Introduction
Economic losses caused by storage pests are high, however, they strongly diverge with the type of crop, country, climatic region and duration of storage (Klys et al., Reference Klys, Malejky and Nowak-Chmura2017). According to the same authors, in general, the global annual losses in the stored products due to insect activity are estimated at 10%. In addition to eating the grains, this pest is also a cause of food contamination by microorganisms (Magan et al., Reference Magan, Hope, Cairns and Aldred2003; Athanassiou et al., Reference Athanassiou, Kavallieratos and Campbell2017).
Sitophilus zeamais (Motschulsky) (Coleoptera: Curculionidae), also known as the maize weevil, is a key pest of grains and grain products in different parts of the world, causing most of the losses in maize grains (Colares et al., Reference Colares, Dionello and Radunz2016; Ojo and Omoloye, Reference Ojo and Omoloye2016). Both the larval and adult stages of this insect devour the grains, causing postharvest significant damages. The control of these insects depends heavily on the use of synthetic insecticides, but their residues pose serious risks to the environment, animals and human, causing lethal effects on non-target organisms and pest resistance (Askar et al., Reference Askar, Al-Assal and Nassar2016; Colares et al., Reference Colares, Dionello and Radunz2016). To avoid such inconsistencies, the search for new alternatives for pest control is required. As a complementary approach or an alternative to synthetic pesticides, phytochemicals, namely essential oils (EOs) constituents are presently under consideration as ingredient of crop protection products, as well as in repellent formulations (Isman and Akhtar, Reference Isman, Akhtar, Ishaaya, Nauen and Horowitz2007; Regnault-Roger et al., Reference Regnault-Roger, Vincent and Arnason2012). Several studies have pointed out the value of Apiaceae plants and their potential application in the context of Integrated Pest Management and Integrated Vector Management (IVM) (Boulogne et al., Reference Boulogne, Petit, Ozier-Lafontaine, Desfontaines and Loranger-Merciris2012; Evergetis et al., Reference Evergetis, Michaelakis, Haroutounian and Soloneski2012; Reference Evergetis, Michaelakis and Haroutounian2013; Pavela and Vrchotová, Reference Pavela and Vrchotová2013; Seo et al., Reference Seo, Jung, Kang, Lee, Kim, Hyun and Park2015). The supporting evidences of Apiaceae pesticidal activities against various types of damaging/noxious organisms, including stored-product insects are substantial (Chaubey, Reference Chaubey2008; Ebadollahi, Reference Ebadollahi2011, Reference Ebadollahi2013; Ebadollahi et al., Reference Ebadollahi, Nouri-Ganbalani, Hoseini and Sadeghi2012; Kim et al., Reference Kim, Kang and Park2013).
In view of their relatively high commercial relevance, we have been studying the pesticidal potential of some of the most important Apiaceae species: Anethum graveolens L. (dill), Cuminum cyminum L. (cumin), Foeniculum vulgare subsp. vulgare var. vulgare Mill. (bitter fennel) and Petroselinum crispum (Mill.) Nyman ex A.W. Hill (parsley) (Sousa et al., Reference Sousa, Rosa, Oliveira, Cunha and Fernandes-Ferreira2013, Reference Sousa, Rosa, Oliveira, Cunha and Fernandes-Ferreira2015a, Reference Sousa, Rosa, Silva, Almeida, Novo, Cunha and Fernandes-Ferreira2015b, Reference Sousa, Rosa, Cunha and Fernandes-Ferreira2017). In the present work, EOs from these four plant species and some EO standard compounds were evaluated for their potential fumigant and contact toxicity, as well as repellent activity towards S. zeamais aiming to prevent damages caused by this pest on stored maize grain. Furthermore, their effects on nutritional physiology were evaluated through analysis of nutritional metrics, to assess to what extent EOs/standard compounds can disrupt insect feeding, metabolism and capacity of conversion of food into body mass.
Material and methods
Essential oils and chemical composition
A. graveolens (dill) plants were grown from commercial seeds while F. vulgare var. vulgare (bitter fennel) germplasm was obtained from a wild population. Voucher specimens of fruits and vegetative parts were deposited at the University of Porto (Portugal) herbarium (accession number PO1000MFF). Dill and bitter fennel green infrutescences (fruits in a pre-ripening phase) were collected from 5 and 14-months old plants, respectively. After 2 h of hydrodistillation in a Clevenger modified apparatus, the recovered EOs were dried with sodium sulphate and stored in brown sealed vials until use (−20°C). The EOs from P. crispum (parsley) and C. cyminum (cumin) fruits were purchased from Sigma-Aldrich, Co. Complete quantitative and qualitative profiles of dill, cumin, bitter fennel and parsley EOs herein tested were previously characterized (Sousa et al., Reference Sousa, Rosa, Cunha and Fernandes-Ferreira2017). The EO extracted from dill infrutescences was mainly constituted by (S)-carvone (66.4%), β-phellandrene + limonene (24.7%) and α-phellandrene (5.3%), while bitter fennel infrutescence EO contained estragole (64.9%), fenchone (15.8%) and β-phellandrene (5.5%). The EO from cumin fruits was rich in cuminaldehyde (39.4%), γ-terpinene (15.8%), β-pinene (12.4%), p-cymene (10.4%) and p-mentha-1,4-dien-7al (9.7%). Major compounds identified in parsley fruit EO were: myristicin (31.5%), apiole (15.9%), α-pinene (16.2%), β-pinene (13.6%) and 1-allyl-2,3,4,5-tetramethoxybenzene with carotol (8.6%).
Chemicals
Four high purity standard volatile compounds were included in all assays based on their relative abundance in the studied EOs (Sousa et al., Reference Sousa, Rosa, Cunha and Fernandes-Ferreira2017). The standards (S)-(+)-carvone (96%), cuminaldehyde (98%), estragole (98%) and (+)-fenchone (99.5%) were purchased from Sigma-Aldrich and Fluka (Aldrich chemical Co., St. Louis, MO, USA).
Ellman´s reagent (DTNB, 5,5′-dithionitrobenzoic acid; 99%), acetylthiocholine iodide (ATChI; ≥99%), berberine and the purified acetylcholinesterase (AChE, EC3.1.1.7) used for enzymatic assay were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA).
Bioassays
Insects
Adults of S. zeamais were obtained from a colony maintained for more than four generations in laboratory conditions in the Biology Department of Azores University (Ponta Delgada, S. Miguel, Azores archipelago, Portugal). Insects were reared with organic yellow corn grains (Zea mays local type) and maintained in plastic cages (35.7 cm × 23.5 cm × 13.4 cm) in the following conditions: 23 ± 1°C, 60% of relative humidity (RH), photoperiod of 14:10 (L: D) h. Zea mays grains were obtained from a biological production and acquired from local farmers with an average moisture content of 14 ± 0.5%. Unsexed adult weevils used in all the experiments were about 2 to 4 weeks old.
Fumigant toxicity
The fumigant insecticidal activity of the EOs and the standard compounds against S. zeamais adults were evaluated at different doses (25, 50, 75, 100, 156, 300, 525 and 600 mg L−1 air). Increasing amounts of EOs/standard compounds (from 1 to 24 mg) were pipetted onto a Whatman no. 1 filter paper disc (Ø 2 cm). Each paper disc was attached to the inner surface of a transparent plastic vial screw cap (total volume of 0.040 L) and a wire sieve was used inside the vial to prevent direct contact of the insects with the treated filter paper (fig. 1). Ten unsexed adult insects taken from the laboratory colony were placed with 1 g of yellow corn grains in a plastic vial (fig. 1). The caps were screwed tightly and hermetically sealed. Vials containing insects were turned upside down over the vials containing the impregnated filter paper disc, to allow saturation of the atmosphere with EO/standard compound vapours. The vials were kept in the dark, in an incubator at 23°C and 60 ± 5% RH. Five replicates were carried out for treatments and negative control groups. The number of dead insects was recorded daily by direct observation until 7 day after the start of treatment. Insects were considered dead if they did not respond to touch stimulation with a blunt needle.
Diagram representing the experimental setup on the fumigant insecticidal activity.
Contact toxicity
The contact toxicity of EOs/standard compounds against S. zeamais adults was evaluated at different doses (0, 30, 50, 100 and 150 µg per insect). One microliter of the dilutions was topically applied to insects’ pronotum using a micropipette. Controls were determined using distilled water. Both treated, and control insects were then transferred to a plastic Petri dish (10 insects/dish) containing corn grains and kept in incubators (23°C, 60 ± 5% RH). Insect mortality was observed daily until endpoint mortality was reached 7 days after treatment. The experiments were repeated in four times with ten insects per replicate.
Repellence bioassays
Area preference bioassays. The repellent effects of EOs/standard compounds on adult maize weevils were assessed as described by Cosimi et al. (Reference Cosimi, Rossi, Cioni and Canale2009) and Chaubey (Reference Chaubey2011). Petri dishes (Ø 9 cm) were used to confine S. zeamais during the experiment. Test emulsions of EOs/standard compounds at different concentrations (2.5, 7.5, 12.5 and 25 mg mL−1) were prepared by diluting in ethanol and then into distilled water (1.5% v/v of ethanol in all the emulsions). Whatman filter paper (Ø 9 cm) was cut in equal halves. A total of 200 µL of the test emulsion was then uniformly applied to one half of the filter paper using micropipette to obtain the following doses per area unit: 16, 47, 78 and 156 µg cm−2. The other half of the filter paper was moistened with 1.5% v/v of ethanol in distilled water, as a control treatment. Treated and untreated halves were placed together at the bottom of Petri dishes and fixed to their opposites. Twenty unsexed adults of S. zeamais were released at the centre of each Petri dishes then covered and kept in the dark. Six replicates were set for each concentration of EO/standard compound emulsion. Repellence evaluation was carried out 1 h after treatment, counting the number of insects present on both treated and untreated halves and expressing it as a percentage of repellence.
Two-choice. In the second assay, repellent activity of EOs/standard compounds against S. zeamais was performed following the method of França et al. (Reference França, Oliveira, Esteves Filho and Oliveira2012) with minor modifications, using a choice bioassay system, which consisted of two 70-millilitre plastic containers connected at their rims by a plastic tube (30 cm of length; 2 cm inner diameter). A circular hole was cut in the middle of the tube to facilitate the introduction of test insects into the bioassay system. Three pre-weighed corn grains were treated with a single dose of EOs/pure compound (1 µg g−1 diet) in glass bottles which were vigorously shaken to ensure proper mixing of corn grains with emulsions. One box contained, treated grains while the other box had untreated corn (negative control). Twenty unsexed adults were introduced into the plastic tube through the circular hole by means of a 0.5 cm diameter funnel. The number of insects present in the control box and the treated box was recorded after 1 and 3 h of treatment. The whole systems were thoroughly cleaned with ethanol and dried after each test to avoid any interference of other allelochemicals. The assay was repeated five times, for each EO/standard compound, using different cohorts of insects, which had not been previously exposed to any treatment.
Nutritional and antifeeding effects
The effects of EO/standard compounds over S. zeamais growth, food consumption and other metabolic parameters were evaluated for a 120-h period experiment. The bioassay was performed with adults (3–3.5 mg of average weight), never exposed to any of the studied allelochemicals. Groups of twenty pre-weighed adults were distributed in plastic containers (volume 110 mL). At least five independent assays with 20 adults per treatment were done (n = 100). After a 6-h starvation period, each group of unsexed adults was fed with three treated and pre-weighed corn grains. Grain treatments were prepared by vigorously shaking three corn grains into a glass vials containing 100 µL of EO/standard emulsion at a single concentration of 3% (w/v), or control solutions, to ensure proper mixing of corn grains with the liquid. Emulsions at the concentration of 3% (w/v) were obtained by a first dilution step of EOs/standard compounds in ethanol, followed by a gradual addition of distilled water up to the final volume (the final concentration of ethanol being 1.5% v/v). Two control groups one with water and another with ethanol (1.5% v/v) were included in the experiment. Corn grains were left for 10 min to evaporate the solvent and weighed before being placed into each container to feed insects. The weights of the non-consumed diet and insect alive, as well as eventual mortality were recorded after 120 h of assay under controlled conditions. All weight measurements were made on an analytical balance with an accuracy of 0.1 mg (Mettler Toledo AB204-S/FACT).
To estimate treatment effects on the food weight that was consumed, assimilated and converted into body mass in the 5 days of the experimental period the following parameters were evaluated: relative growth rate (mg mg−1 day−1): RGR = L/l × t; relative consumption rate (mg mg−1 day−1): RCR = D/l × t; efficiency conversion index of ingested food (%): ECI = 100L/D and antifeeding effect (%), AE = 100 [(C−T)/C], where t is the duration of the experimental period, D the mean dry weight of consumed diet during t, L the mean dry weight gain of maize weevil adults during t, l the mean dry weight of maize weevil adults, C the mean consumption in the control (mg) and T the mean consumption in the treatment (Scriber and Stansky, Reference Scriber and Slansky1981). The calculation on a dry weight basis was used as described by other authors (Koul et al., Reference Koul, Smirle and Isman1990; Senthil-Nathan et al., Reference Senthil-Nathan, Chung and Murugan2005; Yazdani et al., Reference Yazdani, Sendi, Aliakbar and Senthil-Nathan2013), to minimize the influence of water variation, namely induced dehydration of insects possibly caused by some treatments.
Acetylcholinesterase inhibition assay in vitro
The inhibitory effect of EOs and compounds on AChE activity was screened by in vitro assay using a purified AChE from electric eel (Electrophorus electricus) and following the method of Ellman et al. (Reference Ellman, Courtney, Valentino and Feathertone1961). Ellman's colorimetric assay was adapted to 96-well microplates and performed at pH 8.0 in sodium phosphate buffer, using 0.25 U mL−1 of AChE and ATChI as a substrate (75 mM), in the presence of DTNB (3 mM) and different concentrations of EOs/standard compounds (Arruda et al., Reference Arruda, Viana, Rainha, Neng, Rosa, Nogueira and Barreto2012). The isoquinoline alkaloid, berberine, was used as a reference substance of plant origin (Jung et al., Reference Jung, Min, Yokozawa, Lee, Kim and Choi2009). The hydrolysis of the substrate was monitored by repeated spectrophotometric readings (absorbance at 415 nm) for different times of reaction (0, 150, 300 and 450 s) using a Bio-Rad Model 680 Microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). EOs/standard compounds' activity was assessed within a range of 6–7 concentrations (15.6, 31.3, 62.5, 125.0, 250.0, 500.0 and 1000.0 µg mL−1) with four replicates per concentration. The percentage of inhibition was estimated based on the reaction rate (ΔAbs415nm min−1) obtained for each treatment/concentration and the control reaction without inhibitor: Inhibition (%) = 100−100(V sample/V control). Assays were repeated 3 to 4 times to calculate mean inhibition (%).
Statistical analyses
Mean values calculated from dose-response data collected through the mortality, repellence and AChE inhibition assays were used to estimate, respectively, LC50 and LD50 values (the concentration required to kill 50% of the insects in the fumigation test and in topical application, respectively), RD50 values (the concentration required to repel 50% of the insects; area preference bioassays) and IC50 values (the concentration required to inhibit 50% of AChE activity). Each data set and independent group (EOs/standard compounds) was submitted to Probit analysis. Differences between the estimated LC50, RD50 or IC50 values for EOs/standard compounds were considered significant based on the criterion of nonoverlap of the respective 95% confidence intervals (CI). The percentage of adults attracted to treated and untreated corn was analysed by a paired sample t-test (two-choice bioassays). For the nutritional indices, data were submitted to a one-way ANOVA test without previous transformation and, mean multiple comparisons were performed using the LSD test (P = 0.05). All analyses were performed using the statistical software SPSS 23.0 (IBM, 2015).
Results
Fumigant and contact toxicity
The fumigant toxicity of four EOs and four standard compounds against S. zeamais adults was dose-dependent, which allowed the estimation of lethal concentrations within acceptable confidence limits (CLs) through Probit analysis (table 1). In general, most of the data fitted well in the assumptions of this linear model. The LC50 values calculated for Apiaceae EOs/standard compounds ranged from 51.8 to 535.8 mg L−1.
Acute toxicity of Apiaceae EOs and standard compounds against adults of S. zeamais, after 7 days of exposure
a Estimated LC50 and 95% CL were determined by probit analysis based on dose-related adults' mortality.
b LC and LD values within the same column followed by the same letter are not significantly different based on non-overlapping of the 95% CL.
c H, Heterogeneity factor, χ2:df.
Based on LC values and the non-overlapping of CLs we have established two classes of toxicity with a probability of 95%. The first one, with LC50 values <200 mg L−1, which includes the most effective treatments (S)-carvone (LC50 = 51.8 mg L−1), and A. graveolens EO (LC50 = 157.1 mg L−1), and the low toxicity class with LC50 > 200 mg L−1 comprising all the remaining EOs (F. vulgare, P. crispum and C. cyminum EOs) and standard compounds (estragole, (+)-fenchone and cuminaldehyde).
The toxicity of Apiaceae EOs applied topically to the beetles is summarized in table 1. (S)-Carvone shows pronounced contact toxicity against S. zeamais (LD50 = 23 µg per adult) while P. crispum EO had a LD50 value of 128.2 µg per adult. The compound cuminaldehyde, and the EOs of A. graveolens and C. cyminum also revealed contact toxicity against S. zeamais (LD50 = 96.5, 111.3 and 120.4 µg per adult, respectively). Nevertheless, only (S)-carvone exhibited both strong fumigant and contact toxicity against the maize weevils.
Repellence
Results demonstrate that EOs and standard compounds have a good repellent activity against adults of S. zeamais when assessed by two methods (fig. 2 and table 2). In the area preference bioassays, cuminaldehyde, (S)-carvone and estragole are the most repellent (RD50 < 4.9 µg cm−2) and significantly different from the others. Among the EOs tested, the EO extracted from F. vulgare infrutescences was the most repellent (RD50 = 24.0 µg cm−2) (table 2). In the two-choice bioassays, at 1 h after the exposure, the most repellent was P. crispum fruit EO and cuminaldehyde, followed by C. cyminum fruit EO. After 3 hours of exposure, a similar pattern was noted, and no significant difference was observed in the results obtained between the two periods of observation (fig. 2).
Percentage of adults of S. zeamais attracted to untreated and treated corn grain with EO and standard compounds after (a) 1 h and (b) 3 h of experiment. **Statistically significant by the paired sample t-test.
Repellency of Apiaceae EOs and standard compounds evaluated by the area preference bioassays against adults of S. zeamais, after 1 h of exposure
Data are expressed as μg cm−2.
a Estimated RD50 and respective 95% CL were determined by probit analysis based on adults' response to corn grains treated with increasing doses of EOs/standard compounds.
b RD values within the same column followed by the same letter are not significantly different based on non-overlapping of the 95% CL.
c H, Heterogeneity factor, χ2:df.
Nutritional and antifeeding effects
The nutritional parameters determined for adults of S. zeamais when fed with corn grains treated with EOs/standard compounds (3% w/v emulsion), for a period of 120 h are presented in fig. 3. Mean values of RCR did not varied significantly, from 0.20 to 0.27 mg mg−1 day−1 (F 8,36 = 1.08, P = 0.397) and the LSD post hoc test indicated no statistical differences between treatments. Overall, the RCR values were much superior to the RGR mean values obtained (−0.028 to 0.012 mg mg−1 day−1), which reflected on lower ECI values (−16.3 to 5.6%). With the exception of P. crispum fruit EO, none of the treatments showed significant decrease in the maize weevil RGR, when compared to the negative control. Concerning the ECI, only P. crispum EO and estragole presented a significant effect. P. crispum EO significantly impaired the ECI, while estragole seemed to have a promoting effect on this metabolic parameter. Moreover, the inhibition of S. zeamais adults’ feeding behaviour (AE) with relation to the ethanol control group varied significantly, from −23.7 to 28.5% (F 7;30 = 2.35, P = 0.032). Cuminaldehyde was found slightly stimulant (AE = −23.7 ± 10.3%), while P. crispum EO showed slightly deterrent effects on this insect (AE = 28.5 ± 10.9%). Furthermore, P. crispum EO and (S)-carvone exerted some acute toxicity (18 ± 3.0% and 6 ± 2.5% mortality, respectively). The observed percentages of mortality for other treatments were not significantly different from the negative control with ethanol.
Estimated nutritional indices and feeding deterrent activity of Apiaceae EOs and standard compounds on adults of S. zeamais, after 120 h of treatment.
Acetylcholinesterase inhibition assay
Concerning the results obtained by the in vitro assay with purified AChE, all the EOs and the standard compounds showed a dose-dependent inhibitory activity (table 3). With basis on this preliminary study, we found evidences that EOs from parsley and cumin fruits (IC50 = 185 and 235 µg mL−1, respectively) have a more significant inhibitory action over the hydrolytic activity of the AChE, when compared to dill and bitter fennel infrutescence EOs. Moreover, cuminaldehyde and (S)-carvone, previously identified as major volatile constituents of cumin fruit EO (39%) and dill infrutescence EOs (66%), respectively (Sousa et al., Reference Sousa, Rosa, Cunha and Fernandes-Ferreira2017), showed inhibitory effects comparable to their corresponding EOs. Cuminaldehyde and (S)-carvone were also the most effective among the four tested standard compounds (214.5 and 368.1 µg mL−1, respectively). When comparing all results, bitter fennel EO and both its major compounds, estragole and fenchone, showed the lowest anticholinesterase activity in vitro (IC50 = 465.4, 605.7 and 726.5 µg mL−1, respectively).
In vitro inhibitory effect of Apiaceae EOs and standard compounds on AChE activity
a Estimated IC50 and 95% CL were determined by probit analysis based on mean values of inhibition. Values are expressed in μg mL−1 of EOs/standard compounds required to inhibit 50% of the enzymatic activity.
b LC values within the same column followed by the same letter are not significantly different based on non-overlapping of the 95% CL.
c χ2 values were determined for 4 degrees of freedom.
d Berberin was used as the positive control.
Discussion
Due to their high volatility, EOs and their constituents, present a strong fumigant action by penetrating the insect body via the respiratory system. Such exposure through the gaseous phase is acutely toxic to insects and became a relevant handling strategy to control an insect damaging stored-product (Coats et al., Reference Coats, Karr, Drewes and Heden1991; Kim et al., Reference Kim, Roh, Kim, Lee and Ahn2003; Lee et al., Reference Lee, Peterson and Coats2003; Liu et al., Reference Liu, Chu and Jiang2011; Ebadollahi, Reference Ebadollahi2013; Massango et al., Reference Massango, Faroni, Haddi, Heleno, Jumbo and Oliveira2016) and repellence (Bedini et al., Reference Bedini, Bougherra, Flamini, Cosci, Belhamel, Ascrizzi and Conti2016; Lee et al., Reference Lee, Kim, Choi and Park2017). Some EOs also exhibit antifeedant properties (Benzi et al., Reference Benzi, Stefanazzi and Ferrero2009).
In general, contact and fumigant insecticidal activities of plant EOs and monoterpenes against stored product pests have been successfully reported (Tripathi et al., Reference Tripathi, Prajapati, Aggarwal, Khanuja and Kumar2000; Lee et al., Reference Lee, Peterson and Coats2003; Abdelgaleil et al., Reference Abdelgaleil, Mohamed, Badawy and El-Arami2009; Wang et al., Reference Wang, Li and Lei2009; Yang et al., Reference Yang, Zhou, Wang, Du, Deng, Liu and Liu2011; Kordali et al., Reference Kordali, Yildirim, Yazici, Emsen, Kabaagac and Ercisli2012). In the present study, although most of the EOs and compounds were toxic to S. zeamais, their toxicity varied with the type of bioassay. In contact toxicity assays, dill infrutescence EO, cumin fruit EO, (S)-carvone and cuminaldehyde showed the highest toxicity towards S. zeamais. In fumigant toxicity assays, dill infrutescence EO and (S)-carvone were found significantly more effective as fumigants, than other treatments. The relatively high biological activity of the dill and cumin EOs is probably related to the high concentration of (S)-carvone (66%) and cuminaldehyde (39%), respectively (Sousa et al., Reference Sousa, Rosa, Oliveira, Cunha and Fernandes-Ferreira2015a). In general, the toxicity displayed by several plant species EOs against stored pests has been related to their major components, mostly monoterpenes (López et al., Reference López, López, Aragón, Tereschuk, Slanis, Feresin, Zygadlo and Tapia2011; Kumar et al., Reference Kumar, Mishra, Malik and Satya2012). For example, cuminaldehyde exhibited strong contact and fumigant toxicities against Blattella germanica (Yeom et al., Reference Yeom, Kang, Kim and Park2012), and S. oryzae (Chaubey, Reference Chaubey2011; Kim et al., Reference Kim, Kang and Park2013). Another major compound, (S)-carvone also possessed insecticidal activity against S. oryzae, Tribolium castaneum, Rhyzopertha dominica, Cryptolestes pusillus and Callosobruchus chinensis (Abdelgaleil et al., Reference Abdelgaleil, Mohamed, Badawy and El-Arami2009; Fang et al., Reference Fang, Jiang, Wang, Zhang, Liu, Zhou, Du and Deng2010; López et al., Reference López, Contreras and Pascual-Villalobos2010; Kim et al., Reference Kim, Kang and Park2013). Likewise, Yildirim et al. (Reference Yildirim, Emsen and Kordali2013) found that the oxygenated monoterpenes carvone, dihydrocarvone, menthone, terpinen-4-ol, 1,8-cineole, fenchone, linalool and limonene oxide have some insecticidal potential towards S. zeamais adults.
In this study, bitter fennel and parsley EOs showed low fumigant toxicity to S. zeamais. However, Ebadollahi (Reference Ebadollahi2011) previously demonstrated that F. vulgare (seed) EO display fumigant activity against adults of S. oryzae and S. granarius (the wheat weevil), while Maroufpoor et al. (Reference Maroufpoor, Ebadollahi, Vafaee and Badiee2016) observed that P. crispum EO shows significant fumigant toxicity against tree important stored product pests, Callosobruchus maculatus (Coleoptera: Bruchidae), Plodia interpunctella (Lepidoptera: Pyralidae) and Ephestia kuehniella (Lepidoptera: Pyralidae). Such differences of results point to the fact that EO isolated from different plant parts or origin may exhibit distinct fumigant toxicity, which can be attributed not only to their mechanism of action over different targeted arthropods, but also to variable qualitative and quantitative composition, differences in volatile component physical properties (distinct vapour pressure and boiling point), and their equilibrium in the gaseous phase.
The literature survey indicated that EOs obtained from Apiaceae family are active as repellents for Coleoptera and insects of other orders. Based on the comparison of estimated RD50 (table 2) cuminaldehyde, (S)-carvone and estragole, followed by bitter fennel EO, exhibit strongest repellent activity against S. zeamais. On the other hand, it was not possible to establish a clear relationship between EOs biological activities and their main constituents. This could be due to the natural occurring blend of constituents, or due to the presence of other minor compounds, as suggested by other authors (Bertoli et al., Reference Bertoli, Conti, Mazzoni, Meini and Pistelli2012; Ebadollahi, Reference Ebadollahi2013).
Concerning the antinutritional effects towards S. zeamais, the studied parameters indicate that for their most parts EOs/compounds have little impact on the efficiency of the metabolic process and growth of adults, and little or no antifeedant activity at the tested dose. Maize weevils presented similar consumption rates for the 120 h-period of experiment (no alternative food offered) independently of the treatment. Significant effects on RGR, and ECI during the time of the assay were only obtained for parsley EO. This EO significantly decreased the RGR and ECI parameters and caused morbidity, which suggests toxicity through ingestion. In our previous investigation, the same parsley fruit EO caused significant anti-nutritional effects (feeding and growth inhibition with weight loss) to the caterpillar Pseudaletia unipuncta (Lepidoptera: Noctuidae) (Sousa et al., Reference Sousa, Rosa, Oliveira, Cunha and Fernandes-Ferreira2015a). Evidences of EOs activities on the maize weevil feeding behaviour and nutrition, with significant reduction of the nutritional indices and moderate antifeedant effects, have been reported for several plant species, namely nutmeg (Myristica fragrans, Myristicaceae) (Huang et al., Reference Huang, Tan, Kini and Ho1997), Evodia rutaecarpa (Rutaceae) (Liu and Ho, Reference Liu and Ho1999), cardamom (Eletaria cardamomum, Zingiberaceae) (Huang et al., Reference Huang, Lam and Ho2000) and red ginger (Alpinia purpurata, Zingiberaceae) (de Lira et al., Reference de Lira, Pontual, de Albuquerque, Paiva, Paiva, de Oliveira, Napoleão and Navarro2015). However, investigations concerning Apiaceae EOs are scarce. Possible negative effects of EOs/compounds toward other stored-grain pest feeding behaviour, nutrition and metabolism were also studied, as the EOs from leaves and fruits of pepper tree (Schinus molle), and from leaves of Tagetes terniflora, Cymbopogon citratus and Elyonurus muticus on S. oryzae (Benzi et al., Reference Benzi, Stefanazzi and Ferrero2009; Stefanazzi et al., Reference Stefanazzi, Stadler and Ferrero2011) and from eugenol, isoeugenol, methyleugenol and EOs from leaves of Tagetes terniflora, Cymbopogon citratus and Elyonurus muticus on Tribolium castaneum (Huang et al., Reference Huang, Ho, Lee and Yap2002; Stefanazzi et al., Reference Stefanazzi, Stadler and Ferrero2011). In the present study, we conclude that the tested dose of EOs/compounds might not be effective to protect stored maize grain from S. zeamais, since AE were negligible 5 days after the application of emulsions. However, these results might be attributable to the relatively low dose tested when compared to those used in similar studies. For example, EOs or their compounds exhibited potential feeding deterrence when applied at a concentration of 14,400 ppm (Huang et al., Reference Huang, Lam and Ho2000), 13.2 mg g−1 of diet (Huang et al., Reference Huang, Ho, Lee and Yap2002) and 37.5 µL g−1 of diet (de Lira et al., Reference de Lira, Pontual, de Albuquerque, Paiva, Paiva, de Oliveira, Napoleão and Navarro2015). Also, we found that values obtained in the control for the nutritional indices RCR and ECI differed considerably from values described in the literature for S. zeamais probably because of a different diet and longer duration of the experiment. Camaroti et al. (Reference Camaroti, de Almeida, do Rego Belmonte, de Oliveira, de Albuquerque Lima, Ferreira, Paiva, Soares, Pontual and Napoleão2018) and de Lira et al. (Reference de Lira, Pontual, de Albuquerque, Paiva, Paiva, de Oliveira, Napoleão and Navarro2015) reported lower RCR but superior ECI values when assays were performed with wheat flour disks for 7 days. For the RGR index described for S. zeamais, values tend to be very low varying from 0.042 mg mg−1 day−1 after 3 days (Liu and Ho, Reference Liu and Ho1999) to approx. 0.01 mg mg−1 day−1 for a 7-days assessment (Camaroti et al., Reference Camaroti, de Almeida, do Rego Belmonte, de Oliveira, de Albuquerque Lima, Ferreira, Paiva, Soares, Pontual and Napoleão2018). In the present work, The RGR was nearly null for the negative control groups which indicate that, despite the higher consumption rate of maize, adults did not growth during such short period of time. In general, in most studies performed with a flour disk diet, the efficiency of conversion of ingested food into body mass by adults of S. zeamais is not very high (4.2, 11, 17 and 22.4%) (Huang et al., Reference Huang, Tan, Kini and Ho1997, Reference Huang, Lam and Ho2000, Reference Huang, Ho, Lee and Yap2002; Liu and Ho, Reference Liu and Ho1999, respectively).
Similar to several synthetic pesticides (e.g. carbamates and organophosphates), the toxic properties exhibited by EOs and their constituents have been frequently related to a neurotoxic mode of action achieved, in part, via the octopaminergic system and/or through an inhibitory action on the cholinergic synapses, where AChE regulates nerve impulse transmissions by rapidly breaking downs acetylcholine (ACh) into choline and acetate (Coats et al., Reference Coats, Karr, Drewes and Heden1991; Kostyukovsky et al., Reference Kostyukovsky, Rafaeli, Gileadi, Demchenko and Shaaya2002; Tripathi et al., Reference Tripathi, Upadhyay, Bhuiyan and Bhattacharya2009; Rattan, Reference Rattan2010).
EO constituents have been reported for their potential AChE inhibitory effects (Ingkanian et al., Reference Ingkanian, Temkitthawon, Chuenchom, Yuyaem and Thongnoi2003; López and Pascual-Villalobos, Reference López and Pascual-Villalobos2010; Aazza et al., Reference Aazza, Lyoussi and Miguel2011; Arruda et al., Reference Arruda, Viana, Rainha, Neng, Rosa, Nogueira and Barreto2012; Yeom et al., Reference Yeom, Kang, Kim and Park2012; Orhan et al., Reference Orhan, Senol, Ozturk, Celik, Pulur and Kan2013; Seo et al., Reference Seo, Jung, Kang, Lee, Kim, Hyun and Park2015), and the possible correlation between enzyme inhibition in vitro and acute in vivo toxicity in insects was recently examined by Isman and Tak (Reference Isman and Tak2017). In our study, parsley EO and cuminaldehyde, followed by the cumin fruit EO, exhibited the most significant inhibitory effect over AChE in vitro activity (lowest IC50 value). However, in accordance with the categories recently described by Santos et al. (Reference Santos, Gomes, Pinto, Camara and Paes2018) (high potency, IC50 < 20 µg mL−1; moderate potency, 20 < IC50 < 200 µg mL−1 and low potency, 200 < IC50 < 1000 µg mL−1); only parsley EO could be considered of moderate potency. The EO from A. graveolens infrutescences presented a low inhibitory action over AChE activity, which may be attributable to the joined action of (S)-carvone with other major components, such as the cyclic monoterpene hydrocarbons α- and β-phellandrene (not tested). Previous reports established that α- and β-phellandrene, and (S)-carvone, might act as a non-competitive inhibitor of AChE (Bonesi et al., Reference Bonesi, Menichini, Tundis, Loizzo, Conforti, Passalacqua, Statti and Menichini2010; Jankowska et al., Reference Jankowska, Rogalska, Wyszkowska and Stankiewicz2018). The lowest toxicity of estragole and (+)-fenchone (highest IC50 values) may be a possible explanation for the low inhibitory action of bitter fennel EO over AChE. The weak anticholinesterase activity of F. vulgare EO on purified AChE from electric eel was previously reported by Aazza et al. (Reference Aazza, Lyoussi and Miguel2011), although the authors estimated a much higher IC50 value (2.5-fold superior). Contrariwise, López et al. (Reference López, Contreras and Pascual-Villalobos2010) identified fenchone as a competitive inhibitor of AChE with an IC50 of 0.4 mM, which is approximately 10 times inferior to the value we determined in this work (726.6 µg mL−1 ≈ 4.77 mM). Putting into perspective, the potency of the assessed EOs and their constituents as AChE inhibitors might not be so relevant, since these were 97 to 383 times less active than berberine (based on IC50 values). In general, the most notable AChE inhibitors naturally occurring in plant showed effects at a much lower order of magnitude (below 15 µM) (Santos et al., Reference Santos, Gomes, Pinto, Camara and Paes2018). Despite the inhibitory actions herein recorded and evidences of acute effects in the contact toxicity assay for some treatments, the present findings do not permit to establish any correlation of the in vitro AChE inhibitory activity with the acute toxicity results obtained in vivo against S. zeamais. Therefore, conclusions about a possible mode of action are limited. According to Isman and Tak (Reference Isman and Tak2017), the degree of inhibition that has been observed in several studies might be too low to consider AChE inhibition as the major mode of action of EOs and monoterpenoids exhibiting insecticidal properties.
Conclusion
In this study, we have shown the lethal and sublethal effects of EOs from four common Apiaceae species and some of their constituents against S. zeamais, with regard to their toxicity, repellent activity and impact on insect feeding, metabolism and growth. A. graveolens infrutescence EO and the respective major compound, (S)-carvone, demonstrated the highest fumigant and contact toxicity, while cuminaldehyde, (S)-carvone and estragole followed by EO from F. vulgare exhibited high repellent activity. Moreover, P. crispum EO and cuminaldehyde were found to influence maize weevil's nutrition, but only P. crispum fruit EO showed significant negative impacts. Concerning the possible inhibitory effects of EOs/compounds on AChE in vitro activity, the findings suggest that P. crispum and C. cyminum EOs, as well as cuminaldehyde, were more effective (IC50 = 185, 234.9 and 214.5 µg mL−1, respectively) than the remaining tested EOs and volatile compounds, but all were relatively weak AChE inhibitors relatively to other naturally occurring plant products described in the literature. In addition, the present work gives evidences of the potential use of dill and cumin EOs, and their major oxygen-containing monoterpenes (S)-carvone and cuminaldehyde, respectively, as possible natural fumigants for the control of S. zeamais.
Acknowledgements
We would like to thank M.F. Almeida, from the University of Azores for the insect production and the valuable technical assistance.
Financial support
This work was supported by: European Investment Funds and National Funds by FEDER/OE, Project IC&DT: 02/SAICT/2017, Ref. PTDC/BAA-AGR/31131/2017, (Acronim: EOIS-CroProt) and National Funds by FCT – Portuguese Foundation for Science and Technology, through the CITAB research centre, under the project UID/AGR/04033/2019.
Conflict of interest
The authors declare that there are no conflicts of interest.
