Vol. 8(10), pp.103-112, October 2017 DOI: 10.5897/JSPPR2017.0240 Article Number: A2BF99A66534 Journal of Stored Products and ISSN 2141-6567 Copyright ©2017 Postharvest Research Author(s) retain the copyright of this article http://www.academicjournals.org/JSPPR

Full Length Research Paper

Insecticidal activity of four essential oils on the survival and oviposition of two sympatric bruchid : maculatus F. and Callosobruchus subinnotatus PIC. (Coleoptera: Chrysomelidea: Bruchinae)

Seth W. Nyamador1*, Abla D. Mondédji1, Boris D. Kasseney1, Guillaume K. Ketoh1, Honoré K. Koumaglo2 and Isabelle A. Glitho1

1Applied Entomology Laboratory, Faculty of Sciences, University of Lomé, Lomé, Togo. 2Plant Extracts and Natural Aromas Laboratory, Faculty of Sciences, University of Lomé, Lomé, Togo.

Received 6 March, 2017; Accepted 29 September, 2017

Callosobruchus maculatus F. and Callosobruchus subinnotatus Pic. are two pest species of stored cowpeas and bambara groundnuts. Methods of controlling their populations remain the use of chemical insecticides that have ecotoxicological effects. The aim of this work is to look for alternative methods using essential oils extracted from four aromatic plants (Bidens borianiana, Chromolaena odorata, Cymbopogon giganteus and Cymbopogon nardus) to control these pests. Essential oils GC/MS analysis revealed differences in their composition. The major components of the essential oils of the two congeneric Poaceae species C. giganteus and C. nardus are totally different. Limonene (23.03%), cis-p-mentha-2, 8-dien-1-ol (14.26%) and p-mentha-1(7), 8-dien-2-ol isomer (14.06%) were the main compounds in C. giganteus oil whereas citronellal (30.58%) and geraniol (23.93%) were identified in C. nardus oil. In the essential oils of the other two plants, the major components are respectively geyrene (19.44%), α-pinene (15.96%), and germacrene D (14.03%) for Chromolaena odorata essential oil and trans-β-ocimene (31.58%) for Bidens borianiana essential oil. Toxicity tests were performed by fumigation on adult survival and female oviposition in C. maculatus and C. subinnotatus by evaluating the LD10; LD50 and LD90 of the four essential oils. These tests showed that only essential oils extracted from Cymbopogon species are efficient. The essential oil of C. giganteus was the most toxic to adults of both bruchid species. The LD50 were 20.06 and 34.62 µL/L, respectively for C. maculatus and C. subinnotatus while C. nardus essential oil showed the best ovicidal activity with female oviposition reduction in both bruchid species of more than 80% at a lower concentration (10 µL/L). C. giganteus and C. nardus essential oils can thus be used in stocks of cowpea and bambara groundnuts for adult control and prevent female oviposition.

Key words: Callosobruchus maculatus, Callosobruchus subinnotatus, Cymbopogon giganteus, Cymbopogon nardus, Bidens borianiana, Chromolaena odorata, essential oils, survival, oviposition.

INTRODUCTION

Bambara groundnut (Vigna subterranea Verd.) is a food legume cultivated in tropical regions. It is a creeping and

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erect herbaceous annual plant; it measures 20 to 30 cm residues in processed commodities. These are mainly tall (Caburet and Lethève, 2000). Africa is the greatest plant-derived products and microbial biopesticides. producer of food legumes mainly cowpeas and bambara However, interest in these products, some of which have groundnuts. Reliable data on the production of bambara a long-term obvious insecticidal activity in controlling pest groundnuts are difficult to obtain because it is a populations, depends on their mechanisms of action subsistence crop and the seeds are sold in local markets (Ahadji-Dabla et al., 2015). only (Brink et al., 2006). According to the FAO, world Indeed, pests have developed resistance to different production of bambara groundnuts was estimated at classes of insecticides by changing the structure of their 41000 tonnes in 2000 (FAOSTAT, 2007). West Africa sites of action by substitution or by genetic mutation provides 45 to 50% of this production (Brink et al., 2006). (Tabashnik, 1994; Hemingway et al., 2004). Today, we The cultivation and storage of bambara groundnuts note that more than 700 species of have essentially face attack by pests such as leaf eaters, stem developed resistance to a majority of synthetic borers, floral bud and seed pests (Brink et al., 2006). The insecticides (Georghiou and Lagunes - Tejeda, 1991). use of insecticides has allowed the controlling of crop This leads to the loss of an entire class of insecticides pests. However, late arriving at fruiting time, and makes it difficult to develop new products. The especially seed-borne, are mostly not exposed to challenge for the development of new products with insecticides to avoid the presence of residues on the insecticidal activity depends on their competitiveness. pods. These seed-borne insects continue their Thus, only those that will present a new mode of action or development under storage conditions. multiple modes of action, but also a limited In stocks, they cause significant economic losses ecotoxicological risk, and low persistence in treated estimated at between 40 to 80% in four to seven months products may be accepted as new insecticides. of storage (Ngamo et al., 2007; Doumma et al., 2011; Among the alternative products studied, essential oils Huignard et al., 2011). In storage conditions, the main (EO) extracted from aromatic plants are considered food legume pests are weevils. In West Africa, the natural fumigants. They have high toxicity to insects species usually encountered in bambara groundnuts are: (Kéita et al., 2001; Park et al., 2003; Papachristos and Callosobruchus subinnotatus, the main species (Ketoh et Stamapoulos, 2004; Ketoh et al., 2006; Negahban et al., al., 2001) and Callosobruchus maculatus, the sympatric 2007; Nyamador et al., 2010) while they are best known species (Ajayi and Lale, 2000) whose preferred host is to have a low toxicity to vertebrates according to the US the cowpea. Food and Drug Administration (Generally Regarded as The infestation of bambara groundnut by weevils takes Safe, GRAS). This difference in toxicity in vertebrates place mainly during seed drying or storage near old and invertebrates is related to the structure of their stocks of infested bambara groundnut (Nyamador et al., octopaminergic receptors that are the pathways of 2016). The damage done by these pests cause essential oils in the body (Enan, 2001). They are significant losses in seed weight and quality. Attacked biodegradable, non- persistent in the environment, and seeds become unfit for human consumption; suffer a loss act quickly (Don Pedro, 1996). in germination capacity (Odah, 1995) and finally, a This study aims to evaluate, under laboratory substantial reduction in their market value (Siabi, 1996). conditions, the insecticidal activity of EO extracted from The availability of substrate promotes the successive four aromatic herbs (Cymbopogon giganteus Chiov., development of sexually active adult generations of the Cymbopogon nardus L. Rendle, Chromolaena odorata L. two species of in bambara groundnut stocks, and Bidens borianiana L.) on the survival and oviposition important oviposition and a high survival rate of larvae; of females C. maculatus and C. subinnotatus which are leading to the total destruction of bambara groundnut sympatric in bambara groundnut stocks in Togo. stocks within a few months of storage. Pesticides are used in the field to effectively control pest outbreaks, but their use in storage conditions is far MATERIALS AND METHODS from effective in protecting stocks. Thus, the search for alternative methods, using low environmental impact Aromatic plants control agents has grown significantly in recent years Aromatic plants (leaves and inflorescences) were harvested at the (Agboyi, 2009; Nyamador et al., 2010; Mondédji et al., flowering stage in different regions of Togo and dried under shelter 2014, 2015). The choice of these methods is guided by for 72 h at 30 ± 2°C, 80% RH before extraction of EO. The plants the non-persistence of alternative products and their are: C. giganteus Chiov.; C. nardus L. Rendle; C. odorata L. and B. derivatives in the environment and the low toxicity of their borianiana L.

*Corresponding author. E-mail: [email protected]. Tel: (+228) 90 11 55 04; (+228) 22 25 50 94.

Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License

Nyamador et al. 105

Two species of Cymbopogon were harvested in southern Togo in Table 1. Chemical composition of Cymbopogon giganteus the locality of Zévé for C. nardus, and the locality of Assahoun for essential oil (Most abundant compounds are shown in bold). C. giganteus. Samples of C. odorata and B. borianiana were collected from the Dayes Plateau in Kpalime, a town in the West of Togo. Compounds identified Percentage

p-cymene 0.02 Insects Limonene 23.05 Limonene oxide I 0.80 Adults of both species of weevil of the reproductive form of C. Limonene oxide II 1.25 subinnotatus and C. maculatus (Nyamador et al., 2016) aged from 0 to 24 h were used for various tests. These adults come from the p-α -dimethyl styrene 0.10 strain of the Applied Entomology Laboratory (AEL) of the Faculty of cis-p-mentha-2,8-dien-1-ol 14.26 Science of the University of Lomé. C. subinnotatus was reared on trans-p-mentha-2,8-dien-1-ol 5.63 its preferred development substrate, local bambara groundnut 4’-methyl acetophenone 0.03 seeds with a cream ivory integument or beige and black hilum, while C. maculatus was cultivated on local cowpea seeds called p-mentha-1(7),8-dien-2-ol isomer 12.63 "Black eye" or "glei". p-mentha-1(7),8-dien-2-ol isomer 14.06 These seeds, bought in the market, were frozen for a week to cis-carveol 0.75 eliminate any previous contamination, and dried to maintain their moisture content at 13%. Insect mass-rearing was done according trans-carveol 1.40 to the method of Dick and Credland (1984) in Plexiglas boxes in the Carvone 3.32 laboratory at 30 ± 2 °C; 72 ± 2% RH 12: 12 h LD. Photoperiod was Isoamyl hexanoate 0.13 maintained with a programmable timer. Temperature and relative Isoamyl octanoate 0.09 humidity were recorded using a handheld thermohygrometer “Pen – Type”. Perilla aldehyde 0.31 Phenethyl hexanoate 0.03 Phenethyl octanoate 0.01 Extraction and analysis of the chemical composition of Total 77.87 essential oils

The EO was extracted from the aerial part (leaves and inflorescences) of the harvested and dried plants by steam distillation in the Plant Extracts and Natural Aromas Laboratory containing 40 seeds. These seeds were renewed every day to (LEVAN) of the Faculty of Science of the University of Lomé. The evaluate the residual activity of EO on the female reproductive EO was collected by decantation, dried with anhydrous sodium capacity. Every 24 h, the dead individuals in each of the boxes sulfate and stored in amber glass vials in a refrigerator at 4°C. were recovered and counted on the renewal of seeds. The same EO obtained were analyzed by gas chromatography (GC) experiment was performed using 20 couples of C. maculatus with coupled with mass spectrometry (GC/MS) using a chromatograph 100 cowpea seeds. Essential oil doses used for this weevil species system Auto XL chromatograph/mass spectrometer TurboMass were 2.5, 5, 10, 20 and 40 µL/L for C. giganteus and 10, 20, 30, 40 (Perkin Elmer) equipped with a PE-5MS column with 5% phenyl and 50 µL/L for the other three EO, to determine the LD10, LD50 and and 95% methyl polysitoxane size 20 m × 0.18 mm × 0.18 μm. The LD90. The EO doses were compared to the control (0 µL/L). detection of the various compounds was performed according to the method described by Ketoh et al. (2002). Statistical analysis

Bioassay The various tests were repeated four times. The results were expressed as mean ± SD and subjected to variance analysis Toxicity tests were carried out by fumigation in sealed 1L glass jars (ANOVA). The results of the dose - mortality - response test were at room temperature and natural photoperiod. The EO load has analyzed by the Probit method of Finney (1971) for determining the been deposited on a Whatman filter paper disc of 5.8 cm diameter LD10, LD50 and LD90 using the WinDL32 software version 4.6. to facilitate its evaporation into the jars. The various doses applied Discrimination of means was made using the multivariable Newman were expressed by volume of EO per volume of air from the Keuls test at 5% level. enclosure (µL/L) and used in a single application. Essential oil doses used vary according to essential oil activity and sensitivity of the beetle species. RESULTS The tests were performed with 20 couples of C. subinnotatus in the presence of 40 bambara groundnut seeds. These couples were exposed to 5 doses of EO (20, 40, 60, 80 and 100 µL/L) to Analysis of the chemical composition of essential determine the LD10, LD50 and LD90. The EO doses were compared oils (EO) to the control (0 µL/L). In the control, only the Whatman filter paper disc without EO load was placed into the glass jar. Adults and The main constituent of C. giganteus oil was limonene seeds were removed from the jars 24 h after the start of treatment. (23.03%). This EO also contained other important The seeds were then placed in Petri dishes and kept untreated for compounds such as cis-p-mentha-2,8-dien-1-ol (14.26%); five days and then observed under a magnifying glass to determine p-mentha-1,(7),8-dien-2-ol isomer (14.06%); p-mentha- the number of eggs laid by the females during processing. The number of deaths in the treated adults was counted 24 h after the 1,(7),8-dien-2-ol (12.63%); trans-p-mentha-2,8-dien-1-ol end of treatment. (5.63%) and carvone (3.32%) (Table 1). Surviving adults were introduced into untreated Petri dishes each The main compounds in C. nardus EO were citronellal

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Table 2. Chemical composition of Cymbopogon nardus Table 3. Chemical composition of Chromolaena odorata essential essential oil (Most abundant compounds are shown in bold). oil (Most abundant compounds are shown in bold).

Compounds identified Percentage Compounds identified Percentage Limonene 1.39 Dimethyl sulfide 0.01 Linalol 0.27 Tricyclene 0.01 trans-verbenol 0.35 α-thujene 0.11 Citronellal 30.58 α-pinene 15.96 Citronellol 7.65 β-pinene 8.06 Neral 0.42 Camphene 0.14 Geraniol 23.93 Sabinene 2.08 Geranial 0.74 Myrcene 2.08 Geranyl acetate 8.68 α-phellandrene 0.04 Thymyl acetate 3.48 α-terpinene 0.03 β-elemene 2.08 γ-terpinene 0.06 Germacrene D 1.28 Limonene 1.38 Bicyclogermacrene 0.19 cis-β-ocimene 0.43 α-farnasene 0.17 trans-β-ocimene 2.90 trans-β-farnasene 0.33 Terpinolene 0.05 Cardinene 1.18 Linalool 0.06 Elemol 12.04 1,3,7-nonatriene, 4,8-dimethyl 0.08 γ-β-eudesmol 0.26 Geyrene 19.44 β-eudesmol 0.15 Terpinenol-4 0.07 Citryl triglate 1.12 Bornyl acetate 0.53 Total 96.30 Pregeijerene 10.51 λ-elemene 0.20 β-elemene 0.78 γ-elemene 1.47 (30.58%) and geraniol (23.93%), although elemol α-cubebene 0.08 (12.04%), geranyl acetate (8.68%) and citronellol (7.65%) β-cubebene 0.32 were also present (Table 2). The EO of C. odorata α-copaene 1.17 contained as major compounds geyrene (19.44%), α- β-caryophyllene 4.62 pinene (15.96%), and germacrene D (14.03%) (Table 3). α-caryophyllene 1.59 Other compounds were pregeijerene (10.51%), β- Aromadendrene 0.42 pinene(8.06%), β-caryophyllene (4.62%), trans-β- Alloaromadendrene 0.10 ocimene (2.90%), δ-cadinene (2.62%), myrcene (2.08%), Germacrene D 14.03 sabinene (2.08%), α-caryophyllene (1.59%), γ-elemene (1.47%), limonene (1.38 %) and α-copaene (1.17%). Germacrene B 0.13 Analysis of B. borianiana EO revealed that it consists of Germacra 1.6-dien-5-ol 0.10 a single major compound, trans-β-ocimene (31.58%) α-muurolene 0.33 (Table 4). Other important compounds like germacrene D trans-muurorol 0.19 (5.87%), cis-β-ocimene (5.06 %), myrcene (5.05%), the 3e,6e-farnesene 0.03 β-terpinene + limonene (3.80%), p-menthatriene (3.58%) γ-cadinene 0.41 and sabinene (2.14%) were also identified. λ-cadinene 2.62 Calamenene 0.03 Elemol 0.81 Effect of essential oils on adult survival of C. Ledol 0.16 maculatus and C. subinnotatus trans-cadinol 0.19 α-eudesmol 0.22 Effect of C. giganteus essential oil β-eudesmol 0.18 10-epi-γ-eudesmol 0.16 For C. maculatus, with an EO of C. giganteus, the Total 94.48 mortality was 87.50 ± 15.30% of the adults at a dose of 40 µL/L. The LD10, LD50 and LD90 obtained with this EO were 8.31; 20.03 and 48.42 µL/L, respectively (Table 5). The model fits the test data and gives an insignificant 2 2 The regression line obtained, is y = - 4.36295 + 3.35003x. Chi value at 5% (Chi = 2.415, df = 3, P = 0.491).

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Table 4. Chemical composition of Bidens borianiana essential oil LD10, LD50 and LD90 obtained by the Probit method are (Most abundant compounds are shown in bold). 14.69; 39.15 and 104.32 µL/L, respectively (Table 6). The regression line obtained with this essential oil is y = - Compounds identified Percentage 4.79723 + 3,01182x. The model fits the test data and α-thujene 0.02 gives an insignificant Chi2 value at 5% (Chi2 = 1.142, df = α-pinene 0.12 3, P = 0.767). β-pinene 0.27 In the case of C. subinnotatus, mortality observed with Camphene 0.02 the highest dose of C. nardus EO (100 µL/L) was very Sabinene 2.14 low (23.75 ± 15.87%). Moreover, the high standard Myrcene 5.05 deviation showed heterogeneity among individuals cis-3-hexenyl acetate 0.05 tested. The LD10, LD50 and LD90 obtained were 51.26; α-terpinene 0.03 210.09 and 861.12 µL/L, respectively (Table 6). These values were well above the lethal doses observed with C. γ-terpinene 0.05 maculatus. The regression line obtained, is y = - 4.78941 β-terpinene + limonene 3.80 + 3.11143x. The model fits the test data and gives an Paracymene 0.02 insignificant Chi2 value at 5% (Chi2 = 2.137, df = 3, P = cis-β-ocimene 5.06 0.544). trans-β-ocimene 31.58 Terpinolene 0.02 Terpinenol-4 0.05 Effect of C. odorata essential oil 1,3,8-paramenthatriene 3.58 cis-3-hexenyl-2-methyl butyrate 0.03 In C. maculatus adults, the highest dose (50 µL/L) of C. cis-3-hexenyl isovalerate 0.13 odorata EO, showed a low mortality rate (14.37 ± α-copaene 0.06 15.46%). Again, the high standard deviation showed β-ylangene 0.18 heterogeneity of individuals tested. The LD10, LD50 and β-caryophyllene 0.39 LD90 obtained by the Probit method are 42.59; 510.42 α-caryophyllene 0.40 and 6117.44 µL/L, respectively (Table 7). The regression line obtained with this EO is y = - 3.21778 + 1,18828x. A β-caryophyllene oxide 0.24 test of fit of the model to data on observed adults gives Germacrene D 5.87 2 2 an insignificant Chi value at 5% (Chi = 0.4, df = 3, P = γ-elemene 0.09 0.94). 3e,6e-α-farnesene 0.05 This EO also caused a low mortality rate, 18.13 ± γ-cadinene 0.10 13.13% at the highest dose (100 µL/L) in C. subinnotatus, λ-cadinene 0.07 The LD10, LD50 and LD90 obtained were 61.16; 723.02 Calamenene 0.03 and 8546.85 µL/L, respectively (Table 7). The regression trans-nerolidol 0.13 line obtained, is y = - 3.41644 + 1,19491x. The model fits 2 1,6-Germacradien-5-ol 0.08 the test data and gives an insignificant Chi value at 5% 2 trans-cadinol+trans-muurolol 0.12 (Chi = 2.183, df = 3, P = 0.535). trans-muurolol 0.27 Total 60.10 Effect of the essential oil of B. borianiana

B. borianiana EO had a low adulticide activity in C. In the case of C. subinnotatus, the oil caused mortality maculatus and in C. subinnotatus. It caused a mortality comparable to that of C. maculatus (90.62 ± 7.15%) in rate of 8.13 ± 1.13 and 17.5 ± 7.35%, respectively in C. maculatus with 50 µL/L and C. subinnotatus with 100 adults with a dose of 100 µL/L. The LD10, LD50 and LD90 obtained were 13.41; 34.62 and 89.38 µL/L, respectively µL/L. This EO also had the highest LD10, LD50 and LD90 (Table 5). The regression line obtained, is y = - 4.78941 + values obtained by the Probit method (Table 8). 3.11143x. The model fits the test data and gives an insignificant Chi2 value at 5% (Chi2 = 0.63, df = 3, P = 0.889). Comparison of lethal doses of four essential oils tested on both species of weevil

Effect of C. nardus essential oil Comparison of four EO based on their LD50 and LD90, showed that the EO of C. giganteus and C. nardus exhibit With C. nardus EO, the highest dose tested (50 µL/L) strong toxicity. Indeed the LD50 and LD90 values were the caused a mortality of 67% in C. maculatus adults. The lowest. In C. maculatus, the LD50 and LD90 (Table 9) were

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Table 5. Adulticide activity of C. giganteus essential oil upon C. maculatus and C. subinnotatus.

Bruchid species LD C. maculatus C. subinnotatus

LD90 LD50 LD10 LD90 LD50 LD10 Log.Dose 1.685 1.30234 0.91976 1.951 1.53929 1.12735 Standard deviation 0.07252 0.0405 0.0756 0.0709 0.221 0.3947 Dose (µl/L) 48.42 20.06 8.31 89.38 34.62 13.41 Lower limit 37.24 16.49 5.17 26.54 0.0673 0.00014 Upper limit 76.24 24.26 10.91 120.21 60.55 35.555 Regression y = - 4.36295 + 3.35003x y = - 4.78941 + 3.11143x Chi2 = 2.414 ; dd l = 3 ; Chi2 = 0.63 ; dd l = 3 ; Statistical test P = 0.491 P = 0.889 Insignificant test at 5% Insignificant test at 5%

Table 6. Adulticide activity of C. nardus essential oil upon C. maculatus and C. subinnotatus.

Bruchid species LD C. maculatus C. subinnotatus

LD90 LD50 LD10 LD90 LD50 LD10 Log.Dose 2.0183 1.5928 1.1672 2.935 2.3224 1.7097 Standard deviation 0.1333 0.0466 0.1636 0.6253 0.2336 0.1973 Dose (µl/L) 104.32 39.15 14.69 861.12 210.09 51.26 Lower limit 71.51 27.014 1.837 51.226 73.199 21.038 Upper limit 555.59 48.199 23.227 14475.6 602.973 124.879 Regression y = - 4.79723 + 3.01182x y = - 4.85863 + 2.09207x Chi2 = 1.142 ; dd l = 3 Chi2 = 2.137 ; dd l = 3 Statistical test P = 0.767 P = 0.544 Insignificant test at 5% Insignificant test at 5%

Table 7. Adulticide activity of C. odorata essential oil upon C. maculatus and C. subinnotatus.

Bruchid species LD C. maculatus C. subinnotatus

LD90 LD50 LD10 LD90 LD50 LD10 Log.Dose 3.78657 2.70793 1.62929 3.93181 2.85915 1.7865 Standard deviation 3.808 1.694 0.504 3.243 1.266 0.778 Dose (µl/L) 6117.44 510.421 42.588 8546.85 723.021 61.164 Lower limit 0.00021 0.2437 4.3809 0.00375 2.38413 1.8218 Upper limit 1.77.1011 1.07.106 414.006 1.94.1010 2.193 .105 2053.49 Regression y = - 3.21778 + 1.18828x y = - 3.41644 + 1.19491x Chi2 = 0,40 ; dd l = 3 Chi2 = 2.183 ; dd l = 3 Statistical test P = 0.94 P = 0.535 Insignificant test at 5% Insignificant test at 5%

respectively 20.06 and 48.42 µL/L with C. giganteus EO; respectively 34.62 and 89.38 µL/L with the EO of C. they are respectively 39.15 and 104.32 µL/L with C. giganteus while with C. nardus EO; they are 210.09 and nardus EO. In C. subinnotatus, these values are 861.12 µL/L, respectively.

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Table 8. Adulticide activity of B. borianiana essential oil upon C. maculatus and C. subinnotatus.

Bruchid species LD C. maculatus C. subinnotatus

LD90 LD50 LD10 LD90 LD50 LD10 Log.Dose 2.5275 2.12908 1.73065 4.11701 2.9236 1.73019 Standard deviation 0.4945 0.2844 0.0888 4.159 1.5035 1.2277 Dose (µl/L) 336.90 134.61 53.78 13092.1 838.69 53.73 Lower limit 36.157 37.272 36.021 9.23 .10-5 0.9484 0.2107 Upper limit 3139.12 486.049 80.304 1.85. 1012 7.416. 105 1.37.104 Regression y = - 6.84916 + 3.21697x y = - 3.13996 + 1.07401x Chi2 = 0.479 ; dd l = 3 Chi2 = 0.744 ; dd l = 3 Statistical test P = 0.923 P = 0.863 Non-significant test at 5% Non-significant test at 5%

Table 9. Comparison of different lethal doses of four essential oils of C. giganteus; C. nardus; C. odorata and B. borianiana on C. maculatus and C. subinnotatus.

Bruchid species Variables C. maculatus C. subinnotatus Lethal doses (µL/L)

Essential oils LD90 LD50 LD10 LD90 LD50 LD10 C. giganteus 48.42 20.06 8.31 89.38 34.62 13.41 C. nardus 104.32 39.15 14.69 861.12 210.09 51.26 C. odorata 6117.44 510.421 42.588 8546.85 723.021 61.164 B. borianiana 336.90 134.61 53.78 13092.1 838.69 53.73

Effect of essential oils on the spawning capacity of C. females treated with doses ≤ 20 µL/L of EO C. giganteus maculatus and C. subinnotatus females and C. nardus allowed for the observation of oocytes in the ovarioles; some that reached maturity were retained At lower doses (≤ 20 μL/L) of C. giganteus and C. nardus in the oviducts and the copulatory bursa. EO, adults treated, presented an increasing traction difficulty and morbidity which were dose-dependant. These states persist in adults treated with C. giganteus DISCUSSION EO 5 days after treatment, while those treated with the EO of C. nardus, C. odorata and Bidens borianiana Under experimental conditions, the four EO tested regained their mobility and survived within 24 h post showed differential effects on adult mortality of the 2 treatment. The number of eggs laid by females that beetle species. Thus, depending on the effectiveness of regained mobility, increased during the next 2 days and the EO, three (3) groups of EO were found as described showed no significant difference from that of females in by Ketoh (1998). The first group consisted of effective the control group (Figures 1 and 2). EO, that caused up to 70% mortality in the weevil All eggs were fertile. From the third day after the end of population; the second group consisted of less effective treatment, the number of eggs laid by the females EO, causing less than 70% of mortality in adults and the decreased with time up to the ninth day, similarly in the inefficient EO causing no effect on mortality. test and the control groups. However, after treatment with Among the EO tested, only C. giganteus caused over EO of C. giganteus the remaining females of C. 70% adult mortality of C. maculatus at 40 µL/L and C. maculatus laid fewer eggs that diminish in number over subinnotatus adult from 60 µL/L. The effectiveness of this time until the seventh day (Figure 1). The same effect EO has been reported by Seri - Kouassi (2004) on C. was observed in C. subinnotatus females. However, the maculatus where according to their results, C. giganteus latter laid eggs for only 2 days after the treatment ended EO caused mortality in three quarters of the adult (Figure 2). population, at the lowest concentration (6.66 µL/L). The Moreover, the dissection of the abdomen of some difference observed when compared to our results is

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Figure 1. Mean number (X ± SD) of eggs laid daily by C. maculatus females on cowpea seeds after treatment with different essential oils at the dose of 20 µL/l.

Figure 2. Mean number (X ± SD) of eggs laid by C. subinnotatus females on bambara groundnut seeds after treatment with different essential oils at the dose of 20 µL/L.

probably due to the chemotype used. The chemotype piperitone contained in C. schoenanthus showed anti - used in this report contains less limonene (23.05%) than palatable and repulsive effects on ants of the genus that reported by the author (71%). Crematogaster. The low efficiency of other EO would be According to Ketoh et al. (2002), the effectiveness of EO linked to their chemical composition. Indeed according to is usually related to the nature of their main components. Ketoh et al. (2002), the insecticidal activity of plant These authors have shown that it is the terpenes species varies depends on the ecotypes used. This is contained in EO that are toxic to C. maculatus adults. confirmed by Séri - Kouassi (2004), who suggested that The adulticide activity of EO extracted from other species the level of intoxication of insects depends on the of Cymbopogon as C. schoenanthus are due to composition of the EO tested, and composition itself piperitone, the main compound in this EO (Ketoh et al., depends on the plant harvest area and the organ of the 2006). As demonstrated by Bowers et al. (1993), plant used.

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Regarding female spawning capacity of both beetle of the gonadotropin system on the secretion of the egg- species, a significant decrease is found in the number of laying hormone. Retention of eggs would thus induce eggs laid by the females during and/or after exposure to inhibition of ovarian activity. various EO compared to the control at different doses. The EO of C. giganteus and C. nardus reduced the number of eggs laid by females of C. maculatus and C. Conclusion subinnotatus with a rate of reduction greater than 80% for C. maculatus and over 90% for C. subinnotatus relative The EO extracted from the four aromatic plants consisted to the control. mainly of monoterpenes and oxygenated monoterpenes. The spawning reduction observed in both species of These EO were different in their major components and beetle in the presence of EO is probably related to the this difference in composition may influence the biological early death of some females or the morbidity status seen potential of each of these EO. in adults. Similar observations were made by Koumaglo Of the four EO tested, only the essential oil of C. et al. (1996) and Glitho et al. (1997) on the effect of the giganteus showed the greatest adulticide activity against EO of Lippia multiflora and C. schoenanthus, and Seri- C. maculatus and C. subinnotatus. However, adults of C. Kouassi (2004) on the effect of C. giganteus, C. citratus, subinnotatus were tolerant than those of C. maculatus. Ageratum conyzoides, Melaleuca quinquenervia and Essential oils reduced the number of eggs laid during Mentha piperita on C. maculatus. These studies also treatment. The largest reduction was observed with C. confirm those of Schmidt et al. (1991) and Mazibur and giganteus EO and in females of C. subinnotatus. C. Gerhard (1999) who studied the effect of EO from Acorus nardus EO showed a strong ovicidal activity at different calamus (Asteraceae) upon Callosobruchus chinensis doses compared to that of C. giganteus which prevents and C. phaseoli. female oviposition. The oviposition inhibition in C. maculatus and C. subinnotatus females could also be related to a disturbance of oviposition due to EO vapors. Indeed, the CONFLICT OF INTERESTS observation of the reproductive system of some C. subinnotatus females treated with doses ≤ 20 µL/L with The authors have not declared any conflict of interest. the EO of C. giganteus showed in ovarioles, some of the mature oocytes which are in retention in the oviducts and the copulatory bursa. This suggests that EO would cause REFERENCES retention of eggs by physical incapacity for oviposition among females of these beetle species. EO vapors would Agboyi LKBA (2009). 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