Botanical Extracts of the Brazilian Savannah Affect Feeding and Oviposition of Plutella xylostella ( Linnaeus , 1758 ) ( Lepidoptera : Plutellidae )

The indiscriminate use of synthetic insecticides caused an increase in the resistance of Plutella xylostella (Linnaeus, 1758) (Lepidoptera: Plutellidae) to almost all classes of insecticides, as well as affected the environment and non-target organisms. Thus, the search for less harmful alternatives with the purpose of reducing the application of these insecticides has become a priority. A possible alternative to reduce the use of synthetic insecticides is by using botanical insecticides, given the thousands of existing compounds derived from secondary metabolism in plants. In this study, we tested the following hypotheses: (i) the aqueous and ethanolic extracts of native plants reduce the food consumption of larvae and oviposition of adults of P. xylostella; (ii) these botanical species can act as potential plant insecticides. The objective of this work was to evaluate the effect of plant extracts on the feeding preference of larvae and oviposition of adults of P. xylostella. For this, cabbage discs treated with aqueous and ethanolic extracts of Schinus terebinthifolius, Annona coriacea, Annona crassiflora and Serjania marginata were given to larvae. The concentrations used for the aqueous extracts were of 5 and 10 mg mL, and for the ethanolic extracts were of 1 and 5 mg mL. Both extracts of the four tested plant species showed oviposition suppressed. The extract of S. marginata showed the lowest rates of suppression. All treatments with ethanolic extracts showed a phagodeterrant effect being that the aqueous and ethanolic extracts of A. crassiflora and S. terebinthifolius were the most effective as antifeedants on third instar larvae by P. xylostella.


Introduction
Brassicaceae is one of the most important botanical families in the world economy (Vickers et al., 2004).In Brazil, the members of this family are important in most of the national territory, being cultivated both in family farming and by large producers of vegetables (Aragão et al., 2008).However, considerable losses in production occur because of pest attacks, of which Plutella xylostella (Linneaus, 1758) (Lepidoptera: Plutellidae), popularly known as the diamondback moth, stands out as the most important (Li et al., 2016).As a consequence of the high selection pressure due to the indiscriminate use of insecticides, and the high genetic elasticity inherent in the species, P. xylostella has developed resistance to almost all classes of insecticides, making it difficult to control (Khaliq et al., 2007;Li et al., 2016;Mohan & Gujar, 2003;Sarfraz & Keddie, 2005).
The increase in resistant populations and environmental hazards has forced us to continually search for new ways to protect the crop.One possible way to reduce the use of synthetic insecticides is by using botanical insecticides (Dayan et al., 2009;Lestari et al., 2015;Pavela, 2004;Peres et al., 2017).Botanic insecticides are selective for vertebrates and do not persist in the environment (Isman, 1994) and can be easily produced by farmers and small industries (Talukder & Howse, 1994).According to Prakash and Rao (1997), these botanical insecticides do not contribute to the increase in resistance and pest resurgence; they do not have negative effects to natural enemies (Isman, 1994), and thus, plants could be the best source of new chemical structures for the development of new environmentally correct and safe insect control agents (Saxena et al., 1992).
A study by Couto et al. (2016) evaluating the aqueous and methanolic extracts of the leaves of Annona coriacea Mart.and Schinus terebinthifolius Raddi.showed that these plants negatively affected the feeding of P. xylostella, reducing the leaf consumption of third instar larvae (caterpillars).
A decrease in oviposition and food consumption in insects indicates a future population reduction.Hence, knowledge of the mode of action of plant extracts allows bioactive substances from plants to be more efficiently used.According to Shin-Foon and Yu-Tong (1993), this makes them compatible with the intent of integrated pest management (IPM) programs, especially for crucifers whose economic value and lack period restrict the use of synthetic insecticides.
In some situations (for protection of the crop), it may be more appropriate to use substances that only discourage the action of herbivores, since the elimination of some insects can cause ecological imbalance; in addition, such substances limit the development potential of the insect-pest.The advantage of this is its selective action against parasites and pest predators, as well as pollinators.Plant antifeedant substances prepared from secondary metabolites are found in all groups; however, the most effective insect feeding inhibitors come from terpenoids, alkaloids, saponins, and polyphenols (Koul, 2005) and oviposition reducers come from cumarina and rutina (Tabashnik, 1985) andrographolide (Hermawan et al., 1998) and a-terpineno, Limonene, linalool (Zhang et al., 2004), essential oils (Dover, 1985;Zhang et al., 2004) and plant extracts (Abbasipour et al., 2010;Basukriadi & Wilkins, 2014;Chen et al., 1996;Egigu et al., 2010;Kodjo et al., 2011;Medeiros et al., 2005;Patil et al., 2003).These substances also inhibit the oviposition of P. xylostella.
As the extract of a particular plant may contain thousands of compounds, derived from secondary metabolism, in this study, we tested the following hypotheses: (i) aqueous and ethanolic extracts of native plants reduce the feeding and oviposition of P. xylostella; (ii) these botanical species can act as potential plant insecticides.The present study analyzed the effect of aqueous and ethanolic extracts of Schinus terebinthifolius Raddi (Anacardiaceae), Annona coriacea Mart.(Annonaceae), Annona crassiflora Mart.(Annonaceae), and Serjania marginata Casar (Sapindaceae) (which occur in the state of Mato Grosso do Sul, Brazil) on the feeding preference of larvae and oviposition of adults of P. xylostella.

Development of Plutella xylostella
The larvae and pupae of P. xylostella were collected from cabbage plantation areas in the cities of Dourados (22°13′16″ S; 54°48′20″ W) in the state of Mato Grosso do Sul.The collected P. xylostella were reared at the Insect-Plant Interaction Laboratory of the Faculty of Biological and Environmental Sciences at the Federal University of Grande Dourados (UFGD), Mato Grosso do Sul, Brazil.Individuals were maintained under laboratory conditions of 25±2 °C, 12 hours of photoperiod, and relative humidity of 55±5%.The pupae were placed in transparent plastic cages until the adults emerged.Adults were fed with a 10% honey solution and were provided with an oviposition substrate of cabbage discs measuring 8 cm in diameter placed on moist filter paper.
After oviposition, the leaves holding the eggs were placed in sterile plastic containers measuring 30 cm in length, 15 cm in width, and 12 cm in height.Once hatched, the larvae remained in this container until reaching the pupal stage.The larvae were fed on leaves of organic cabbage (Brassica oleracea var.acephala DC) that were first treated with 5% sodium hypochlorite solution and then washed in tap water.
Young cabbage leaves, located in the third and fourth node, were collected from plants that had 90 days of growth in the organic garden of the UFGD.Healthy cabbage leaves were placed with the adaxial face against the plastic container.The larvae were placed on the abaxial face, and then covered with another cabbage leaf that was oriented with the abaxial face covering them.This procedure was repeated daily or when the leaves were wilted, whichever occurred first, and continued until pupae formation (Barros et al., 2012).For the experiments carried out in this work were used individuals of the first generation obtained from the laboratory creation.

Botanical Material
Healthy and fully expanded leaves of S. terebinthifolius, S. marginata, A. coriacea, and A. crassiflora were collected from the garden of medicinal plants at the Federal University of Grande Dourados and from the farms Coqueiro (forest) and Santa Madalena (Cerrado) in the city of Dourados-MS (22°14′ S, longitude 54°9′ W and altitude 452 m), between 7 am and 9 am, between March and May of 2012.Authorization for collection of botanical material was granted by the Brazilian National Research Council (CNPq)/Council of Genetic Heritage Management (CGEN/MMA, number 010220/2015-1).
The plant species were identified by a specialist from the laboratory of Applied Botany, and exsiccated samples were deposited at the Herbarium of the Federal University of Grande Dourados, Mato Grosso do Sul, Brazil, with the following registration numbers: Annona coriacea: DDMS 5419, Annona crassiflora: DDMS 5499, Schinus terebinthifolius: DDMS 5688, and Serjania marginata: DDMS 5561.

Preparation of Aqueous and Ethanolic Extracts
The leaves were dried in a forced circulation oven for three days at a maximum temperature of (40±1 °C).After this period, the dried leaves were crushed until a fine powder was obtained.
The maceration technique was used to prepare the aqueous extract (AE), in which 10 g or 5 g of vegetable matter (leaf powder) were dissolved in 100 mL of distilled water and after manual shaking, they were allowed to rest for 24 hours in a refrigerated place in order to extract the hydrosoluble compounds.Which were then strained through a voile fabric to obtain the AE solutions at concentrations of 10% and 5%.
To prepare the ethanolic extract (EE), samples of 100 g of the powder were placed in a beaker along with 1000 mL of solvent (ethanol).Filtrations were performed every 2 days for 15 days.The filtered extract was concentrated in a rotary evaporator at 60 °C under reduced pressure.The product obtained in this process was dissolved in distilled water at concentrations of 1 mg mL -1 and 5 mg mL -1 for subsequent tests.

Test with Aqueous and Ethanolic Extracts on the Feeding of P. xylostella
The free choice tests occurred in an environment at 25±1 °C, 55±5% RH and photoperiod of 12 hours.The cabbage discs (4 cm in diameter) were placed in a Petri dish cross-shaped distributions and equidistantly (Figure 1), two of them immersed in extract and the others immersed in distilled water.Three larvae of third instar of P. xylostella were placed on each plate.The instar was identified by the width of the cephalic capsule of the larvae (0.33-0.44 mm).After 24 hours, the insects were removed and the leaf area was scanned, and the images measured using the software ImageJ (Schneider et al., 2012).The leaf consumption was obtained by the difference between the initial area of the leaf and the area that remained after larval feeding.

Test With Aqueous and Ethanolic Extracts on the Oviposition of P. xylostella
Three adult pairs (one male and one female) of P. xylostella of up to 12 h of age were introduced into the cages and kept there for four days for oviposition.They were fed a 10% honey solution.Egg counting and disk exchange were performed daily for a period of 4 days.The same experimental procedure was performed for the ethanolic extracts.The experiment was kept in laboratory in the following conditions: temperature of 25±1 °C, relative humidity of 55±5%, and 12 h photoperiod.Note.*Means followed by the same letter in the column do not differ from each other by the Tukey test at 5% probability.

Discussion
Plant compounds can act in several ways, especially in a complex mixture of active molecules that can affect insects.Studies have shown that organic compounds of plants with insecticidal activity can act as inhibitors of insect feeding, and can affect growth, development, reproduction, and diapause (Freitas et al., 2014;Menezes, 2005).In this study, the active substances acted as antifeedants, that is, they inhibited the larvae feeding activity and oviposition suppressants, that is, they inhibited the moth to oviposit.
The results obtained from the food preference tests carried out in this study showed that the aqueous extract of S. marginata served as a feeding stimulant for the larvae.On the other hand, the aqueous and ethanolic extracts of S. marginata showed the highest indexes of suppressed oviposition.The species of the genus Serjania and their biological properties are little studied.Nevertheless, the scientific literature describes Serjania species to possess different biological properties, such as gastroprotective, antifungal, analgesic, anti-inflammatory, antioxidant, anticancer, and antiprotozoal activities.Phytochemical studies on plants of the genus Serjania reveal the presence of a range of compounds, including saponins, flavonoids, terpenes, steroids, tannins, alkaloids, and fatty acids (Heredia-Vieira et al., 2015).
In the current study, a phagostimulant effect and suppressed oviposition by S. marginata was observed when the concentration was increased; hence, it is assumed with increasing concentration there was an increase in stimulant substances, which may have caused a greater feeding stimulus and reduction in oviposition in leaves treated with the extract in question.Périco et al. (2015) reported that plants of the Sapindaceae family have saponins and tannins.The high saponin content of Sapindaceae fruits has foam-forming properties and can be used as an insecticide (Fernandes et al., 2007), which may cause an inhibitory effect on P. xylostella oviposition.Saponins are substances mainly related to the defense system.The amphiphilic behavior of saponins and their ability to form complexes with steroids, proteins, and phospholipid membranes allow for diverse biological actions (Schenkel et al., 2001).
Tannins are metabolites that act to significantly reduce the growth and survival of many herbivores.They act as repellents to a wide variety of animals; their toxicity is attributed to the ability to form nonspecific proteins in the digestive tract of herbivores, negatively affecting their nutrition (Taiz & Zeiger, 2013).Simpson and Simpson (1990) claim that high consumption of certain extracts is not always advantageous to the insects, because an increase in the inhalation of allelochemicals can result in harmful effects on their development.Thus, we can suppose that the food stimulation caused by S. marginata on P. xylostella larvae and the induction to reduce oviposition, could lead to a decrease in the populations of this pest, since it can result in a reduction of the insect population.
The leaf extracts of S. terebinthifolius, A. coriacea, and A. crassiflora were not as effective as the extract of S. marginata, but they also showed an oviposition-inhibiting effect.The suppression of oviposition occurred due to the presence of secondary metabolites extracted from these plants.
The oviposition of P. xylostella occurs through olfactory and gustatory stimuli (Ang et al., 2016) through chemoreceptors in the ovipositor, tarsus, or mouth (Feng et al., 2017).The secondary metabolites present in the tested plant extracts act to reduce the oviposition stimuli of moths.Secondary metabolites belonging to the Brassicaceae family that stimulate diamondback moth oviposition, mask the action of glucosinolates and cause irritability in females when they are in contact with treated surfaces (Mordue & Blackwell, 1993).Egigu et al. (2010) showed that oviposition by P. xylostella moths is significantly reduced when using the methanolic extract of Cordeauxia edulis (Fabaceae), suggesting that this extract also regulates the pest population, protecting the plants against possible damages.Previously Qiu et al. (1998) and Charleston et al. (2006) reported deterrent effects of neem (Azadirachta indica A. Juss.) and syringa berrytree (Melia azedarach L.) extracts on P. xylostella oviposition.There are receptor cells present in the olfactory sensilla of the antennae that are sensitive to inhibitory compounds (Qiu et al., 1998).These receptor cells prevent oviposition by signaling to the moths that the substrate is not suitable for oviposition and development of their offspring.
In this study, the inhibition of oviposition of P. xylostella was directly related to the increased concentration of aqueous extracts, independent of the plant species used, demonstrating that the inhibiting effect on oviposition is accentuated by the quantity and type of extracted substances.
In relation to food preference, the aqueous and ethanolic extracts of A. crassiflora and S. terebinthifolius are more effective as antifood for P. xylostella third instar caterpillars.
In a recent study, Krinski et al. (2014) showed the effectiveness of Annona as insecticidal plants for different insects.The secondary metabolites corresponding to the insecticidal activities of Annona belong to the class of acetogenins.These substances inhibit NADH in the mitochondria, provoking mortality in the insects (Zafra-Polo et al., 1996).The bioactivity of acetogenins can vary significantly depending on the plant species as well as the solvent used in the extraction (Chirinos et al., 2007;Shaalan et al., 2005).
The effects observed in S. terebinthifolius can be explained by the presence of secondary metabolites, including tannins and flavonoids (Johann et al., 2010).Tannins inhibit digestion by deactivating digestive enzymes, creating a tannin-protein complex that is difficult to digest, thereby affecting the growth and survival of insects (Mello & Silva-Filho, 2002).Procópio et al. (2015) showed that the leaf extract of S. terebinthifolius caused damage to the midgut of Aedes aegypti (Linnaeus, 1762) (Diptera: Culicidae) larvae, interfering with survival and development.
Thus, the ethanolic and aqueous extracts of the leaves of A. coriaceae, S. terebinthifolius and A. crassiflora acted to reduce feeding of P. xylostella larvae; The oviposition preference tests showed that the aqueous and ethanolic extracts of S. marginata were more effective as a suppressor of the oviposition of this insect pest.
Therefore, the knowledge of how plant extracts act on oviposition and feeding can be an important tool in IPM for reducing the population of these insects before economic damage occurs.Consequently, this study serves as a basis for future studies on P. xylostella that aim to reduce the applications of synthetic inputs and the damage caused by this pest without promoting the selection of resistant individuals.

Table 1 .
Index of oviposition suppression (IOS) and egg mean by treatment of aqueous extractNote.*Means followed by the same letter in the column do not differ from each other by the Tukey test at 5% probability.

Table 2 .
Index of oviposition suppression (IOS) and egg mean by treatment of ethanolic extract