Effects of Saponins Extracted from Passiflora passion fruit of Passiflora family on the development of Spodoptera rugiperda (J.E. Smith) (Lepidoptera, Noctuidae)
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https://www.eduzhai.net International Journal of Plant Research 2012, 2(5): 151-159 DOI: 10.5923/j.plant.20120205.03 Effect of Saponin Extracted from Passiflora alata Dryander (Passifloraceae) on development of the Spodopterafrugiperda (J.E. Smith) (Lepidoptera, Noctuidae) Marianna Pilla D’Incao1,2, Gre ice Gosmann2, Vilmar Machado1,*, Lidia Mariana Fiuza1, Gilson R. P. Moreira2 1Laboratório de M icrobiologia e Toxicologia Universidade do Vale do Rio dos Sinos , UNISINOS, Av. unisinos, 950 CEP 93022-000, São Leopoldo, RS. Brasil 2Instituto de Biociências, Universidade Federal do Rio Grande do Sul, UFRGS. Av. Ipiranga, 2752, 90610-000, Porto Alegre, RS, Brasil 3Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, UFRGS.Av. Ipiranga, 2752, 90610-000, Porto Alegre, RS, Brasil Abstract Thesaponins are glycosides with wide distribution among plants which may be to xic for herbivorous arthropods. Their insecticidal activ ity may be associated to the ability of producing alterat ions in the feeding behavior, in the mo lting process and causing death. This study evaluated the lethal and sub lethal effects of a saponins extract, obtained fro m Passifloraalata on Spodopterafrugiperda, by ingestion tests with artificial diet. Nine solutions with increasingextract concentration and a control solution with sterile distilled water were used in the test. In total 1500 insects were used in 5 repetitions, 30 replicates per concentration. Considering the total mo rtality, all treat ments differed statistically fro m the control one, but not among them. Most of the observed effects were sub lethal, in which 68.3% insects presented deformation. The nu mber of deformed insects per treatment increased as the extract o f saponins concentration increased. In conclusion the mortality revealed significant difference between control and other treat ments, which indicated the potential of saponins present in P. alata as a control agent of S. frugiperda. This potential needs to be better evaluated, especially if the facilities of raising and large scale production of the plant are considered. Keywords Saponin, Spodopterafrugiperda, Lethal Effects, Fall Army worm, Passifloraalata 1. Introduction Th e res earch es d es ign ed at id en t ify in g n ew t o xic substances in o rder to con tro l ag ricu ltu ral p lagu es are const ant in th e act iv it ies relat ed to man ag emen t an d integrated control of plagues nowadays. This is partially a resu lto f t he kno wledg e o f t he negat iv e imp acto f th e chemical p roducts used in the ecosystem as well as the possibility of evolution of the resistance of the target insects to the products used, whether these are chemical and/or biological. A great number of these researches aredirected to the evaluat ion o f to xicity of secondary metabo lite of plants , s uch as phenols , alkaloids , glucos inolates , cyan ogen ic g lycos id es and sapon ins .1M ore th an 2000 species of plants are kno wn to possess some insecticidal * Correspondingauthor: email@example.com (Vilmar Machado) Published online at https://www.eduzhai.net Copyright © 2012 Scientific & Academic Publishing. All Rights Reserved activity. In many cases plants have a history as traditionalmedicines or are used to kill or repel insects. In spite of the various studies available about the entomotoxic effects of extracts of different species of plants, their application is still incip ient.2 The saponins are glycosides with detergent properties, due to the presence of hydrophobic (aglycones) and hydrophilic (sugar) co mponents with wide distribution among plants which may be to xic forherb ivorous arthropods such as mites, beetles and Lepidoptera, among others.3-7Their insecticidal activ ity may be associated to the ability of producing alterations in the feeding behaviour, in the molt ing process, of interacting with hormones that regulate the growth and causing death in the different stages of development.8-11Their wide spectrum of action reaches a great number of herbivores and its amplitude of physiological impacts makes these substances an excellent model for the study of the effect of natural substances with insecticidal activ ities.8 The passion fruit,Passifloraalata, is one of the main species of economic importance of the gender cultivated in 152 M arianna Pilla D’Incao et al.: Effect of saponin extracted from Passiflora alata Dryander(Passifloraceae) on development of the Spodoptera frugiperda (J.E. Smith) (Lepidoptera, Noctuidae) Brazil, wh ich is used mainly as fruit and for juice and sweets production as well as a med icinal plant.1216Thesaponins are the main substances of its secondary metabolism. Five types of saponins were isolated and identified in its leaves: one is a steroid and four are triterpenic(3-6).17-18 The fall armyworm Spodopterafrugiperda (J. E. Smith) (Noctuidae) is a polyphagous insect, using more than 50 species of plants as food supplies, distributed in over 20 families,19 predominantly in g rasses, among them corn and rice, which are important cultures for Brazilian and world economy. In rice,S. frugiperda is considered a pestof the initial phase, since it attacks the seedlings in the beginning of their develop ment, feeding on the leaves and cutting the new stalk close to the ground,20causing losses of 14 to 24% of the grains. Depending on the population level, the destruction of the crop can be total.21In maize, the attack can occur fro m the seedling stage until the tasseling and the formation of spikes. In the later attacks specimens between the stalk and the cob can be found, where they penetrate the female inflorescence destroying the grain.22 The effect of saponins extract of some plants on the development of the Spodopteraspecieswas demonstrated in studies with S. litura.2,4,23-27The results of these studies indicate alterations in the feeding behaviour, lengthening of the larvae and/or pupa stages, increased mortality and reduced fertility. The study of plants with potential insecticidal act ivity is important to discover new active molecules, which may be isolated fro m p lants or synthesized, or a molecu le-prototype for structural changes to obtain a more act ive compound. This work evaluated the effects of saponins extracts of PassifloraalataonSpodopterafrugiperda, identifying the sub lethal and lethal effects of these substances in different development stages of this species. 2. Material and Methods collaboration of researchers at the Estação Experimental do Arroz (EEA) of the InstitutoRiograndense do Arroz (IRGA), located in Cachoeirinha, RS. The insects were reared in the room for the creation of insects of the Laboratory of Microbiology of the Un iversity of Vale do Rio dos Sinos (UNISINOS), heated to 26°C, 75% RH and 12h photo phase. The adults were kept in plastic cages, fed with a glucose solution of 10%. Eggs were collected three times a week and put in gerbo x with d iet. Five or six days after hatching, the larvae were individualized in plastic cups, and maintained with art ificial Po itoutdiet,28 where they remained until they became pupae. As a routine procedure adopted, the pupae were identified, separated by sex, kept in sleeves with moistened filter paper and covered with tulle, until e mergence of adults. 2.3. Bioassay For the test with S. frugiperda nine solutions with increasing concentrations of saponin ext ract were used due to the 2x (312, 625, 1250, 2500, 5000, 10000, 20000, 40000 and 80000pp m) and a control of sterile distilled water. A solution of 20ml was prepared for each concentration, which, after use, was stored at 4°C. In this case, the extracts were diluted in sterile d istilled water. The second instar larvae of S. frugiperda were maintained for seven days in mini acry lic plates (35mm diameter) containing Po itout diet, where 100μL of the solutions were applied on the diet. After thisthey were transferred to plastic cups containing only artificial diet, where they were observed until emergence of adults for the evaluation of possible sub lethal effects (Figure 1). Damages were evaluated under stereoscopic microscope and classified according to intensity (low, mediu m and high) (Figure 2). Bioassays were kept at 26°C, 75% RH and 12h photo phase. Five repetitions of 30 larvae/concentration, totalizing 1500 insects were performed. 2.4. Data Analysis 2.1. Preparati on and Characterizati on of the Saponin Extr acts To obtain saponin ext ract the dried leaves were submitted to macerat ion with 70% ethanol. The ethanol was filtered and placed in a rotary evaporator; the aqueous residue was extracted successively with chlo roform, ethyl acetate and nbutanol. The evaporation of n-butanol fraction resulted in a fraction consisting main ly of saponins, which will be called henceforth saponin extract.17Thesaponin extract is characterized as a yellow-orange powder. 2.2. Obtenti on and Rearing of Insects Data were tested for normality using the chi-squared and Shapiro-Wilks and the homogeneity with the tests of Hartley and Bart lett. When these criteria were satisfied, they were compared by an ANOVA followed by mult iple tests of Tukey. Otherwise, they were co mpared by KruskalWallis follo wed by Dunn's mult iple test. Statistical analysis was performed with the aid of TOXTAT Soft ware version 3.3.29To compare mo rtality rates between larvae and pupae of S. frugiperda the Student t test was conducted for different means and to analyse the deformation of the same species a simp le linear regression was made.30 The establishment of S. frugiperda began with immaturelarvae co llected in rice plantations, with the 3. Results International Journal of Plant Research 2012, 2(5): 151-159 153 Figure 1. Characterization of the sublethal effects observed in Spodopterafrugiperda. A) Larva with abdominal distension; B) Larva with the point of necrosis in the integument; C) larva with incomplete molting process; D) Pupa with globular evagination in the integument; E) dead adults emerging from pupae; F) Pupa with failure in the process of sclerotinization of the integument; G) Pupa with morphological features of larvae; H) Pupa with the point of necrosis in the integument; I) Adult with the wings unfolded e J) Adult with wings slightly distended and pupae attached on the wing Figure 2. Characterization of the intensity of deformation in Spodopterafrugiperda. A) Larva without deformation; B) Pupa without deformation; C) Adult without deformation; D) Larva with deformation of low intensity; E) Pupa with deformation of low intensity; F) Adult with deformation of low intensity; G) Larva with deformation of medium intensity; H) Pupa with deformation of medium intensity; I) Adult with deformation of medium intensity; J) Larva with deformation of high intensity; L) Pupa with deformation of high intensity e M) Adult with deformation of high intensity 154 M arianna Pilla D’Incao et al.: Effect of saponin extracted from Passiflora alata Dryander(Passifloraceae) on development of the Spodoptera frugiperda (J.E. Smith) (Lepidoptera, Noctuidae) Table 1. Summary results of the experiments indicating mortality levels by treatments and stage of development Treatment s Larvae Mortality tot al % Pupae Mortality tot al % Cumulative Mortality tot al % Larval Period - days Pupal Period - days mean mean Control 5 3 0 0 0 0 Treatment 1 58 37 26 17 84 56.0 Treatment 2 44 29 23 15 67 44.7 Treatment 3 59 39 25 17 84 56.0 Treatment 4 59 39 22 15 81 54.0 Treatment 5 54 36 39 26 93 62.0 Treatment 6 40 27 31 21 71 47.3 Treatment 7 48 32 39 26 87 58.0 17.61 29.18 20.23 26.41 21.1 25.59 18.78 20.19 10.2 10.69 9.79 9.92 10.51 8.87 9.57 9.48 Treatment 8 44 29 24 16 68 45.3 18.47 9.31 Treatment 9 30 30 39 26 69 46.0 18.78 9.75 Table 2. Summary results indicating the number of individuals with deformations by stage of development and treatments Larvae P upae Adult s Tot al Single Deformations T1 T2 T3 T4 T5 T6 T7 T8 T9 1 1 3 4 9 7 8 8 10 25 4 46 3 46 1 20 32 32 25 36 24 30 29 42 23 38 39 33 51 34 42 43 53 Mult ip leDefo rmat ion s T1 T2 T3 T4 T5 T6 T7 T8 T9 0 00 00 0 0 0 0 1 10 02 6 9 2 1 0 0 1 0 1 10 2 2 3 1 1 1 0 3 16 11 4 4 The quantitative and qualitative results of the experiments are indicated on tables 1 and 2, respectively Considering the total mo rtality (larvae and pupae) all treatments differed statistically fro m control but not among them (Figure 3) (p<0.05). Co mparing the mortality of larvae with the mortality of pupae in each treatment, it was observed that the rate of larval mortality was higher than that of the pupae from the control concentration until the concentration 2500pp m. At higher concentrations (5000 to 80000 pp m), these rates showed no significant difference (Figure 4). There were 441 o f larvae and 268 of pupae deaths, i.e. 8.1% and 29.4% of o rganisms exposed to saponin, respectively, totalizing 17.9% of the insects used in bioassays. Of the 441 larvaethat died during the tests, 121 (27%) died with some deformity, of which 45 (37.2%) died during the process ofmolt, unable to release in who le or in part of mo lting, 8 (6.6%) had points of necrosis and 5 (4.1%) some parts of the body distended Of the 268 dead pupae, 107 (39.9%) died with some deformity, of which 27 (25.2%) had retention of morphological characters of larvae, 88 (82.2%) d ied during the emergence of the adult failing to release in whole or in part, 5 (4.7%) had deformit ies in the form of globular evaginations of the cuticle, 23 (21.5%) had points of necrosis in the cuticle and 11 (10.3%) sclerotinizat ion failures, leaving the internal organs exposed. Some specimens had more than one type of deformation, such as evaginations and points of necrosis (2.93%), or points of necrosis around the failu res of the cuticle (0.97%). Often, when reviewing the experiments, the pupae with failure sclerotinization were found still alive, being dead in 70% alcohol immediately to avoid the suffering of the indiv idual. The main deformation observed in 599 adults obtained after the bioassays was in the reg ion of the wings, where 200 (33.4%) insects showed little or no distension of the wings. A total of 1025 insects (larvae, pupae and adults) were observed withdeformations (76% of organis ms) (Figure 5). These organisms were screened according to the intensity of their deformat ions: 612 (59.7 %) had deformities of low intensity, 144 (14.05%) of average intensity and 269 (26.2%) of high intensity (Table 3) The linear reg ression obtained from the number of deformit ies observed in insects exposed to the saponin extracts, was a straight up line, wh ich shows that there is a tendency to increase the number of deformed insects as the concentration of extract saponins increases (Figure 6).. International Journal of Plant Research 2012, 2(5): 151-159 155 Figure 3. Total mortality (mean ± standard error) of Spodopterafrugiperda after bioassay of ingestion in different concentrations of saponin.Columns followed by different lett ers are significant ly different (ANOVA, followed by Tukey's mult iple t est s α=0.05) Figure 4. Mortality of larvae and pupae of Spodopterafrugiperda after ingestion test of saponin at different concentrations. Columns followed by different letters differ significantly at a given concentration (Student'st test, α = 0.05) 156 M arianna Pilla D’Incao et al.: Effect of saponin extracted from Passiflora alata Dryander(Passifloraceae) on development of the Spodoptera frugiperda (J.E. Smith) (Lepidoptera, Noctuidae) Figure 5. Deformations (mean ± standard error) observed in Spodopterafrugiperda, after ingestion test of saponin, divided into three categories (low, medium and high), by concentration of saponin Fi gure 6. Linear regression of the number of deformat ions observed in Spodopterafrugiperda aft er ingest ion t est of different concentrat ions of saponin Table 3. Summary results of the damage levels by each treatment Damage Level Low T1 T2 T3 T4 T5 T6 T7 T8 T9 82 69 65 65 62 61 59 74 75 Medium 19 20 13 9 11 18 16 16 12 High 8 9 26 24 37 39 46 32 44 Total 109 98 104 98 110 118 121 122 131 In summary the deformit ies were observed in 76% of organisms exposed to saponin. These deformations were lin ked, primarily, to the process of mo lt, as 45 larvae of S. frugiperda died during mo lting process, failing to release in whole or in part of molted cuticle. Besides the problems related to the process of molt, the organis ms that had points of necrosis in the larvae stage were located mainly in the thorax and the pupae stage and adults, in most cases, in the region of the wings. Moreover, problems were observed in sclerotinization of pupae, which opened up large holes in the region of the wings, leaving the interior of organis ms exposed. In organisms of control treatment no deformities were observed. International Journal of Plant Research 2012, 2(5): 151-159 157 4. Discussion and Conclusions This study identified the impact o f saponins extract on the different stages of development of S.frugiperda.Besides the mortality of larvae and pupa, the presence of structural deformit ies was observed in all stages. When considered the total impact of the treatments 47.4%of the adults emerged without structural problems identified by optical microscopy. The effects of the saponin extract on S. frugiperdamay be associated to a deterrent toxic act ion and/or by its interactions with substances responsible for the different stages of development of the specie. The deterrent effect reduces the consumption of food producing nutritive deficiency causing deficient growth or deformit ies, wh ich may lead to death or inhibit the progress for the ne xt stage.26,27,31,32Among the alterations observed, the presence of necrosis points stood out, sclerotinizat ion problems and retention of larvae characters and tumors.These results are similar to the ones registered,21for the biotypes of corn and rice of S. frugiperdausing different extracts of different species of plants.It is important to highlight that alterations in the length of the larvae and pupal stages were not observedin this study, as registered for S.littoralis23e for S. frugiperda.33The differences in these results may be associated to the variation in the composition of saponins among the plant species used for the acquisition of the e xtracts.3,34 The physiological basis of the to xicity of saponins has not been fully elucidated, but we know that there is a great interaction between them and the cell membranes, with effects on the hydrophobic-lipophilic balance and permeab ility of these because they are capable of forming complexes such as sterols, for instance, cholesterol.10,11,35The toxicity of saponins on arthropods may derive fro m their ability of interacting with free steroids of the intestine and/or of inhibit ing the digestive proteases, reducing the rates of digestion and absortion.8,35,36 The mortality and deterrence caused by saponin were also observed in experiments that evaluated the effects of diffe rent types of saponin for aphids,38,39,42nematodes,4042beetles31,43,44 and Spodoptera sp.2,5,6,25,27 A fact that drew attention was the appearance of specimensin the intermediary period between larvae and pupae (1.8% o f exposed organisms). Apparently, the organisms began the process of becoming pupae and could not complete it, which led to death. The appearance of intermediate indiv iduals between pre-pupae and pupae can occur when the activity of juvenile hormone, which controls the metamorphosis, is affected.32 Furthermore, the metamorphosis may have been affected because of saponins form co mplexes with sterols, like cholesterol, both in the intestine in cell membranes of insects. These complexes formed by saponin make sterols unavailable, influencing the synthesis of ecdison, one of the hormones involved in the process of molt. It has been shown that insects are unable to produce these sterols, re moving them entire ly fro m the diet.10,11,34,36,39 The statements of these authors may account for the large number of deaths of larvae and pupae during the processes of molt and emergence of pupae malformed, beyond the failures of sclerotinizat ion observed. In conclusion the mortality of S. frugiperda showed significant difference between control and other treatments. It was observed that the death of the specimens was not immed iate after the ingestion of saponin, but slow and gradual, which apparently occurred under the influence of saponin in the metabolism of the larvae, pupae and adults. These results indicate the potential of saponins present in P. alataas a control agent of S.frugiperda. This potential needs to be better evaluated, especially if the facility of raising and large scale production of the plant is considered. ACKNOWLEDGMENTS We acknowledge the CNPq by the financia l support to do this research at Biology Postgraduate Program of UNISINOS REFERENCES  Tagliari M S, Knaak N and Fiuza LM . Plantas inseticidas: interações e compostos. PesquisaAgropecuáriaGaúcha 10: 101-111, 2004.  Ulrichs C, M ewis I, Adhikary S, Bhattacharyya A and Goswami A. 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