1 Introduction

Botanicals of different species have been noted for their insecticidal efficacy. Many of them are believed to contain myriads of chemicals that are insecticidal in nature (Zibaee, 2011). The management of insect pests based on the use of plant extracts and powders have long been made known as potential substitute to synthetic chemical insecticides because they are believed to have lesser or no effect on human and environmental health (Isman et al., 2011). Many of these botanicals have antifeedant and growth reducing effect on insects while many of them have been proven to have toxic effect on the survival of the insects and their ability to oviposite (Berenbaum and Zangerl, 1996; Isman, 2006; Zibaee, 2011; Akinneye and Ogungbite, 2013).

Before the discovery of many nowadays popular synthetic chemical insecticides, botanical powder and extracts have been reported of being used extensively by farmers to protect their farm produce (Isman, 2006; Martins et al., 2012; Forim et al., 2012). Capsicum annum dust for example has been used extensively by ancient farmers to protect their cowpea (Oni, 2014). Thacker (2002) and Isman (2006) traced back the use of botanicals and their derivatives as crop protectant to ancient China, Egypt, Greece and India before they were being downgraded by many synthetic chemical insecticides in the early 1930s. However, many adverse effects associated with these chemical insecticides have made the government of many developed countries of the world to ban their use (2006). Therefore, this has created a chance for botanical insecticides to gain back their first position in the global world insecticide market.

Different factors have been reported to be responsible for the effects of botanical powders and extracts against insects. Oni et al. (2015) reported the different drying methods as a key factor in effectiveness of botanicals. Also, the period of harvest of botanicals and their age have been reported to have considerable effect on their effectiveness as insecticide Doughari (2012). Furthermore, different parts of botanicals have been reported to have different effects against insects (Ashamo et al., 2013). Extraction methods have been opined to have significant effect on botanical insecticides (Ashamo and Ogungbite, 2014; Akinneye and Ogungbite, 2016).

Anacardium occidentale (Linn.) is a medicinal plant which has been used for the treatment of many diseases (Fazali et al., 2011). It belongs to the family anacardiaceae. It has been proven insecticidal against wide range of insects. Ileke and Olotuah (2012) reported its insecticidal potential against Callosobruchus maculatus. However, the comparative efficacy of the different parts of the plant extracted with different methods has not been well established. Therefore, there is necessity to investigate the effect of extraction methods on the efficacy of different parts of the plant. This present work investigated the effect of different extraction methods on the biocidal efficacy of different parts of Anarcadium occidentale against Callosobruchus maculatus infestation on cowpea.

2 Results

2.1 Percentage mortality of C. maculatus treated with different concentrations of different parts of A. occidentale

The effect of different parts of A. occidentale extracts on the survival of C. maculatus on protected cowpea seeds were presented in Table 1. There were statistically significant differences within and between the treatments throughout the period of observation. The mortality of the insect was the function of the part of the plant used, the extraction method, the concentrations of the extract and the period of application. The ethanol extract (4% concentration) of the nut achieved the highest mortality of 46% within 24h post treatment and it was significantly different from others at F= 79.058, df= 48, 98, p < 0.0001 except 3% and 4% ethanolic extract of the nut and the stem bark which respectively recorded 43 and 44% mortality of the insect. At 48 and 72 h post treatment, ethanol extract of the nut recorded the highest mortality of 67.67 and 75.33% mortality respectively and were significantly different from other treatments at F= 93.943, df= 48, 98, p < 0.0001 (48 h) and F=89.389, df=48, 98, p < 0.0001(72). Irrespective of the extraction method and concentration used, the nut of the plant significantly affected the survival of the insect than other parts. Also, making the plant parts and the concentrations used constant, ethanolic extract of the plant recorded high mortality of the beetle compared to cold extract and hot extract of the plant. At 96h post treatment, only ethanolic extract of the nut achieved 100% mortality and its effect was significantly different from other treatments at F= 101.751, df= 48, 98, p < 0.0001. There was significant difference between the control and the plant parts regardless of the period of exposure. Table 2 showed the interactive effect of the plant part, extraction method used and the concentration of the oils on survival of the insect. There was significant interactive effect between the plant part used, the extraction method used and concentration on the mortality of the insect.

2.2 The lethal concentration required to achieve 50 and 95% mortality of C. maculatus by the extract of A. occidentale within 72 h post treatment.

Table 3 showed the estimated concentration of extract of different parts of A. occidentale required to achieve 50 and 95% mortality of C. maculatus within 72 h post treatment. The dosage required varied with the plant part used and the extraction method. With the extraction method being constant, nut extracts appeared most effective among the plant part used as reflected by their Fiducial limits. Thus, the order of effectiveness of the extracts in term of the plant part used could be arranged as nut > stem > root > leaf. Nevertheless, irrespective of the plant part used, the ethanol extracts of the plant appeared most effective as reflected by their Fiducial limits. Hence, the order of effectiveness of the plant in term of extraction method could be arranged as ethanol > hot > cold extraction. The ethanolic extract of the A. occidentale nut was the most effect extract of the plant as it required only 0.42% (0.09-0.75%) and 1.00 % (0.82-1.56%) to achieve 50 and 95% mortality of the beetle within 72 h of application. However, the chi square values that were greater than 3.81 reflected high level of relationship between the concentration used and the mortality of the insect except leaf extracted by hot water which had a chi square value of 3.20. The relationship between the concentrations of the extracts and the mortality of the insect was significant as reflected by the p-values of the chi square at df=13, p < 0.0001 except the leaf extracted by hot water (df= 13, p < 0.07)

2.3 Oviposition and adult emergence of C. maculatus exposed to different concentrations of different parts of A. occidentale

Figure 1 and Figure 2 respectively presented the oviposition and adult emergence of C. maculatus exposed to different concentrations of different parts of A. occidentale extracted by different methods. The oviposition and adult emergence of the insect varied with the part of the plant, extraction method used and concentration of the extract. The extracts statistically significantly reduced the oviposition and adult emergence of the beetle at F=35.086, df =48, 98; p < 0.0001 (oviposition) and F= 287.066, df= 48, 98; p < 0.0001 (adult emergence). Irrespective of the plant part and the extraction method used, none of the concentrations of A. occidentale used was able to prevent the oviposition of adult O. maculatus. Ethanolic and cold extract of the nut and the ethanolic extract of the root at 4% concentration achieved the lowest oviposition of 7.33 and their effect was significantly (p < 0.05) different from other treatments. Regardless of the plant part used, the ethanolic extract of the plant significantly reduced the oviposition of the beetle compared to extraction methods. Furthermore, only the ethanolic extract of the nut at all concentration, 2, 3 and 4% ethanolic stem extract, 3 and 4% ethanolic root extract and 4% ethnolic leaf extract used as well as all concentrations of the nut extracted with hot water prevented the emergence of the adult C. Maculatus.

2.4 Damage and weight loss of cowpea seeds treated with different concentrations of different parts of A. occidentale

Figure 3 and Figure 4 respectively presented the damage and weight loss of cowpea seeds protected with different concentrations of different parts of A. occidentale extracted by different methods. The damage and weight loss of the cowpea seeds varied with the part of the plant, extraction method used and concentration of the extract. The extracts statistically significantly reduced the damage and weight loss of the protected cowpea seeds at F=384.792, df =48, 98; p < 0.0001 (damage) and F= 3592.567, df= 48, 98; p < 0.0001 (weight loss). Regardless of the plant part used, the ethanolic extract of the plant significantly reduced or prevented the ability of the beetle to cause damage and weight loss of the protected cowpea seeds. Furthermore, only the ethanolic extract of the nut at all concentration, 2, 3 and 4% ethanolic stem extract, 3 and 4% ethanolic root extract and 4% ethnolic leaf extract used as well as all concentrations of the nut extracted with hot water prevented the emergence of the adult beetle.

2.5 Correlation between mortality and oviposition as well as between adult emergence and weight loss

The correlation between adult mortality at 96 h post treatment and oviposition as well as adult emergence and weight loss of the protected cowpea seed were presented in Table 4 and Table 5 respectively. The R values of the regression that tend to 1 reflected high correlation between the mortality of the insect and the oviposition rate. However, hot water extract of the nut recorded the highest R value of (0.982). Also, the R² value of the hot extract of the nut showed that only 96.4% of the oviposition rate of the insect are being determined by the mortality of the insect. Nevertheless, after the adjustment of the value only 96.1% of the oviposition can be explained by the insect mortality. The t-value of -18.675 of the hot water extract of the nut that was more negative than -1.98 indicated that there was a great statistically significant relationship between the mortality and oviposition. Irrespective of the plant part used, all the extracts of A. occidentale recorded statistically significant relationship between the insect mortality and oviposition at F=261.987, df =1, 13, p < 0.0001 (NC); F=236.372, df =1, 13, p < 0.0001 (SC); F= 109.123, df =1, 13, p < 0.0001 (RC); F= 67.562, df =1, 13, p < 0.0001 (LC); F= 348.742, df =1, 13, p < 0.0001 (NH); F=150.433, df =1, 13, p < 0.0001 (SH); F=103.689, df =1, 13, p < 0.0001 (RH); F=90.381, df =1, 13, p < 0.0001 (LH); F=301.730, df =1, 13, p < 0.0001 (NE); F=258.375, df =1, 13, p < 0.0001 (SE); F=270.390, df =1, 13, p < 0.0001 (RE) and F=48.275, df =1, 13, p < 0.0001 (LE). In addition, the F-value of the Nut extracted with water showed that it recorded the highest relationship between the insect mortality and the oviposition. There was a great relationship between the adult emergence of the insect and the weight loss of the protected cowpea grains as reflected by their R value which tends to 1. However, the nut extracts of the plant recorded the highest R value (0.999) regardless of the extraction method used. Also, The R² value of the nut extracts showed that adult emergence of the beetle was responsible for 99.8% of the seed weight loss of the cowpea even after adjustment. The t-values of the regression that were greater than 1.98 showed that there was correlation between the adult emergence of the beetle and the weight loss of the cowpea with the nut extracts recorded the highest t-value (87.847). Regardless of the plant part used, all the extracts of A. occidentale recorded statistically significant relationship between the adult emergence of the insect and weight loss of the cowpea at F=7717.107, df =1, 13, p < 0.0001 (NC); F=1024.332, df =1, 13, p < 0.0001 (SC); F= 1091.932, df =1, 13, p < 0.0001 (RC); F= 712.071, df =1, 13, p < 0.0001 (LC); F= 7717.107, df =1, 13, p < 0.0001 (NH); F=3072.066, df =1, 13, p < 0.0001 (SH); F=2598.284, df =1, 13, p < 0.0001 (RH); F=5250.411, df =1, 13, p < 0.0001 (LH); F=7717.107, df =1, 13, p < 0.0001 (NE); F=302.003, df =1, 13, p < 0.0001 (SE); F=195.893, df =1, 13, p < 0.0001 (RE) and F=248.729, df =1, 13, p < 0.0001 (LE). In addition, the F-value of the Nut extracts showed that it recorded the highest relationship between the adult emergence and the seed weight loss.

3 Discussion

The result obtained in this research showed that all the parts of A. occidentale have insecticidal properties and their effectiveness varied with their extraction method, concentration used and period of application. All the parts of the plant recorded high mortality of the insect irrespective of their extraction method. The ethanolic extracts of the plant however, appeared more effective than the hot water extract and cold extract of the plant. Also, it was observed that the nut of the plant was more effective than the three other parts of the plant regardless of the extraction method used. The probit regression analysis however showed that the ethanolic extract of the nut was the most effective extract of the plant as it required the lowest concentration to achieve 50 and 95% death of the insect within 72h post treatment. The high mortality of the insect recorded by the ethanolic extract of the plant parts could be due to the ability of the ethanol to dissolve the phytochemicals present in the plant parts as suggested by Ashamo and Ogungbite (2014). Neoliya et al., (2007) and Al-Qahtani et al., (2012) reported that the effectiveness of plant materials varied with respect to the part of the plant and the extraction method used. Epidi and Udo (2009) opined that the degree of effectiveness of botanical extract varied with the type of solvent used for their extract because different solvents have different degree at which that can extract the active compound present in the botanicals. Ashmo et al., (2013) reported variation in the potency of Newbouldia laevis parts on C. maculatus. These authors reported that the variation in the potency of N. laevis parts could be due to the varying phytochemicals present in the them. Tannin, saponins, alkaloid and flavonoid have been reported as the major phytochemicals present in different parts of A. occidentale but in different proportion (Fazali et al., 2011). However, alkaloid, tannin and saponin are being reported of being absent in the leaf extract of A. occidentale (Fazali et al., 2011) and could be the major reasons why the leaf extract of A. occidentale was not as effective as other parts regardless of the extraction method used. It has been known that adult C. maculatus don’t feed thus lead to their short life sperm. However, if being provided with sugary substance they can feed and live for longer period. Therefore, the high mortality of the beetle recorded by the extracts of A. occidentale reflected that the extracts have no sugary substance upon which the insect can feed on, hence lead to their starvation.  Botanicals have been known for their ability to block the voltage-gated sodium channels in the nerve axons or electron transport chain (in the mitochondrion, leading to inhibition of energy production) (Hollingworth et al., 1994; Schmutter, 2002; Isman, 2006; Zibaee, 2011). So, the high insect mortality recorded in this work could be due to ability of the extracts from different parts of the plant to cause total knockdown of the insect or inhibit the production of energy in the insect. Also, these extracts may have blocked the breathing pores (spiracle) on the insect and thereby caused asphyxiation and subsequent death of the insect. The result obtained on mortality of the insect was in agreement with the findings of Ashamo et al., (2013) in which different botanicals extracted with different solvent recorded high mortality of C. maculatus. The general linear analysis done on the mortality showed that there was significant interactive effect of the plant parts, the method of extraction and the concentration of the extracts on the mortality of C. maculatus.

It was noted in this study that the extracts of A. occidentale significantly reduced the oviposition and adult emergence of C. maculatus and its ability to cause damage and weight loss of the protected cowpea seeds. However, the oviposition and adult emergence of the beetle and its ability to cause damage and weight loss of the cowpea changed with the parts of the plant used, method of extraction and concentration of the extracts. The ability of the extracts to reduce oviposition of the insect could be due to inability of the insect to mate because many of the insects must have been experiencing hyperactivity and convulsions as suggested by Isman (2006). Botanicals have been reported to have ability cause sterility in insects (Isman, 2006; Zibaee, 2011). Therefore, both the sexes of the beetle must have been sterilized because of their exposure to the extracts of A. occidentale and thereby reduced their oviposition rate. The regression analysis showed that mortality of the insect was responsible more than 80% oviposition. So, low oviposition rate recorded could be due to high insect mortality. However, the nut extracts of the plant recorded the lowest oviposition rate of the insect. The reduction or prevention of the adult emergence of C. maculatus recorded in the study might be due to inability of the insect to lay many eggs. Isman (2006) reported that botanicals have ability of inhibiting the synthesis and release of ecdysteroids from their prothoracic gland and this in turn causes incomplete ecdysis in their larvae. Therefore, the prevent or reduction in adult emergence of C. maculatus by the extracts of A. occidentale could be due to inability of the insect larvae to castoff their exoskelecton which remained connected to their posterior abdomen. Digestion of food is playing an important role in the existence of living organisms and it involves assistance of many enzymes. The disruption of the normal activities of these digestive enzyme causes inability of insects and other living organisms to provide their nutrients for biological requirements (Zibaee, 2011). Botanical insecticides have been noted for the inhibitory effect on wide range of digestive enzymes in insect (Zibaee and Bandani, 2010). Several botanical extracts have been reported of inhibiting the a-amylase, glycosidases, lipases and proteases (Terra and Ferriera, 2005; Senthil et al., 2006; Shekari et al., 2008; Zibaee et al., 2008; Zibaee et al., 2009). Therefore, no or low percentage adult emergence of C. maculatus exposed to different extracts of different parts of A. occidentale could be due to inability of the insect larvae to digest the treated cowpea seeds which in turn lead to their death. The result of this work acquiesced with the findings of Epidi and Udo (2009) in which different extracts of Ricinodendron heudelotii reduced the emergence of C. maculatus. Also, the findings of Abd-Elhady (2012) concise with the results of this study as the essential oil of Artemisia Judaica was found to significantly affected the life stages of C. maculatus.

The prevention or reduction of the damage and weight loss of the protected cowpea seed achieved by the extracts of A. occidentale may due to inability of the insect larvae to fed on the protected cowpea seeds as suggested by Ogungbite (2015). The linear regression analysis showed that more than 98% of the seed weight loss was determined by the adult emergence. Consequently, no or low weight loss recorded by the seeds reflected no or low adult emergence of the beetle.

4 Conclusion

The results of this research have proven biocidal efficacy of extracts of different parts of A. occidentale against C. maculatus and its ability to cause damage and weight loss of cowpea seeds. The result showed that only little concentrations of the extracts were required to achieve high mortality of the insect. Also, it was revealed that the mortality of the insect had significant effect on oviposition and weight loss of the seeds respectively as reflected by the linear regression analysis. However, the nut extracts of the plant showed more biotoxic efficacy against the insects than other parts of the plant while the ethanolic extracts of the plants recorded the highest biocidal efficacy on the insect.  Since, the extracts of A. occidentale have proven insecticidal against C. maculatus it could be introduce to management system of the insect. Moreover, the ethanolic extracts of the plant appeared more promising than other extracts while the nut of the plant showed more biocidal efficacy.

5 Materials and Methods

5.1 Insect culture

The initial culture of the insect used was gotten from an existing culture in the food storage research laboratory of the Department of Biology, Federal University of Technology, Akure. The insect was reared on an uninfested cowpea collected from National seed research institute, Ibadan, Nigeria. The insects were cultured at temperature of 28±2ºC and relative humidity of 75±5% inside plastic container covered with muslin cloth to disallow the escape of the insect and as well disallow the entry of other insects that may be a parasitoid of C. maculatus. The culture was maintained by replacing the devoured seeds with new uninfested seeds.

5.2 Collection of Plant Materials

The culture nuts used were collected from an open field from a mature cashew tree in Ado-Ekiti, Ekiti State, Nigeria. The nuts were sun dried between 8.00 am to 12.00 pm, in order to ensure their phytochemicals are not denatured by extreme temperature. The leaves, stem bark and the root bark were collected from an open field inside Ekiti State University, Ado-Ekiti, Nigeria. These parts were air dried in the laboratory of Department of Plant Science of the University. After drying, the samples were separately pulverized into fine powder and were kept inside separate airtight plastic containers.

5.3 Extraction of the Plant Extracts

The ethanolic extracts of the plant powders were made by separately soaking 50g of each of the plant powders in 100ml of ethanol for 3 days. The hot water extracts of each of the plant parts were made by separately soaking 50g of the powders in hot water of 60^oC temperature for 30 minutes in a heating mantle. Then the extracts were removed from the heating mantle. Cold water extracts of the plant parts were made by separately soaking the powders of the plant parts inside water of 3^oC temperature stored for 3 days inside refrigerator to maintain the temperature. The extracts were stirred early in the morning for the period of extraction in order to ensure uniform extraction of the extracts. Irrespective of the extraction method used, the extracts were separated from the solvents used by sieving using muslin cloth. The following extracts were made:

Nut cold extract (NC), stem cold extract (SC), root cold extract (RC), leaf cold extract (LC), nut hot extract (NH), stem hot extract (SH), root hot extract (RH), leaf hot extract (LH), nut ethanolic extract (NE), stem ethanolic extract (SE), root ethanolic extract (RE) and leaf ethanolic extract (LE).

5.4 Effect of Cashew Plant Parts Extracts on Mortality of Callosobruchus maculatus.

Aliquot of 0.2, 0.4, 0.6 and 0.8 ml which corresponds to 1.0 %, 2.0 %, 3.0 % and 4.0 % v/w plant extracts were separately pipetted into Petri dishes (9 cm diameter) containing 20 g of disinfested cowpea seeds. Three replicates per treatment were prepared. Untreated cowpea seeds were set as control in triplicate. The cowpea seeds and the extracts were thoroughly mixed with the aid of a glass rod to ensure uniform coating of the extracts on the seeds. The seeds were then air-dried for 4 hours before introducing the insect. 20 adult C. maculatus were introduced into each petri-dish and they were observed daily for 4 days for mortality. After every 24 hours, the numbers of dead beetles were counted. The percentage adult mortality was calculated using the formula below:

All the insects (both dead and live) were removed on the fifth day of observation and oviposition was recorded. The treatments were left for 20 days and adult emergence was counted till no emergence was observed for five days. The percentage adult emergence was calculated with the formula below:

The severity of damage and weight loss of the protected cowpea was done by counting the number of seeds with hole and weighing them after no emergence of adult was observed. The percentage seed damage and weight loss were calculated using the formulae below:

5.5 Statistical analysis

Data obtained were subjected to One-Way analysis of variance and means were separated with Duncan’s Multiple Range Test. Also, data obtained from mortality were subjected to Probit analysis to calculate the lethal concentration required to achieve 50 and 95% mortality within 72 h (LC₅₀ and LC₉₅). General Linear Model (GLM) was used to determine the interactive effect of the plant part used, extraction methods and concentrations. Linear regression analysis was done to determine interaction between adult mortality and oviposition as well as between adult emergence and weight loss.

Authors’ contributions

Both authors designed and carryout the research, author 2 carryout the statistical analysis while the two authors wrote the paper.

Refrences

Abd-Elhady 2012, Insecticidal activity and chemical composition of essential oil from Artemisia judaica L. against Callosobruchus maculatus (f.) (Coleoptera: Bruchidae). Journal of Plant Protection Research, 52(3):347-352. DOI: 10.2478/v10045-012-0057-9

https://doi.org/10.2478/v10045-012-0057-9

Akinneye J.O., and Ogungbite O.C., 2013, Insecticidal activities of some medicinal plants against Sitophilus zeamais (Motschulsky) (Coleoptera: Curculionidae) on stored maize, Arch Phytopathol. Plt Protect., 46:(10):1206-1213

https://doi.org/10.1080/03235408.2013.763614

Akinneye J.O., and Ogungbite O.C., 2016, Entomotoxicant potential of some medicinal plant against Ephestia cautella infesting cocoa bean in storage. Intl J Appl Sci and Eng, 14(1): 59-68

Al-Qahtani A.M., Al-Dhafar Z.M., and Rady M.H., 2012, Insecticidal and biochemical effect of some dried plants against Oryzaephilus surinamensis (Coleoptera-Silvanidae). The J Basic Appl Zoo, 65: 88–93

https://doi.org/10.1016/j.jobaz.2012.10.008

Ashamo M.O., and Ogungbite O.C., 2014, Extracts of medicinal plants as entomocide against Sitotroga cerealella (Olivier) infestation on paddy rice, Medicinal Plant Research, 4(18): 1-7

Ashamo M.O., Odeyemi O.O., and Ogungbite O.C., 2013, Protection of cowpea, Vigna unguiculata L. (Walp.) with Newbouldia laevis (Seem.) extracts against infestation by Callosobruchus maculatus (Fabricius). Arch Phytopatho Plt Protect, 46(11): 1295-1306

https://doi.org/10.1080/03235408.2013.765136

Berenbaum M.R., and Zangerl A.R., 1996, Phytochemical diversity: adaptation or random variation? Recent Adv Phytochem (in press)

https://doi.org/10.1007/978-1-4899-1754-6_1

Doughari J.H., 2012, Phytochemical: Extraction Methods, Basic Structures and Mode of Action as Potential Chemotherapeutic Agents, Phytochemical - A Global Perspective of Their Role in Nutrition and Health, Dr Venketeshwer Rao (Ed.), ISBN:978-953-51-02960

Epidi T.T., and Udo I.O., 2009, Biological Activity of Ethanolic Extract Fractions of Dracaena arborea Against Infestation of Stored Grains by Two Storage Insect Pests. Pakistan J. Bio Sci., 12(13): 976-980. DOI: 10.3923/pjbs.2009.976.980

https://doi.org/10.3923/pjbs.2009.976.980

Fazali F., Zulkhairi A., Nurhaizan M.E., Kamal N.H., Zamree M.S., and Shahidan M.A., 2011, Phytochemical screening, in vitro and in vivo antioxidant activities of aqueous extract of Anacardium occidentale Linn. and its effects on endogenous antioxidant enzymes in hypercholesterolemic induced rabbits. Research Journal of Biological Sciences, 6 (2): 69-74

https://doi.org/10.3923/rjbsci.2011.69.74

Forim M.R., Da-silva M.F.G.F., and Fernandes J.B., 2012, Secondary metabolism as a measurement of efficacy of botanical extracts: The use of Azadirachta indica (Neem) as a model. In: Perveen F (Ed.), Insecticides-Advances in Integrated Pest Management, 2012, p367-390

Hollingworth R., Ahmmadsahib K., Gedelhak G., and McLaughlin J., 2010, New inhibitors of complex I of the mitochondrial electron transport chain with activity as pesticides. Biochemical Society and Transgenesis, 1994; 22, 230–33

https://doi.org/10.1042/bst0220230

Ileke K.D., and Olotuah O.F., 2012, Bioactivity Anacardium occidentale (L.) and Allium sativum (L.) powders and oil extracts against cowpea bruchid, Callosobruchus maculatus (Fab.) (Coleoptera: Bruchidae). Intl J Bio, 4:8–13

Isman et al., 2011,Commercial opportunities for pesticides based on plant essential oils in agriculture, industry and consumer products Phytochemi. Rev. 10:197-204

Isman M.B., (2006) Botanical insecticides, deterrents, and repellents in modern agriculture and an increasingly regulated world. Annal Rev Entomo, 51: 45-66

https://doi.org/10.1146/annurev.ento.51.110104.151146

Martins C.H.Z., Freire M.G.M., Parra J.R.P., Macedo M.L.R., 2012, Physiological and biochemical effects of an aqueous extract of Koelreuteria paniculata (Laxm.) seeds on Anticarsia gemmatalis (Huebner) (Lepidoptera: Noctuidae), SOAJ of Entomolog. Studies, 1:81-93

Neoliya N., Dwijendra S., and Sangwan R., 2007, Azadirachtin-based insecticides induce alteration in Helicoverpa armigera Hub. head polypeptide. Current Sci., 92 (1): 94–99

Ogungbite O.C., 2015, Entomopoison efficacy of fume of different parts of Newbouldia laevis against Callosobruchus maculatus in Storage. Intl. J. Res. Stud. Microbio. Biotech, 1(1):6-14

Oni M.O., 2014. Entomotoxic efficacy of cayenne pepper, sweet pepper and long cayenne pepper oil extracts against Sitophilus zeamais infesting maize grain. Mol Entomo, 2014: 5(5): 37-44 doi: 10.5376/me.2014.05.0005

https://doi.org/10.5376/me.2014.05.0005

Oni M.O., Ogungbite O.C., and Akindele A.K., 2015, The Effect of Different Drying Methods on Some Common Nigerian Edible Botanicals. International Journal of Advanced Research in Botany, 2015, 1(1):1-8

Schmutter H., 2002, The Neem Tree. Mumbai: Neem Found. 892 pp

Senthil Nathan S., Chunga P.G., and Muruganb K., 2006, Combined effect of biopesticides on the digestive enzymatic profiles of Cnaphalocrocis medinalis (Guenee) (the rice leaffolder) (Insecta: Lepidoptera: Pyralidae). Ecotoxicol Environ Safety, 64, 382–389

https://doi.org/10.1016/j.ecoenv.2005.04.008

Shekari M., Jalali Sendi J, Etebari K, Zibaee A, Shadparvar A. Effects of Artemisia annua L. (Asteracea) on nutritional physiology and enzyme activities of elm leaf beetle, Xanthogaleruca luteola Mull (Coleoptera: Chrysomellidae). Pest., Biochem.Physiol, 2008; 91:66-74

https://doi.org/10.1016/j.pestbp.2008.01.003

Terra W.R., and Ferriera C., 2005, Biochemistry of digestion. In: Comprehensive molecular insect science by Lawrence I. Gilbert, Kostas Iatrou, and Sarjeet S. Gill, Elsevier, 3:171-224

https://doi.org/10.1016/b0-44-451924-6/00053-3

Thacker J.M.R., 2002, An Introduction to Arthropod Pest Control. Cambridge, UK: Cambridge Univ. Press. 343 pp

Zibaee A, Bandani A.R., and Ramzi S., 2008, Lipase and invertase activities in midgut and salivary glands of Chilo suppressalis (Walker) (Lepidoptera, Pyralidae), rice striped stem borer. Invert. Survi. J. 5:180-189

Zibaee A., 2011, Botanical insecticides and their effects on insect biochemistry and immunity, pesticides in the world. Pests Control and Pesticides Exposure and Toxicity Assessment, pp. 55-68. Dr. Margarita Stoytcheva (Ed.) ISBN: 978-953-307-457

Zibaee A., and Bandani A.R., 2010. Effects of Artemisia annua L. (Asteracea) on digestive enzymes profiles and cellular immune reactions of sunn pest, Eurygaster integriceps (Heteroptera: Scutellaridae), against Beauvaria bassiana. Bull Entomolo Res, 100:185-196

https://doi.org/10.1017/S0007485309990149

Zibaee A., Bandani A.R., and Ramzi S., 2009, Characterization of and glucosidases in midgut and salivary glands of Chilo suppressalis Walker (Lepidoptera: Pyralidae), rice striped stem borer. Comptes Rendus Biologies,:633–641

https://doi.org/10.1016/j.crvi.2009.02.009