herbivores by affecting their digestive system with toxic or ovicidal substances, in addition to influencing the photosensitivity of insects (Zagrobelny et al., 2004). Oliveira (2012) reported the presence of phenolic compounds, flavonoids, tannins, steroids, terpenoids and coumarins in aqueous, hydroalcoholic and crude extracts of Dicksonia sellowiana hexane fractions. However, phytochemical tests conducted by Xavier et al. (2016) indicated the presence of alkaloids, steroids, tannins, flavonoids, cardiac glycosides and phenolic compounds in N. cordifolia.

Conclusion
N. cordifolia and D. sellowiana show potential for research on selective biodegradable substances for use as green insecticides. New tests using extracts of these species are needed in order to better understand the physiological mechanisms of insect interaction with the extract components and acquire data that can contribute to research on obtaining more selective substances.