RESEARCH ARTICLE: PROTECTION AND NUTRITION OF PLANTS. REVISTA DE CIENCIAS AGRÍCOLAS

Volumen 36(1):5-15 p-ISSN 0120-0135 e-ISSN 2256-2273

doi: http://dx.doi.org/10.22267/rcia.193601.94

Fumigant toxicity of eugenol and its negative effects on biological development of maculatus L

biológico de Callosobruchus maculatus L Toxicidad fumigante del eugenol y sus efectos negativos en el desarrollo

Maria José González Armijos.1; Luis Oswaldo Viteri Jumbo.2; Lêda Rita D’Antonino Faroni3; Eugenio Eduardo Oliveira4; Adolfo Fernando Flores5; Fernanda Fernandes Heleno.6; Khalid Haddi.7

1 Ing. Agropecuaria, Universidad Nacional de Loja, Loja, Equador, [email protected]. 2 Dr. Departamento de Entomologia, Universidade Federal de Viçosa, Viçosa, MG, Brazil, [email protected]. 3 4 Dr. Departamento de Entomologia, Universidade Federal de Viçosa, Viçosa, MG, Brazil, [email protected]. 5 Dr. Departamento de Engenharia Agrícola, Universidade Federal de Viçosa, Viçosa, MG, Brazil, [email protected]. 6 com.M.Sc. Universidad Nacional de Loja, Loja, Equador, [email protected]. 7 Ph.D. Director, Serviço Autônomo de Água e Esgoto – SAAE, Senador Firmino, Minas Gerais, Brasil. fernandafhg@gmail..

Dr. Departamento de Entomologia, Universidade Federal De Lavras, Lavras, Minas Gerais, Brazil, [email protected] Cite: González Armijos, M.J., Viteri Jumbo, L.O., Faroni, L.R., Oliveira, E.E., Flores, A.F., Heleno, F.F. &

Callosobruchus maculatus L. Revista de Ciencias Agrícolas rcia.193601.94Haddi, K. (2019). Fumigant toxicity of eugenol and its negative effects on biological developmente of . 36(1):5-15. doi:http://dx.doi.org/10.22267/

Received: July 20 2018. Accepted: May 16 2019. ABSTRACT

The protection of stored products from damages, when accomplished, is mostly relying on the application of synthetic insecticides with serious health and environmental issues in addition to risks of selection of resistant insect populations associated with such practice. The use of plants derived compounds has been presented as sound strategy for sustainable insect management. Eugenol is UNIVERSIDAD DE NARIÑO Rev. Cienc. Agr. Enero - Junio 2019 36(1): 5 - 15 6 González et al.- Toxicidad fumigante del eugenol.

an aromatic component of natural occurrence in essential oilsCallosobruchus of numerous maculatus plants and known for its repellent and insecticidal bioactivities against different insect species. Here, we investigated the fumigant application of eugenol to control the (Coleoptera: Chrysomelidae: Bruchinae) by assessing toxicity, effects on biological developmentC. (includingmaculatus emergence inhibition) and repellency to these weevil. Besides a good fumigant insecticidal activity, eugenol exposure resulted in a dose-dependent decrease of the growth rate of associated with a steady reduction in bean mass losses. Additionally, significant inhibition of the) offspring emergence was achieved after the exposure of parental adults to lethal and sublethal doses99 of eugenol. Furthermore, adult were repelled away from beans exposed to high50 dose (LD of eugenolC. maculatus. in contrast with their attraction to the beans treated with lower dose (LD ). Our findings suggest that eugenol has potential as control tool to be used in sustainable management startegies Key words: compound.

bean; bioactivity; cowpea weevil; natural RESUMEN

La protección de productos almacenados contra daños causados por insectos, cuando se realiza,

medioambientales, además de riesgos de selección de poblaciones de insectos resistentes asociadas se basa principalmente en la aplicación de insecticidas sintéticos con graves problemas de salud y estrategia sólida para el manejo sustentable de insectos plagas. Eugenol es un componente aromático decon ocurrencia dicha práctica. natural El en uso aceites de compuestosesenciales de derivados varias plantas, de plantas conocido ha por sido su presentadobioactividad como repelente una e insecticida contra diferentes especies de insectos. En este estudio se investigó la aplicación vía fumigante del eugenol en el control del gorgojó de fríjol caupi, Callosobruchus maculatus

(Coleoptera: víaChrysomelidae: fumigante, la Bruchinae), exposición evaluandoal eugenol la resultó toxicidad, en una efectos disminución en el desarrollo de la tasa biológico de crecimiento (incluyendo de C.inhibición maculatus de la emergencia) y repelencia a estos gogojos.. Además de una buena actividad insecticida

dependiente de la dosis; y una reducción constante en la pérdida de masa del fríjol. losAdicionalmente, gorgojos adultos una fueroninhibición repelidos significativa cuando de los la granosemergencia de fríjol de lafueron descendencia tratados secon logro dosis cuando altas los adultos fueron expuestos a concentraciones letales y subletales de dosis de eugenol. Además, 99 50). Nuestros resultados

de(LD manejo) de eugenol sustentable y atraídos de C. maculatus cuando se. aplicaron dos dosis más bajas (LD sugieren que eugenol tiene potencial como una herramienta de control para ser usado en estrategias Palabras llaves: fríjol, bioactividad, gorgojo do fríjol, compuesto natural.

INTRODUCTION et al et Vigna unguiculata L. Walp, is a Leguminosae al ., 2013; Mkenda and Ndakidemi, 2014; Purohit Cowpea, ., 2013). However, cowpea production especially at species widely cultivated in tropical and subtropical small level farmers can suffer high losses of weight, countries. With protein content of about 23%-25%, nutritional value and viability of stored grains due to cowpea is a good protein source suitable in the diet various insect pests in field as well as during storage UNIVERSIDADof many people DE NARIÑO in third world countries (Kaliramesh (Mkenda and Ndakidemi, Rev. Cienc. Agr. 2014). Enero - Junio 2019 36(1): 5 - 15 González et al.- Toxicidad fumigante del eugenol. 7

Callosobruchus maculatus Viteri Jumbo et al The cowpea seed pest management (Polatoğlu and Karakoç, 2016; (Coleoptera: Chrysomelidae: ., 2014; Chang S. and Cheng mostBruchinae), serious apests cosmopolitan in stored product pest ofin tropical legume S. 2002, Enan E. 2001), the effects of isolated seeds both in storeset andal., 2018; in the Lopesfield, is et among al., 2018; the eugenol on stored grains pests especially when Massango et al., 2017) being able to cause losses applied as fumigant has been rarely studied. countries (Haddi et al., 2013; Melo et al Here, we evaluated the fumigant effect of up to 100% of stored cowpea beans within few eugenol by assessing the insecticidal (lethal months (Kang ., 2015). The toxicity, effects on biological develpomentC. maculatus and in control of this insect pest, when accomplished, is fumigantemergence applications inhibition). and repplency activities mainly achieved by theet alapplication of synthetic of eugenol on the cowpea weevil risksmolecules of selecting such as phosphineresistant populations and pyrethroid as insecticides (Freitas ., 2016), with serious MATERIAL AND METHODS et al., 2016; Iturralde- Garcíawell as et increased al., 2016). hazard to human health and Callosobruchus maculatus populations. environment (Gbaye of C. maculatus used in all experiments

Plant products such as essential oils and their were originally field-collected from small farms major constituents are known to possess in the Viçosa region (Minas Gerais State, Brazil). insecticidal or repellent activities and have long Populations of these insects were maintained been portrayed as safe to the environment or to under laboratory conditions (27 ± 2°C, 75 ± 5% human health leading to research effortset al., 2017;being RH, 12 h scotophase) on cowpea beans. To avoid Ismancurrently and focusedGrieneisen, on 2014; the use Pavela of theseand Benelli, plant possible infestations from the field and to reduce 2016).products in pest control (Haddi the potential effects of residual insecticides, the beforecowpea being beans offered were storedad libitum at -18 to C.°C maculatus for 15 days.. The cowpea beans moisture content was 11%, essential oils extracted from numerous plants of Fumigant toxicity bioassay. Comercial Eugenol is a naturally occurring componentSzygium of aromaticum, Eugenia aromatica, or Eugenia caryophyllusthe Lauraceae family includingCinnamomum cloves ( spp.), Brazil).formulation of liquid eugenol (99% pure) was Ocimum Pimenta dioica), acquired from Sigma-aldich (Sao paulo, SP State, Laurus), cinnamon nobilis ( Curcuma longabasil ( spp.), allspice ( bay laurel ( ) and turmeric ( The bioassay of fumigantet al.,toxicity 2017 of the eugenol ). Essential oils ofet some al., species2016; Viteri of this Jumbofamily was performed following previously described ethave al concentrations of this compound higher methods (MassangoC. maculatus ). Briefly, in than 90% (El-Maati 0.8L glass jars, 50 g of beans and twenty2 unsexed Sitophilus., 2014). granarius Although, Acanthoscelides essential oils withobtectus, this 1–2 days old adults were placed. Coptotermescompound are formosanus, toxic to various Periplaneta Americana (i.e., The eugenol was applied to 5 cm of filter paper Camponotus pennsylvanicus (Wattman N°1) clips, using a 100 µL micro- syringe (Hamilton, Reno, NV, USA), that were considered as alternative strategies) with promisingfor insect attached to a cotton wire and hung to the caps´ results under laboratory conditions and are being inner surface to serve as oil diffuser. The jars

UNIVERSIDAD DE NARIÑO were hermetically Rev. Cienc. sealed Agr. withEnero - metalJunio 2019 screw 36(1): 5 cap - 15 8 González et al.- Toxicidad fumigante del eugenol.

-1 air reinforced with silicone adhesive in a completely four doses with four replicates were used: 0, corresponding to control, LD20, LD60, and LD99. eugenolrandomized L-1 design. The doses used were 0.0 13.46, 29.80 and 134.13C. maculatus μL of eugenol obtained L from (control), 12.5, 18.75, 25.0, 37.50, 50,0 μL of The air and five replications were used progeny of the adult per each dose. The insect mortality was recorded the beans wereth assessed every two days starting after 24 hours exposure period. Insects were Repellencefrom the 22 bioassay. day after the treatments. considered dead if they do not respond to a The repellency experiment Instantaneousstimulus with a finerate paintbrush. of increase (r ) and grain i was conducted in an apparatus consisting of five losses bioassays. circular plastic containers (12 cm diameter, 8 cm r i height), with a central container (E) connected to The instantaneousC. maculatus rate of 1-2 of increase ( ) test was carried out in 0.8 L glass jar the other four containers (A, B, C and etD) al by., 2014).plastic where 20 unsexed adults of cylinders (12 cm long, 1 cm in diameter), as previously described (Viteri Jumbo day old were allowed to colonize 50 g of exposed Containers A and B were arranged diagonally-1 beans with eugenol based on the dose-mortality 50 =and 28.80 filled or with LD 50 = g134.13). of cowpea Containers beans exposed C and toD results previously obtained (see results-1 99 air eugenol (two doses were used [in µL/L ]: LD section). Five doses were used: 13.46, 20.64, 20, LD40, L60, LD80 e LD99, 29.80, 45.66, and 134.13 μL of eugenol L were filled with 50 g of cowpea beans untreated that corresponded to LD (control). In the center container, 50 unsexed respectively; applied in the same conditions adults were released, and after 24 h, the total of bioassays toxicity. Five replicates were used Statisticalnumber of insectsanalyses. per container was recorded. for each concentration. All the glass jars were curves maintained at 27 ± 1 °C, 75 ± 5% relative humidity Concentration-mortality and 12 h of scotophase. The control treatment were estimated according to probit did not receive any eugenol application. The analyses using the PROC PROBIT SAS software, number of insects emerged (F1) was counted version 9.2, of the SAS System for Windows (SAS ri and grain after 45 days and the instantaneous= rate of; Institute, Cary, NY). Regression analyses were ri (Nf /Ni )/ t increase in each population was calculated for performed to detect trends in the Nf each concentration as follows: Δ mass losses, cumulative and daily emergence adults; Ni C. maculatus Where:t is the final number of observed live that resulted in each treatment through time. is the initial number of Regression analysis was performed using the and Δ is the duration of the experiment curve-fitting procedure of SigmaPlot 12.0 (Systat 2 (Walthall and Stark, 1997). standardSoftware, errors, San Jose and CA,steep USA). increases The regressionin R model was chosen based on parsimony, lower with The grains masses provided for the insect model complexity. Paired t test was used for the colonization were weighed using an analytical analysis of repellence. scale (GEHAKA bk 800, Shimadzu, Tokyo, RESULTS AND DISCUSION Japan) at the start and the end (after 45 days) grain losses. Fumigant toxicity of eugenol to C. maculatus. of the bioassays to calculate the percentage of Emergence bioassay. The dose-mortality data 2 were satisfactorily same experimental proceduresThe bioassays described for thefor describedP by the probit model values [goodness-of- are given in daily emergence were conducted using the ri Tablefit tests 1. exhibiting low χ -values (<10) and high -values (>0.05)]. The LD

UNIVERSIDADthe instantaneous DE NARIÑO rate of increase ( ). Only Rev. Cienc. Agr. Enero - Junio 2019 36(1): 5 - 15 González et al.- Toxicidad fumigante del eugenol. 9

Table 1. C. maculatus.

Insect Slope LD LD LD LD LD LD 20 Fumigant40 toxicity50 of eugenol60 to 80 99 X2 P number 1)

(±3.17 SD (FI13.46 95%) (FI20.63 95%) (FI24.80 95%) (FI29.80 95%) (FI45.77 95%) (FI134.11 95%) 5.49 0.24 600 + 0.25

1SD = Standar (11.76-15.02) (18.84-15.01) (22.79-27.03)-1 (27.33-32.85) (40.62-52.96) (105.35 – 185.27)

d deviation, LD = Lethal dose (μL /50g cowpea), FI 95% = Fiducial intervals at 95% of probability, χ2 = Chisquare,­ P = Probability.

Instantaneous rate of increase (ri) and mass losses. r ) i the final number of live insects almost equal The instantaneous rate of increase ( C. to zero (Figure 1A). In a similar trend,df = the 5; maculatus.was used to estimate the eugenol fumigant mass losses of the cowpea beans exposed to ofeffects C. maculatus on the biological development of eugenol were significantly (F = 145.73; The instantaneous rate of increase developmentP <0.001) smaller of C. thanmaculatus those of the untreated wasdf significantly reduced in control (Figure 1B). The losses caused by the a dose-dependent manner in the presence of were up to 78% ri 60 eugenol (F = 27.35; = 5; P<0.001). Although less in bean masses treated with doses higher 99 than the LD compared to the losses found for causethe valuesC. maculatus decreased extinction linearly with resulting increasing in doses of eugenol, only the LD was able to the untreated beans.

The symbols represent the mean of five replicates and the vertical bars represent the SD. Figure 1. Instantaneous rate of increase ri C. maculatus -1 (A) of and mass losses of cowpea beans (B) exposed to different fumigant doses of eugenol (µL /50g cowpea beans).

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Emergence bioassay. LD caused The daily emergence of eugenol.20 Indeed, even the smallest tested dose adult weevils was significantly different when a smaller peak of emergence (more the grain masses were infested by unexposed than 50% smaller) and a delay in the time of or eugenol exposed insects (Figure 2). Both maximum emergence of 1- 2 days compared to the number of progeny individuals (Figure 2A) the control while higher doses almost abolished

and the time to maximum emergence (Figure the progeny emergence. 2B) were negatively affected by exposure to Cumulative emergence (%) Cumulative emergence Daily emeregence (No. insects)

Figure.The 2. symbols represent the mean of five replicates and the vertical barsC. represent maculatus the exposed SD. to -1). Daily emergence (A) and cumulative emergence (B) of progeny of increased fumigant concentration of eugenol (µL Repellence bioassay. Repellence to C. maculatus

-1 traitsfumigant of application of eugenol at lethal and 50 ) C. maculatus ofwas eugenol dependent presented of theattracting fumigated effects dose to ofC. sublethal doses and its effects on biological maculatuseugenol (Figure3), t =-12.05, while df the = LD4, P(24.80 μL L , a key pest of stored -1 beans. Our results showed, good fumigantC. 99 ( C. maculatus<0.001),t = 3.26, the maculatusinsecticidal activityand a reduction of eugenol in acompainedbean mass dfbeans = 4, treatedP = 0.031). with LD (134.13 μL L ) were with a decrease of the growth rate of significatively repellent to ( losses. Additionally, significant inhibition of the offspring emergence was achieved after As part of the continuous and active quest for the exposure of parental adults to lethal and cheaper, safe and eco-friendly alternatives to sublethal doses of eugenol. Interestingly, the chemical insecticides, several plant oils and behavior of the adult weevil in front of eugenol their major constituents have been screened exposed beans balanced between attraction at as potential tools preventing post-harvest low dose and repellency at higher ones. losses due to insects. Here, we evaluated the

UNIVERSIDAD DE NARIÑO Rev. Cienc. Agr. Enero - Junio 2019 36(1): 5 - 15 González et al.- Toxicidad fumigante del eugenol. 11

et al The bars represent the mean of five replicates and the that are effective on a wide variety of insects vertical bars representP<0.05 the). SD. *Significant difference between the number of individuals in the treated and and mites (Dayan ., 2009), working by untreated by test t ( disrupting the octopaminergic (or via its precursor tyramine) transmission on the insect nervous system (Enan, 2001; Enan, 2005). As a fumigant compound, the respiratory system should be the main entrance route of eugenol and other monoterpenes into the insect body, making the respiratory system the potential major target site for eugenol. In a recent study, it has been shown that eugenol have a strong activitiesinhibition in Sitophilus of acetylcholinesterase oryzae, (AChE) and adenosine triphosphatases (ATPases) Figure 3. C. maculatus after but also that the fumigant toxicities of the monoterpeneset al., 2014; such Repellences (%) to-1 50 ) and LD99 Saadas eugenol et al are higher than their contact and/ 24 h -1of) ofexposure eugenol to in cowpea fumigant beans application. untreated and or oral toxicities (Athanassiou treated with LD (24.80 μL L (134.13 ., 2018). Furthermore, eugenol-rich μL L essential oils (e.g., clove essential oil) that exhibited insecticide activity similar to the C. maculatus activity recorded for deltamethrin (Viteri Although a majority of the studies on the 2013;Jumbo Kadacol, et al., 2018) J. & Kamanavalli,can be easily C. biodegraded 2010). toxicity of essential oils; against by microorganisms (Mishra, S. & Sachan, S. and other stored products pests; have mostly tested crude essential oils; different works with individual synthetic compounds suggest that In addition to the good toxicity of eugenol, the insecticidal activities of these essential oils fumigant application of this compound also are due principally to their primary constituents Negativeresulted effects in a significanton biological inhibition traits including of the such as eugenol.et al Indeed, various studies haveet offspring emergence even at sublethal doses. reported that this terpenoid has acaricidal al., 2017; Saad et al et emergence of C. maculatu (Ribeiroal ., 2016), insecticidalet (Larson al., 2014) rates of growth, grain consumption and progeny activities. ., 2018), termiticidal (Xie s can be the results of ., 2015) and fungicidal (Koeduka direct mortalityet alof., adults, 2014; Germinara repellency, et oviposition al., 2015). Eugenol belongs to monoterpenes, a large group or progeny deterrence and growth inhibitionSitophilus zeamais(Athanassiou Similar effects have been reported in et of volatile and lipophilic compounds which are al., 2017; when Silva etexposed al., 2017). to other In fact, essential monoterpenes oils or etable al to rapidly penetrate inside insects and their monoterpenes constituents (Nattudurai interfere with their physiological functions (Saad ., 2018). The mode of action of monoterpenes such as eugenol are volatile and can penetrate etin alinsects is still not well understood although it between the grains, enhancing the exposure of has been studied by various authors (Soujanya different stages of the pest to the treatment. . (2016). In contact application, eugenol and Such easy circulation between the grains is an other monoterpenes are fast-acting insecticides UNIVERSIDAD DE NARIÑO advantage to these Rev. Cienc. compounds Agr. Enero - thatJunio makes2019 36(1): them 5 - 15 12 González et al.- Toxicidad fumigante del eugenol.

et al., 2014). strategies. useful as fumigants (Athanassiou which favors environment friendly pest control Furthermore, the inhibition of emergence of new insects was positivelyC. maculatu correlateds larvae with or ACKNOWLEDGMENTS lower grain consumption, probably due to a lower feeding activity of due to the progeny deterrence. Agrícola, Universidade Federal de Viçosa, Viçosa MG,We Brazil. thank to Departamento de Engenharia In this study, the repellence activity of eugenol was dependent of the fumigation dose. Indeed, This work was supported by grants from the Secretaria Nacional de Educación Superior being able either to repel or attract insectsC. Ciencia y Tecnología of Ecuador (SENESCYT), maculatus(Atkinson, insects 2018; Bakerat a dose and of Grant, LD 2018), the 50 Grants from the CAPES Foundation (Finance Leugenol-1 exposed beans were attractive to code 001), the National Council of Scientific and (24.80 μL-1 99= ) ) while eugenol presented repellent action Technological Development (CNPq), the Minas Gerais State Foundation for Research at high fumigant doseet al. (LD, 2016;134.13 Viteri μLJumbo L ConflictAid (FAPEMIG). of interest: etin al accordance., 2014; Zeringóta with previous et al studies (Kafle and Shih, 2013; Reis The authors declare that ., 2013). Such for use in formulations for trapping there is no conflict of interest. attractant activity of eugenol has been suggested BIBLIOGRAPHIC REFERENCES Popilliaattacking japonica horticultural crops, particularly those in the scarab familyProtaetia such brevitarsisas the Japanese beetle, Grant, 2018). Newman and the white-spotted Athanassiou, C.G., Rani, P.U. & Kavallieratos,Advances N.G. in(2014). plant flower chafer, (Baker and biopesticidesThe use of plant extracts for stored product protection, In: Singh, D. (Ed.). . pp. 131-147. New Delhi: Springer. CONCLUSIONS Journal of Atkinson,Agricultural R.G. (2018). and Food Phenylpropenes: Chemistry Occurrence, Distribution, and Biosynthesis in Fruit. . 66(10): 2259- Eugenol exhibited good insecticidal activity 2272. doi: 10.1021/acs.jafc.6b04696 and increments in the fumigation dosages C. maculatus Baker, B.P. & Grant, J.A. (2018). Eugenol Profile. proportionately decreased the growth rate of Recovered from https://ecommons.cornell.edu/ . Additionally, eugenol delayed bitstream/handle/1813/56125/eugenol-MRP- C.the maculatus biological development and significantly NYSIPM.pdf?sequence=1&isAllowed=y. reduced the bean weight losses caused by and showed repellent activity at high Chang, from S. T. Cinnamomum & Cheng, S. osmophleum S. (2002). . AntitermiticJournal of Agriculturalactivity of leaf and essential Food Chemistry oils and. 50 components doses against the cowpea bean weevils whereas its activity in low doses may serve as an attractant (6): 1389- in lures for other pests and could be subject 1392. doi: 10.1021/jf010944n integratedof study C. object. maculatus Thus, management our results program revealed in products in crop protection. Bioorganic & that eugenol can be part of a comprehensive Dayan,medicinal F. E., Cantrell, chemistry C. L., &, Duke,17 S. O. (2009). Natural (12), 4022-4034. doi: storage facilities (especially for small farmers), 10.1016/j.bmc.2009.01.046. UNIVERSIDAD DE NARIÑO Rev. Cienc. Agr. Enero - Junio 2019 36(1): 5 - 15 González et al.- Toxicidad fumigante del eugenol. 13

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