DOI: 10.13102/sociobiology.v63i2.936Sociobiology 63(2): 770-776 (June, 2016) Open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology ISSN: 0361-6525 Toxicity of plant extracts from Bahia, Brazil, to Atta sexdens sexdens (Hymenoptera: Formicidae) workers Introduction Ants of the Atta and Acromyrmex genera (Formicidae: Myrmicinae: Attini) are the true leaf cutting-ants with their species utilizing fresh plant parts, mainly leaves, to cultivating fungus of the Leucoagaricus and Leucocoprinus genera (Agaricaceae: Leucocoprinae) (Della Lucia et al., 2014). The leaf cutting-ants are one of the most significant pests in Neotropical America damaging almost all cultivated plants and causing losses to agriculture, forest and pasture plants (Zanetti et al., 2000a; Zanuncio et al., 2002). Leaf cutting-ants use green leaves what justifies their importance by reducing survival, growth and reproduction of plant of economic value (Zanetti et al., 2000b; Zanetti et al., 2014). Abstract Ants of the Atta and Acromyrmex genera (Formicidae: Myrmicinae: Attini) are the true leaf cutting-ants with species of economic importance in America Neotropical and mainly controlled by toxic baits. There are few active ingredients for use in baits, being necessary studies to indicate molecules with insecticide potential. The aim of this study was to evaluate the toxicity of Aspidosperma spruceanum Benth ex. Mull Arg. (leaf and bark), Casearia arborea (Rich.) Urb. (leaf and branch), Casearia sylvestris Sw. (leaf and bark), Erythroxylum affine A.St.-Hil. (leaf and branch), Esenbeckia grandiflora Mart. (leaf and bark), Ocotea brasiliensis Coe-Teix (bark and branch), Simarouba amara Aubl. (bark), Tabernaemontana bracteolaris Mart. ex Müll.Arg. (leaf, bark and branch) and Zanthoxylum rhoifolium Lam. (leaf and branch) extracts to workers of Atta sexdens sexdens L. (Hymenoptera Formicidae). The contact and ingestion toxicity of all extracts to this ant by was evaluated by topical application and addition in their diet, respectively. Data of contact application were submitted to analysis of variance and Tukey test while those from ingestion were compared by survival curves using the statistical test ¨log rank¨. Through contact, the leaf and branch extracts of Z. rhoifolium and of that of bark of S. amara were the most toxic ones. Through ingestion, four extracts were toxic and showed delayed action. The extract of Z. rhoifolium branches presented the slowest action (S 50 = 10 days). This characteristic is crucial for toxic baits. The Z. rhoifolium leaf and branch extracts were the only ones with contact and ingestion toxicity to A. sexdens sexdens workers. Sociobiology An international journal on social insects MCAR Gomes1, VF Paula1, AA Moreira2, MA Castellani2, GEL Macedo1 Article History Edited by Evandro Nascimento Silva, UEFS, Brazil Received 16 October 2015 Initial acceptance 21 February 2016 Final acceptance 11 June 2016 Publication date 15 July 2016 Keywords Atta sexdens sexdens, bioassays, insecticidal activity, Zanthoxylum rhoifolium. Corresponding author Maria Aparecida Castellani Department of Plant and Animal Science State University of Southwest Bahia BOX 95, 45.083-900 Vitória da Conquista-BA, Brazil E-Mail: castellani@uesb.edu.br Leaf cutting-ants are the main pests in cultivated forests of the Pinus and Eucalyptus genera (Zanetti et al., 2014). Damages by leaf cutting-ants is common in forest commercial cultivation in 2005 in 3.4 million of hectares planted with eucalyptus, 1.8 million with Pinus and 326 thousand with other plant species (Pereira & Santos, 2008). The leaf cutting-ants can be controlled with different methods, but toxic baits are the most practical and efficient one (Laranjeiro et al., 1995; Zanuncio et al., 2000). On the other hand, thermal fogging with plant extracts could be an alternative to these baits, but the costs of equipments with this method is much higher compared to toxic bait application (Zanetti et al., 2014). The active ingredient must be attractive to ants even distant from the nest to assure the efficiency of toxic baits. 1 - Universidade Estadual do Sudoeste da Bahia (UESB), Jequié-BA, Brazil 2 - Universidade Estadual do Sudoeste da Bahia (UESB), Vitória da Conquista-BA, Brazil RESEARCH ARTICLE - ANTS Sociobiology 63(2): 770-776 (June, 2016) 771 Initial rejection of these baits cannot occur and its toxic compound should have delayed action at the right time to completely contaminated the ant colony besides having low toxicity to non- target organisms (Nagamoto et al., 2004; Verza et al., 2006). The sulfuramids (N-ethyl perfluoroctane sulfonamide) and fipronil are the most used active ingredients in ant baits (Zanuncio et al., 2003; Della Lucia et al., 2014). The sulfuramid, categorized in annex B of the Stockholm Convention on Persistent Organic Pollutants (POPs) as an organic pollutant (United Nations Treaty Collection, 2009), is the most used active ingredient in baits to control leaf cutting-ants, what makes urgent searching for active ingredients to replace it. The leaf-cutting ant [(Atta sexdens Forel) (Hymenoptera: Formicidae)] has attracted the attention of researchers (Zanetti et al., 2003) and studies have been developed to searching for plants with toxic substances to this pest (Peñaflor et al., 2009; Gouvêa et al., 2010). The toxicity is due to secondary metabolites in plant that can be toxic to ants, to its fungus (Morais et al., 2015) or to both. Oil of Ricinus communis L. and Jatropha curcas L. seeds with different concentrations were toxic, through ingestion and topical application to A. sexdens, in laboratory tests, thus, suggesting the toxicity of these oils to this ant (Alonso & Santos, 2013). The search for alternatives that are effective to control leaf cutting-ants is important. The objective of this study was to evaluate the toxicity of extracts of several plant species collected in the Bahia State, Brazil, to A. sexdens sexdens workers, through topical application and ingestion, in laboratory conditions. Material and Methods Plant species were collected in June 28th of 2012, in a forest fragment in Brejo Novo Farm (13º56’41”S and 40º06’33.9”W) between 617 m and 755 m of altitude at 9 km from Jequié, Bahia Sate, Brazil. The plant material was dried in Tecnal drying oven (TE 394\2 Model) at 40 °C for 48 h and submitted to cold maceration with methanol. Extracts from different plant parts of the following species represented by their respective abbreviations were prepared: leaf (EFAS) and branch (EGAS) of Aspidosperma spruceanum Benth. ex Müll. Arg.; leaf (EFCA) and branch (EGCA) of Casearia arborea (Rich.) Urb.; leaf (EFCS) and bark (ECCS) of Casearia sylvestris Sw.; leaf (EFEA) and branch (EGEA) of Erythroxylum affine A. St.-Hil.; leaf (EFEG) and bark (ECEG) of Esenbeckia grandiflora Mart.; bark (ECOB) and branch (EGOB) of Ocotea brasiliensis Coe-Teix; bark (ECSA) of Simarouba amara Aubl.; leaf (EFTB), bark (ECTB) and branch (EGTB) of Tabernaemontana bracteolaris Mart. Ex Müll. Arg.; and leaf (EFZR), branch (EGZR) and root (ERZR) of Zanthoxylum rhoifolium Lam. The extract solution was filtered and concentrated under vacuum, in rotary evaporator (Fisatom, 801 Model), at 50 °C. The masses of dry plant materials and their respective extracts and their yields were obtained (Table 1). The biological tests were conducted using A. sexdens sexdens workers collected from several nests in the Myrmecology Laboratory of the Southwestern Bahia State University. Foraging workers of the similar size and width of head capsule between 1.7 and 3.0 mm were selected. Family Species Plant Abbr. Mat. (g) Ext. (g) Yield (%) Salicaceae Casearia arborea Leaf EFCA 91.45 29.91 33 Branch EGCA 135.79 11.58 9 Casearia sylvestris Leaf EFCS 91.08 34.77 38 Bark ECCS 147.4 19.37 13 Erythroxylaceae Erythroxylum affine Leaf EFEA 56.50 13.23 23 Lauraceae Ocotea brasiliensis Branch EGOB 704.75 30.41 4 Bark ECOB 388.48 55.14 14 Apocynaceae Tabernaemontana bracteolaris Leaf EFTB 81.38 26.54 33 Bark ECTB 281.07 32.99 12 Aspidosperma spruceanum Leaf EFAS 54.76 19.16 35 Branch EGAS 152.57 8.35 5 Simaroubaceae Simarouba amara Bark ECSA 362.27 13.07 4 Rutaceae Esenbeckia grandiflora Leaf EFEG 79.57 23.45 29 Bark ECEG 138.78 18.98 14 Zanthoxylum rhoifolium Leaf EFZR 117.04 22.16 19 Branch EGZR 179.78 19.75 11 Root ERZR 122.23 14.58 12 Table 1. Family, species, plant parts (Plant), abbreviation (Abbr.), mass of dry material (Mat.), mass of extract (Ext.) and yield of plant species and extracts used in bioassays with Atta sexdens sexdens (Hymenoptera: Formicidae) workers. MCAR Gomes – Toxicity of plant extracts to Atta sexdens sexdens772 The contact toxicity test (Araújo et al., 2008) was conducted in a completely randomized design with 21 treatments (19 extracts and two controls) with three replications and each parcel having 10 ants. The extracts (Table 1) were diluted in ethanol at the concentration of 1.0 mg.mL-1. Each leaf cutting-ant worker was treated topically with 1.0 µL of this solution on its pronotum using a dosing micro syringe of 10 µL (Hamilton, 701N Model). Two controls were prepared: one without application and another with the application of 1.0 µL of solvent (ethanol). The ants were conditioned in Petri dishes per treatment with a cotton ball soaked in distilled water. The ant mortality was evaluated after 24, 48 and 72 of treatment application. In the ingestion toxicity test the ants were fed on a solid diet composed of (g.L-1): glucose (50), bacteriological peptone (10), yeast extract (1.0) and agar (10) dissolved in distilled water and autoclaved at 120 °C for 15 minutes (Bueno et al., 1997). The experimental diets, except the controls, were obtained by adding the extracts EFEG, ECEG, EFEA, EFZR, EGZR, EFCA, ECCS, ECSA, EFAS (concentration of 0.2 mg.mL -1) to the diet still hot, immediately after its removal from autoclave. The liquid was poured in Petri dishes previously identified and kept under refrigeration. The ant mortality was evaluated in a daily basis during 25 days. The Petri dishes were covered with paper filter, previously damped with distilled water. The diet was replaced daily when 0.4 g of the artificial diet was put per Petri dish. The experimental design was completely randomized with 10 treatments, nine with the extracts and one control (diet without extract) with five replications using 10 ant workers each one. In both tests, the Petri dishes (90 mm diameter) were kept at a temperature of 25 ± 1 °C and relative humidity from 70 to 80%. The data from the topical application test were submitted to analysis of variance and the averages compared by the Tukey test (p< 0.05) using the SAS Institute program (2002). The data from ingestion tests were graphically analyzed comparing the survival curves of each treatment with that of the control by the statistical test “log-rank” using the PRISMA 6.0 program (GraphPad Software). For each treatment, the average survival period of ants (S50) was determined considering the day that 50% of them were still alive. Results and Discussion The topical application of the extracts EFZR, EGZR, ECSA, EGAS and EFCA caused higher mortality of ants than the controls. Ants treated with ECSA, EFCA, EGZR, EFZR and EGAS presented cumulative mortality between 33% and 37% (Table 2). This may be due to secondary metabolites toxics to Treatment Mortality (%) 1º day 2º day 2ºday (cumulative mortality) 3º day 3ºday (cumulative mortality) Control with solvent 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00a Control without solvent 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00a EFTB 0.00±0.00 6.67±9.43 6.67±9.43 15.00±10.80 21.67±16.41a EFEG 3.33±4.71 0.00±0.00 3.33±4.71 3.33±4.71 6.67±4.71 a EGTB 0.00±0.00 3.33±4.71 3.33±4.71 3.33±4.71 6.67±4.71a EGZR 16.67±9.73 24.34±9.55 41.01±15.38 0.00±0.00 41.01±13.21 b ERZR 0.00±0.00 6.67±4.71 6.67±4.71 0.00±0.00 6.67±4.71 a ECEG 6.67±9.43 4.17±5.89 10.83±15.32 3.33±4.71 14.17±12.47 a ECCS 0.00±0.00 0.00±0.00 0.00±0.00 6.67±4.71 6.67±4.71 a EFCA 10.00±8.16 8.33±11.79 18.33±19.29 19.26±7.93 37.59±13.18 b ECSA 6.67±4.71 14.44±5.52 21.11±9.07 16.93±12.25 38.04±13.33 b EFZR 6.67±9.43 21.67±8.50 28.33±14.34 11.11±15.71 39.44±5.47 b EGCA 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 a EFCS 0.00±0.00 0.00±0.00 0.00±0.00 3.33±4.71 3.00±4.71 a ECTB 0.00±0.00 0.00±0.00 0.00±0.00 3.33±4.71 3.00±4.71 a ECOB 0.00±0.00 3.33±4.71 3.33±4.71 7.04±5.00 10.37±8.71 a EGOB 0.00±0.00 0.00±0.00 0.00±0.00 6.67±4.71 6.67±.4.71 a EGEA 0.00±0.00 0.00±0.00 0.00±0.00 6.67±4.71 6.67±4.71 a EFEA 6.67±4.71 3.70±5.24 10.37±8.62 0.00±0.00 10.74±8.17 a EFAS 3.33±4.71 7.41±10.48 10.74±15.19 0.00±0.00 10.74±14.16 a EGAS 3.33±4.71 14.44±13.97 17.78±18.53 21.48±18.17 39.26±12.74 b Table 2. Mortality (%) of Atta sexdens sexdens (Hymenoptera: Formicidae) workers (mean and standard deviation) treated with plant extracts at concentration of 1 mg.mL-1 through topical application. Different letters after the mortality value on the 3rd day, show significant difference in relation to the control (Tukey at 5%). Sociobiology 63(2): 770-776 (June, 2016) 773 A. sexdens sexdens and that these plants need further studies in field with thermal fogging. Besides, secondary metabolites are usually found at low concentrations in extracts and their isolation is necessary to prove their respective activities. The quassinoids, the most active substances used in traditional medicine, is the main chemical components of S. amara and present inhibitory effect (Fiaschetti et al., 2011). Simarouba versicolor was toxic to cutting-ants at concentrations of 2.0, 1.6 and 0.3 mg.mL1 (Peñaflor et al., 2009). Species of Casearia genus present therapeutic properties and active substances as diterpenes, called casearins (Bento et al., 2013). Aspidosperma has species, including A. spruceanum, important sources of indole alkaloids with therapeutic properties (Oliveira et al., 2009). Insecticidal activity for C. arborea, S. amara and A. spruceanum has not been found, but plant extracts may be active against leaf cutting-ants. For instance, Ruta graveolens L. and Ageratum conyzoides L. extracts employed in traditional medicine, induced mortality of A. sexdens workers through topical application, at concentration of 1 mg.mL-1 (Araújo et al., 2008). In tests through ingestion, the extracts of leaves and branches of Z. rhoifolium (EFZR and EGZR), leaves of E. grandiflora (EFEG) and of bark of C. sylvestris (ECCS) caused higher mortality of ants than the control (Figure 1). Results for the EFCA, ECEG, EFAS, ECSA and EFEA extracts were similar to those of the controls. The average survival period (S50) of A. sexdens sexdens workers per treatment varied from 8 to15 days (Table 3). The average survival period (S50) of ants in the EFEG, ECCS, EFZR and EGZR treatments was seven, eight, eight and 10 days, respectively, evidencing their mortality after ingesting them. The values of S50 showed that these extracts had delayed action and therefore, they may be considered for future use in the management of cutting-ants. The delayed toxic action occurs when the mortality of ant workers is lower or equal to 15% up to the first day of evaluation, and higher or equal to 90% after the twentieth first day of evaluation (Nagamoto et al., 2004). The accumulated mortality showed that extracts with difference from control had this characteristic. Further tests need to be develop to implement these extracts in field. An extract, to be used in field in toxic bait, must present action preferably through ingestion, delayed toxic action, lethality at low concentrations, environmental safe odorless and non repellent (Boaretto & Forti, 1997). In ingestion tests, the toxicity of plants studied to leaf cutting-ants are not found in literature, but other plants such as Ricinus communis L. (Bigi et al., 2004), Sesamum indicum L. (Morini et al., 2005), Cedrela fissilis Vell (Bueno et al., 2005), Helietta puberalla RE Fr. (Almeida et al., 2007), Simarouba versicolor St. Hil. (Peñaflor et al., 2009), Jatropha curcas L. and Ricinus communis L. (Alonso & Santos, 2013) Treatment Accumulated Mortality (%)/day S50 1º 2º 3º 6º 8º 10º 14º 17º 21º 25º Control 2 4 8 14 16 23 42 56 75 87 15 a EFZR 2 2 4 30 56 72 86 92 94 94 8 b EFEG 4 6 22 51 57 71 87 89 94 96 7 b EFCA 2 4 12 24 30 38 68 78 85 85 12 a ECCS 4 10 28 48 60 86 92 92 94 98 8 b ECEG 2 2 8 23 31 42 66 83 85 87 11 a EFAS 6 6 8 14 22 51 69 78 82 90 11 a ECSA 0 2 4 14 22 32 50 62 84 94 15 a EFEA 10 12 12 16 22 52 80 90 92 92 11 a EGZR 6 16 26 32 34 58 80 86 98 98 10 b Table 3. Mortality (%) and average survival period (S50) in days of Atta sexdens sexdens (Hymenoptera: Formicidae) workers fed on diet containing several plant extracts at the concentration of 0.2 mg.mL-1 Fig 1. Survival curves for Atta sexdens sexdens (Hymenoptera: Formicidae) workers in ingestion test with plant extracts. Average survival period (S50) is displayed besides the names of extracts, in brackets. Different lower case letters indicate difference in relation to control (data obtained with the application of “log-rank” test). S50= average survival. Letters after the value of S50 showed significant differences according to the “log-rank test” (b = 0.01