Asian Pac J Trop Biomed 2014; 4(9): 695-700 695

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Document heading doi:10.12980/APJTB.4.2014APJTB-2014-0178 2014 by the Asian Pacific Journal of Tropical Biomedicine. All rights reserved. 襃 Toxicity and antifeedant activity of essential oils from three aromatic grown in Colombia against Euprosterna elaeasa and Acharia fusca (Lepidoptera: Limacodidae)

Ricardo Hernández-Lambraño, Karina Caballero-Gallardo, Jesus Olivero-Verbel* Environmental and Computational Chemistry Group, Faculty of Pharmaceutical Sciences, University of Cartagena. Cartagena, Colombia

ARTICLE INFO ABSTRACT

Article history: Objective: ( ) nardus Cymbopogon To determi nflexuosuse the biologica lCymbopogon effects of ess emartiniintial oil s EOs isolated from Received 3 Apr 2014 , and grown in Colombia against two 4 2014 Received in revised form May LMethods:epidopter a larvae, common pests in the oil palm. A 8 J 2014 ccepted un Specimens were captured in thAchariae field a nfuscad the antifEuprosternaeedant activ ielaeasaty and dermal contact A 24 J 2014 EO (L vailable online un lethality of s were measured against μ and 2 μ epidoptera: ) 0 002 0 600 0 002 8 LResults:imacodi dae at various concentrations . - . L/cm and . - AchariaL/g, res pfuscaectively. Keywords: EuprosternaAl lelaeasa EOs exhibitedCymbopogon strong antif emartiniiedant a nd toxicity activity toward and Elaeis guineensis larvae. oil was the most active against both pest insect species, although all tested EOs were better than the synthetic repellent IR3535 on both insects. Pest Conclusions: Colombian EOs have potential for integrated pest management programs in the oil Lepidoptera palm industry. Defoliators

1. Introduction parenchyma. These negatively affect the competitiveness of oil palm sector, by causing declines in yield, an increase in Elaeis guineensis All the organs of the African oil palm ( the use of agricultural inputs and then increasing costs[2,8]. Euprosterna elaeasa E. Jacquin 1763) can be attacked by insects. Although this Dyar (Lepidoptera: Limacodidae) ( elaeasa Acharia fusca species was originally found in West Africa, the majority of ) and Stoll (Lepidoptera: Limacodidae) A. fusca the pests of economic importance that attacks the are ( ) highlight as insect crops that cause extensive from Tropical America, which adapted to the new crop[1-5]. defoliation in the palm areas[5,6]. The main damage is E. The leaves constitute the main source of food for a diverse caused by the larvae. In fact, larva one specimen of elaeasa 2 number of insect pests. Most of these belong to the order can consume during its larval stage, 50 cm of Lepidoptera but also include various species of Coleoptera leaflet, leaving just the midrib, and an entire colony can A. fusca and some Orthoptera[2,6]. cause up to 80% defoliation whereas a larva of 2 In Colombia, the Lepidoptera insects attack the majority of can consume 350 cm of foliage throughout their lives[1,5]. African oil palm crops[5,7]. All of them are phytophagous in These pests are commonly controlled using chemical the larval stages and are considered as the most important insecticides, but over time, insects have acquired some pests of agricultural crops, by feeding on the leaves and the physiological and behavioral resistance. This has forced ú *Corresponding author: Jes s Olivero Verbel, PhD, Environmental and many plantations to increase the doses of insecticides and Computational Chemistry Group Campus of Zaragocilla, Faculty of Pharmaceutical application frequencies, with serious implications in terms Sciences, University of Cartagena, Cartagena, Colombia. Tel: +57-5-6698180. of production costs, environmental pollution and natural Fax: +57-5-6699771. agroecosystem imbalance[6]. E-mail: [email protected] Foundation Project: Supported by the Vice-Presidency for Research of the Over the recent years, essential oils (EOs) have long been University of Cartagena for the financial aid through the Program to Support Research touted as attractive alternatives to synthetic chemical Groups (2012-2013) and K. Caballero is sponsored by the National Program for Doctoral Formation (Colciencias, 567-2012). insecticides for pest management. This arises from the fact Ricardo Hernández-Lambraño et al./Asian Pac J Trop Biomed 2014; 4(9): 695-700 696 2.2.2. Antifeedant assay that these botanical mixtures reputedly pose little threat to the environment or to human health[9]. A significant The antifeedant activity was assessed through the binary et al number of authors have studied the antifeedant effect of EOs choice method described by Wellsow . using leaves in Lepidoptera[10-14], as well as their toxicity on larvae[15- of oil palm impregnated with EOs[20]. The leaves were cut 17], even though they have also been used as oviposition disc shaped of 2 cm in diameter and weighed using an deterrents[18]. analytical balance to the nearest 0.1 mg (Ohaus Pioneer). μ In this study, EOs from three species of the Colombian EOs were dissolved in acetone and 60 L of respective flora were tested for toxicity and antifeedant activity against solutions where applied on the leaves to produce final A. fusca E. elaeasa μ 2 and , two common defoliators of African concentrations of 0.002, 0.020, 0.200, 0.400 and 0.600 L/cm oil palm plantation in Colombia. on 2 cm discs. A commercial repellent formulation (Stay off Colombia), which contains a 150 mL/L solution [ethyl 3-(N- acetyl-N-butylamino) propionate] (IR3535), was employed 2. Materials and methods as positive control. Ten larvae were individually placed in Petri dishes (9 cm伊1.2 cm) with a single treated or vehicle 2.1. EOs μ control (60 L acetone) leaf disc. After 12 h, the remained leaf fraction was weighted and used to calculate the feed- Cymbopogon nardus (C. nardus) Cymbopogon flexuosus , rate using formula[21]: (C. flexuosus) Cymbopogon martinii C. martinii) and ( EOs FI (%)=[1-(T/C)]伊100 were obtained from plant material (300 g in 0.3 L of water), by Where T=consumption on treated dish; C=consumption microwave assisted hydrodistillation and were characterized of control dish. An FI=100% indicates complete feeding as previously reported[19] at the Research Center of inhibition. Three replicates were used for each tested n Excellence, CENIVAM, Industrial University of Santander, concentration of EO ( =30), and the assays were repeated B T D E ucaramanga. he oils we°re provided by r. lena twice. Stashenko, and stored at -4 C until used for conducting 2.2.3. Contact toxicity assays experiments. Each extraction was repeated in triplicate. The chemical composition of the EOs were presented in the The contact toxicity of the EOs was evaluated using a supplementary information. topical application test[14,17]. Dilutions of the tested EOs (0.1-30.0 mL/L) were prepared using acetone as a solvent. 2.2. Test procedures Each larva was individually weighed using an analytical μ balance (Ohaus Pioneer) and received 40 L of solution per 2.2.1. Experimental units treatment, with acetone alone as the control. Doses used A. fusca E. elaeasa μ μ Third instar larva specimens of and were between 0.02 L/g and 8.00 L/g of larva, and solutions were collected directly from oil palm plantations in the were applied topically to the dorsal surface of the larvae ° ’ ” municipality of Maria La Baja, Bolivar-Colombia (9 58 52 N, using a micropipette. After 24 h exposure dead larvae ° ’ ” 75 17 55 W) where used for the assays (Figure 1). Organisms were counted and data tabulated for mortality assessment. T were stored in glass containers covered with a p°lastic mesh o determine whether the larva was alive or dead, the with a diet of fresh oil palm leaflets at (26依2) C, relative palpation method was utilized (the larva was touched humidity of 70%-85% and photoperiod 10:14 h (light: dark) with a soft painting brush; if it makes any movement, it is and kept under these conditioning until used for assays, considered alive, otherwise it is considered dead)[17]. Five within 96 h after collection. replicates were used for each tested concentration of EO n A B ( =50), and each assay was repeated twice. 2.3. Statistical analysis

The results are presented as mean依SE. The sign obtained in the calculation of FI (%) was employed to qualify the antifeedant (positive) or phagostimulant (negative) action of the EO. FI50 and median lethal dose (LD50) of EOs and their confidence intervals at 95% were calculated using Probit Analysis[22]. Normal distribution and equality between ’ variances were checked by Kolmogorov-Smirnov s and Figure 1. A. fusca E. elaeasa ’ Third instar larva of (A) and (B). Bartlett s tests, respectively. Comparisons of the FI (%) and mean mortality between evaluated EOs and positive control Ricardo Hernández-Lambraño et al./Asian Pac J Trop Biomed 2014; 4(9): 695-700 697 ’ μ 2 were performed using ANOVA, with Dunnett s post-test At concentrations between 0.020 and 0.600 L/cm , there ’ used to compare treated with control group, Tukey s post- were statistical differences between the antifeedant A. fusca μ 2 test to compare between the concentrations of the EOs properties of tested EOs against (0.020 L/cm , t F P μ 2 F P and -test to compare between the concentrations of EOs =16.15; =0.000 2; 0.200 L/cm , =22.26; <0.000 1; μ 2 F P μ 2 F for both pest insects. Statistical analysis was performed 0.400 L/cm , =21.53; <0.000 1; 0.600 L/cm , =34.67; P E. elaeasa with Statgraphics Plus 5.1[23], and Graph pad Prism 5 for <0.000 1; Table 1). However, for , these μ 2 F Windows[24]. differences occurred only at 0.600 L/cm ( =20.57; P <0.0001; Table 1). Post-test analysis revealed that for some concentrations at which there were statistical 3. Results A. fusca C. martinii differences between EOs, for , showed C. nardus C. flexuosus significant differences against and ; 3.1. Antifeedant activity of EOs E. elaeasa whereas for , the only detected difference was C. martinii C. flexuosus μ observed between and at 0.600 L/ 2 A. fusca vs. E. The results of the antifeedant activity assays for tested cm . On the other hand, when comparing elaeasa C. martinii EOs are presented in Table 1. Data showed that at all tested , only the EO of presented greater activity μ concentrations, the EOs presented antifeedant properties on the first, and this happened at 0.020, 0.400 and 0.600 L/ 2 T P T P T P against both examined organisms, with a clear dose- cm ( =3.54; =0.005; =2.97; =0.01; =4.29; =0.002; Table dependent activity (Tables 1 and 2). The maximum feed 1). The activities of tested EOs were compared to that elicited C. martinii A. fusca rate inhibitions were obtained for at the highest by the commercial repellent IR3535. For , only the μ 2 C. martinii C. nardus tested concentration (0.600 L/cm ) with values of 98% and oils from and were significantly greater A. fusca E. elaeasa 88% for and , respectively. than the positive control at concentrations greater than μ 2 E. elaeasa 0.002 L/cm , whereas for , such difference was Table 1 C. martinii A. fusca E. elaeasa registered for at the greatest tested concentration. Feed rate inhibition (%) on and exposed to EOs of three Finally, based on the FI-values (Table 2), the antifeedant different leaves and IR3535. A. fusca C. nardus C. flexuosus C. martinii Pest insect Concentration IR3535 properties of the EOs against decreased in the order μ 2 C. martinii≈C. nardus C. flexuosus E. elaeasa ( L/cm ) > , whereas for A. fusca ≈ 0.002 20依3 18依6 26依10 7依4 C. martinii C. nardus C. flexuosus a d bcd it was > . In both cases, the 0.020 38依3 25依6 59依4 21依4 C. flexuosus a d bd EO 0.200 51依3 37依4 64依1 36依3 isolated from was the least potent. a d bcd 0.400 73依4 54依2 84依4 43依3 a d bcd 0.600 80依3 69依3 98依1 63依6 3.2. Contact toxicity of EOs E. elaeasa 0.002 14依7 9依4 20依5 6依7

0.020 39依6 28依6 43依3 28依4

0.200 49依6 41依3 51依5 38依7 The results of the contact toxicity assays for examined 0.400 65依4 58依3 64依6 44依5 a d bd EOs are shown in Figure 2. All EOs showed toxicity activity 0.600 84依1 71依3 88依2 65依6 A. fusca E. elaeasa n against and , with a clear dose-dependent Values are mean依SE, =6. a: Significant difference between activities of EOs P at a particular concentration, ANOVA ( <0.05); b: Significant difference when toxicity (Table 3). The maximum mortality percentage C. martinii ’ P A. fusca C. martinii compared to , Tukey s post-test ( <0.05); c: Significant difference obtained for was reached with 70%, between pest insects for the activity elicited by an EO at a particular t P E. elaeasa concentration, -test ( <0.05); d: Significant difference between the activity of whereas for it was 63%, also with the same EO at ’ P an EO and the positive control (IR3535); ANOVA, Dunnett s post-test ( <0.05). the highest applied concentration.

Table 3 Table 2 A. fusca E. elaeasa A. fusca E. elaeasa Lethal doses (LD50) at 24 h after of and were exposed with FI50 at 12 h after of and exposed with three EOs and three EOs and positive control (IR3535) at five concentrations. positive control (IR3535) at five concentrations. A. fusca E. elaeasa A. fusca E. elaeasa EO EO χ2 χ2 χ2 χ2 LD50 95% CL Slope LD50 95% CL Slope 50 95 50 95 FI % CL Slope FI % CL Slope μ μ μ μ μ 2 μ 2 μ 2 μ 2 ( L/g) ( L/g) ( L/g) ( L/g) ( L/cm ) ( L/cm ) ( L/cm ) ( L/cm ) C. nardus C. nardus 7.35 6.47-8.61 0.18依0.02 77.5 4.83 4.33-5.52 0.36依0.04 95.5 0.19 0.13-0.25 2.52依0.36 55.53 0.24 0.18-0.29 2.75依0.36 65.07 C. flexuosus C. flexuosus 6.70 5.99-7.64 0.21依0.02 100 5.52 4.83-6.57 0.30依0.04 66.3 0.36 0.29-0.45 2.19依0.34 43.86 0.34 0.28-0.42 2.51依0.35 56.39 C. martinii C. martinii 4.34 3.73-5.05 0.19依0.02 93.0 4.00 3.60-4.52 0.37依0.04 114 0.08 0.02-0.13 3.42依0.43 77.34 0.20 0.14-0.26 2.62依0.36 58.70 IR3535 8 7 03 6 02 8 85 0 33 0 05 46 1 IR3535 0 45 0 37 0 55 2 31 0 35 46 67 0 42 0 35 0 53 2 18 0 34 42 58 > - - - . . - . . 依 . . . . - . . 依 . . . . - . . 依 . . n n 依 500 D 依SE 300 CL C Data of slope are expressed as mean SE. = larvae; CL: confidence limit; ata of slope are expressed as mean . = larvae; : onfidence limit; χ2 Chi χ2 Chi : -square. : -square. Ricardo Hernández-Lambraño et al./Asian Pac J Trop Biomed 2014; 4(9): 695-700 698 A B 80 C. nardus 80 C. flexuosus 80 C. nardus 80 C. flexuosus a 60 a 60 60 a a 60 a a 40 40 a 40 a 40

) a ) a % % ( 20 a 20 a ( 20 a 20 a a a a 0 0 0

t a l i y t a l i y 0

o r -2 -1 0 1 2 -2 -1 0 1 2 o r -2 -1 0 1 2 -2 -1 0 1 2 M 80 C. martinii a 80 M 80 C. martinii IR3535 80 IR3535 a a 60 a 60 60 60 a 40 a 40 40 40 a a 20 20 20 20

0 0 0 0 -2 -1 0 1 2 μ -2 -1 0 1 2 -2 -1 0 1 2 -2μ -1 0 1 2 L ( L ) Log dose ( L/g) Figure 2. A. fuscaog dose E. /elaeasag C. nardus C. flexuosus C. martinii Mortality of (A) and (B) after 24 h exposure to EOs from , , and the positive control (IR3535). P a: Significant difference between activities of EOs and the positive control ( <0.05). Error bars represent the SE.

Based on LD50 values (Table 3), the dermal toxicity of their diverse mode of action, including repellency and A. fusca C. martinii C. EOs against decreased in the order > antifeedant activity, disruption of molting and cuticle, as flexuosus≈C. nardus E. elaeasa . However, for , although the well as retardation of growth and fecundity[27,28]. Recent C. martinii LD50 was lower for , the variability of the data reports indicated a strong antifeedant effect of plant was greater within EOs, with clear overlapping between derivatives and recommended their widespread use as confidence intervals. Interestingly, the positive control, they showed great environmental safety[29-31]. However, IR3535, was not only less potent but also presented lower it should be kept in mind that some EOs may posse efficacy than the examined EOs. neurotoxic effects, evident from their rapid action against A. fusca E. elaeasa After 24 h exposure to EOs, and larvae some pest insects[26]. depicted characteristic behavioral changes, consisting of The present study demonstrated that the EOs isolated C. nardus C. flexuosus C. martinii extreme agitation, random walking and wandering, dieresis from , and , exhibited A. fusca and convulsions, finally leading to paralysis and death. strong toxicity and antifeedant activity toward and A. fusca E. elaeasa C. martinii However, only larvae exhibited discoloration, larvae. The EO from was the most changing their body color to a dark brown (Figure 3). Larvae active against both species, whether it was evaluated as treated with vehicle-control did not show any change. a larvicidal or as a feeding deterrent. In terms of acute EO toxicity and antifeedant properties, tested s were better A B C D E than the synthetic repellent IR3535 on both insects. Cymbopogon The EOs extracted from the genus have been evaluated by numerous authors as repellents and insecticides for protecting crop as well as for preventing from bites[32-36], making this genus a great Figure 3. A. fusca Representative specimens of larva exposed for 24 h [37- C. nardus C. flexuosus C. martinii source of natural repellents of worldwide popularity to vehicle-control and EOs from , , and 39]. The composition of these oils has been previously IR3535. C. nardus C. flexuosus C. martinii [19] A: Vehicle-control; B: ; C: ; D: ; E: IR3535. published , and some of their components, such as citronellal and citronellol have been reported for their ability as contact insecticides, repellents and antifeedant chemicals[12,14,17,37,40-44]. 4. Discussion It should be pointed that synergistic effects of complex mixtures such as EOs are thought to be important in plant Plants with insecticidal properties have been traditionally defense against herbivore predators. Plants usually present used for crop protection, but only recently, the potential for defenses as a set of compounds, thus, complex EOs may be the development of products utilized in pest management more efficient than individual pure compounds[45,46]. applications has been recognized[25]. In general, EOs Although several extracts and EOs isolated from this are mostly considered nontoxic to vertebrates[26]. On the genus have been evaluated against other insect species. other hand, they act as broad spectrum pesticides due to This is the first report showing the use of EOs to control Ricardo Hernández-Lambraño et al./Asian Pac J Trop Biomed 2014; 4(9): 695-700 699 A. fusca E. elaeasa Schinus and . These promising results should essential oils from leaves and fruits of pepper tree ( molle Sitophilus oryzae Chil J encourage the development of field tests to validate these L.) to control rice weevil ( L.). Agric Res 69 results, with the aim of to be included together with other 2009; (2): 154-159. effective control options, in the management of defoliator [10] B hathal SS, Singh D, Singh S, Dhillon RS. Effect of crude root Inula racemosa Saussurea lappa insect in crop of African oil palm. oils of and on feeding, survival Spodoptera litura and development of (Lepidoptera: Noctuidae) Eur J Entomol 90 larvae. 1993; : 239-240. Conflict of interest statement é [11] L arocque N, Vincent C, B langer A, Bourassa JP. Effects of Tanacetum vulgare tansy essential oil from on biology of Choristoneura rosaceana J Chem We declare that we have no conflict of interest. oblique-banded leafroller, . Ecol 25 1999; (6): 1319-1330. [12] I sman MB. Plant essential oils for pest and disease Acknowledgements Crop Prot 19 management. 2000; (8-10): 603-608. á í í [13] G onz lez-Coloma A, Mart n-Benito D, Mohamed N, Garc a- The authors would like to thank the Scholarship Program Vallejo MC, Soria AC. Antifeedant effects and chemical “ é for Young Researchers and Innovators Virginia Guti rrez composition of essential oils from different populations of ” Lavandula luisieri Biochem Syst Ecol 34 de Pineda from Colciencias, the Vice-Presidency for L. 2006; (8): 609-616. Research of the University of Cartagena for the financial [14] K oul O, Singh R, Kaur B, Kanda D. Comparative study on the aid through the Program to Support Research Groups behavioral response and acute toxicity of some essential oil Helicoverpa (2012-2013). K. 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