Bioactivity of iridoids of Genipa americana against the coconut mite Aceria guerreronis Keifer (Acari: Eriophyidae)

In April 2016, fresh leaves of G. americana of the CR2 genotype were collected from the Genipap Genebank located in the Nossa Senhora das Dores municipality (10°29’30”S-37°11”36”W- 204 m.a.s.l.), Sergipe State, Brazil. To obtain the leaf methanolic extract (LME), 500 g of the leaves were macerated in 1 liter of MeOH for 48 hours. Afterwards, the extract was filtered and concentrated at 40°C. For the identification of the iridoids in the leaves, thin layer chromatography (TLC) plates on Si gel (MERCK-Germany, 105553) were developed by the solvent systems: EtOAc-HCOOH-AcOH-H₂O (100: 11: 11: 26, v:v), and EtOAc-HCOOH-AcOH-H₂O (100: 0.5: 0.5: 0.5, v:v). Briefly, an aliquot of 10 µl of genipin, geniposide, and LME was developed on the plate of chromatography. The detection was performed using vanillin-sulphuric acid reagent. The compounds genipin (690277-8) and geniposide (24512-63-8) used in the additional bioactivity bioassays were purchased from Sigma-Aldrich (98 % purity).

Young and healthy coconut fruits (from Embrapa Tabuleiros Costeiros) were collected for the confection of arenas; the mites, likewise, were obtained from the young infested fruits. The epidermis of the perianth was covered by a mixture of 5 % agar, 0.3 % methylparaben (Nipagim®) as a fungicide, and distilled water. Discs (1 cm diameter) were opened with the help of a strainer to expose the area of the fruit epidermis, which served as an experimental units.

The concentrations used were selected in the initial concentration-mortality bioassays and ranged between the lower (0 %) and higher (100 %) limits of mortality (15). According to the recommendations of the IOBC/WPRS (International Organization for Biological Control of Noxious Animals and Plants/West Palearctic Regional Section) (16), increasing concentrations of LME (0.06, 0.1, 0.2, 1.0, 2.0, 4.0, and 5.0 mg.ml^-1) were dissolved in acetone and subsequently sprayed through a Potter Tower (Burkard, Rickmansworth, UK) at a pressure of 5psi, with a 9.3 ml spray aliquot, which resulted in a residue of 1.38 mg cm²; the arenas of the control treatment were sprayed only with acetone. The sprayed discs were exposed to the environment for 30 minutes to dry; subsequently, 20 A. guerreronis adults were transferred to each arena with the help of a fine brush. Eight arenas were used for each concentration of LME and they were maintained at 28 ± 2°C, with a relative humidity of 65 ± 5 %, and a 12 h photoperiod. Mortality was assessed after 24 h of exposure and the mites that did not respond to the brush stimulus were considered dead. Based on the estimated LC₅₀ value of LME, the toxic effects of standard concentrations (0.6 mg.ml^-1) of either genipin or geniposide were assayed, aiming at investigating the role of these iridoids in the acaricidal activities of the extract. Abbott’s formula (17) was used for mortality correction. The corrected mortality (PT) values were computed as: PT = [(Po - Pc)/(100 Pc)] × 100, where Po = observed mortality and Pc = control mortality.

The persistence of LME was evaluated by spraying LC₉₀ on the arenas using the same procedures as described above and adapting the methodology of others bioassays (18,19). Twenty adults of the coconut mite were placed on an arena at 6 h intervals (i.e., 6, 12, 18, 24, 30, 36, 42, and 48 h after spraying), with five replications. The control group only used the solvent acetone. Mortality was recorded 24 h after mite transference.

For the repellency bioassays, the arenas were prepared as previously described; however, each arena consisted of a half treated and a half untreated area, this latter area was covered with two layers of adhesive tape during the LME spraying (20). The arenas were sprayed with LC₅₀ and LC₉₀ of the LME. After the drying of the solution, a white glue point was placed in the center of the disc (1.0 x 1.0 x 0.5 mm). The adult coconut mites were individually positioned onto the white glue point and their positions were evaluated after 1 h, 24 h, and 48 h. Three replicates were performed for each treatment, and the replicates consisted of 20 mites, totaling 60 mites per treatment.

The lethal concentrations of LME to the A. guerreronis were determined by the Probit analysis using SAS software (SAS Institute, 2002). The data on mortality of mites when treated with the main compounds were analyzed using an analysis of variance (ANOVA); the means were compared with Tukey test (p<0.005). We conducted a linear regression to assess mortality persistence of the LME lethal doses, and the binomial test, at 5 % probability, was used to analyze the differences of the mite numbers choosing the treated or the untreated areas (repellency).

Due to the negative effects related to using synthetic products, especially halogenated and organophosphorus pesticides, as well as to the increasing attention given to the use of natural products to assist in pest management and the demonstrations of phytochemicals as a great source for pest control, various research groups have profiled secondary metabolites from a variety of plant species (13,14). Data of these current studies have indicated that LME is toxic to the coconut mite (LC₅₀ = 0.60 mg.ml^-1, LC₉₀ = 16.69 mg.ml^-1) and show genipap as promising to control this important coconut pest (Table 1).

In previous screening studies, extracts of lipidomic profiles from the families Arecaceae, Fabaceae, and Malvaceae , such as babassu, coconut, cottonseed, and degummed soybean oils, were assayed against A. guerreronis; and they all demonstrated toxicity to this mite (21, 22). Interestingly, linoleic and oleic acids, which have been shown to be bioactive against the coconut mite (21, 22), were the major compounds in the lipidomic profiles of soybean and cottonseed oil.

The phytochemical profiles of the LME of genipap detected the presence of iridoids as main compounds. After 24 h of exposure, the iridoids genipin and geniposide, at 0.6 mg.ml^-1, inflicted 48 % and 52 % mortality on A. guerreronis, respectively, which differed significantly from mortality in the control (p<0.05) (Figure 1). The biological and insecticidal activities of genipap are associated with several molecules, such as steroids, iridoids, and monoterpenoids, and they have been demonstrated by other authors (23, 7, 2, 8). These findings suggest that the major iridoids genipin and geniposide contributed to the bioactivity of LME towards A. guerreronis.

The persistence bioassays revealed over 60 % mortality of A. guerreronis when exposed to the LC₉₀ of LME after 6 h of spraying. In general, the loss of toxic effects was evident as time after spraying elapsed, falling to less than 50 % only after 18 h, and the acaricidal effects disappeared completely after 42 h. This contrasted with the plain mortality in control (Fig 2). The low persistence may be explained by the compound oxidation, since the presence of phenolics was significant in the extract (24). The acaricide residual activities are crucial for the management of A. guerreronis. Indeed, the understanding of persistence is important because, as this pest inhabits the perianth of the fruits, its control is difficult and frequent applications are needed (25, 26).

The LC₅₀ and the LC₉₀ of LME were repellent to the coconut miteafter 1, 24, and 48 hours after application (Figure 3A; Figure 3B), indicating that the mites were able to detect and avoid the treated areas. The observed repellent activities may be attributed to the rich chemical composition of the iridoids and to the fact that they act as a defense against the herbivores (2, 3). Plant repellency has been pointed out as an efficient strategy for avoidance of pest infestations in agroecosystems, being capable of reducing oviposition, injuries, and productivity losses; with the consequent reduction of the costs in the control of some of these pests (27, 14).

Thus, in addition to toxicity, it is important to understand the sublethal effects, such as repellency, in order to control pest dissemination. Several studies have demonstrated the repellant activities of extracts of plants against pest mites (14, 21, 22).

The results of this study suggest that the major iridoid compounds of G. americana play an important role in toxicity, with high repellency and low persistence to A. guerreronis. Other studies implicating genipin and geniposide also showed a variety of biological properties (28, 29, 30). Overall, the results indicate that LME is a potential candidate for the development of plant-based pesticides to be used in the integrated management of A. guerreronis. Also, the major iridoids genipin and geniposide explain, at least in part, the bioactivity of LME towards the mite A. guerreronis. Further laboratory studies are necessary to assess the possible sublethal effects of LME on the natural enemies of A. guerreronis.