Insecticidal Activity of Ethanolic Leaf Extracts against the Maize Weevil, Sitophilus zeamais Motsch (Coleoptera: Curculionidae)

Synthetic insecticides have been commonly used for controlling pests in stored products [1-4]. However, this has resulted in problems such as pest resurgence and increase in costs of application arising from the development of resistance to insecticides [5,6]. This has led to the search of alternative, eco-friendly methods of controlling insect pests of storage such as the use of botanicals [7,8]. In this regard the toxicity of many plant materials has been evaluated against S. zeamais in stored grains with recorded success [7,9-12].

One of the ways used to evaluate the toxicity of insecticides is by measuring their mortality effects on insects. Ethanolic, methanolic and acetonic extracts of various plant materials have been reported to cause high mortality of S. zeamais [9,12-14]. It was reported that some medicinal plants such as olax subscorpioidea Oliver (Santalales: Olacaceae), aframomum melegueta k. schum (Zingiberales: Zingiberaceae) and zingiber officinale Rosce (Zingiberales: Zingiberaceae) caused adult mortality of S. zeamais which ranged from 18.35 to 88.35% after 72 hours of exposure [15]. Tilahun and Daniel reported the toxicity effects of seed powder of Azadirachta indica a. Juss (Sapindales: Meliaceea) against S. zeamais and recorded 23.00 to 28.67% adult mortality after 45 days of treatment [16].

Another way of assessing toxicity of insecticides is by evaluating their effects on biochemical parameters of insects [17]. Kumar and Michael noted that the carbohydrates, proteins and lipids play an important role in the biochemical process underlying growth and development of insects [18]. Fox et al., reported that the reproductive capacity of adult female insects is affected largely by their body protein content [19]. Piri et al., noted that proteins are important for individual-level fitness-associated traits such as body size, growth rate and fecundity [20]. At high levels of organization, proteins have been linked to population dynamics, life cycles and even biological diversification [20]. Some of the biochemical composition of Acanthoscenlides obtectus Say, S. oryzae, S. granarius and T. confusum have been investigated [17-19,21,22]. However, little is known with regards to toxicity of botanicals against body protein of S. zeamais.

In addition to body protein, enzymes such as Acetycholinesterase (AChE) play a vital role in insect survival. AChE is a key enzyme of the cholinergic system regulating the level of acetylcholine and terminates nerve impulses by catalyzing the hydrolysis of acetylcholine [23]. Some botanicals were reported to inhibit AChE activity in some insect pests of storage such as S. oryzae, S. granarius and T. castaneum , but little is known on effects of botanicals in inhibiting AChE activity in S. zeamais [24-28]. This study was carried out to investigate the ability of E. balsamifera, L. inermis, M. hirtus and E. balsamifera, L. inermis, M. hirtus to cause adult mortality, reduce concentration of body protein and inhibit AChE activity in S. zeamais.

One hundred adults of S. zeamais were obtained from infested sorghum grains at Katsina Central Market and then introduced into 500ml rearing bottles containing 250g of the disinfested sorghum grains. The bottles were covered with muslin cloth and secured with rubber bands. They were then kept in an incubator for oviposition at 30°C and 70% RH for seven days, after which the parents were removed. The bottles were maintained in the incubator under the same condition for adult emergence. Progeny of 7 days old were sieved and used for the laboratory experiments.

Fresh leaves of E. balsamifera, L. inermis, M. hirtus and E. balsamifera, L. inermis, M. hirtus were shade-dried and ground into powder using a laboratory blender. One hundred grams of each plant powder was dissolved in 400ml of ethanol in conical flasks. Mouths of the conical flasks were properly corked and kept in a refrigerator for 48 hours. The extract was separated using muslin cloth and filtered with Whatman No.1 filter papers as described by Khaliq, et al., [29]. The filtrate was separately concentrated by evaporating excess solvents using rotary evaporator with rotary speed of 3 to 6 rpm for 8 hours. The resulting extract was air-dried to remove traces of the solvent and stored in refrigerator at 4°C [30].

The method of de Oliveira, et al., was adopted to assess adult mortality of S. zeamais in sorghum grains treated with ethanolic leaf extracts of the test plants [31]. Crude extracts were diluted with ethanol to make different concentrations of 2.5, 5.0 and 10.0 x 10⁴ ppm. Twenty grams of sorghum grains were weighed and put into 250 ml plastic bottles. The grains were impregnated with 2ml of the ethanolic extracts at three concentrations, while the control contained the grains. Ten weevils were released into each of the bottles after complete evaporation of the solvent. The grain mass was mixed thoroughly with the aid of glass rod and air-dried until complete solvent evaporation. All treatments were arranged in a Completely Randomized Design (CRD) in the incubator and replicated four times. The set-ups were inspected daily and dead weevils in each treatment were removed and recorded daily for three days.

To evaluate LC₅₀ of the ethanolic extracts of the botanicals against S. zeamais, methods of Ebadollahi and Mahboubi were employed [32]. The number of dead weevils at the end of 24 hour exposure to the extracts was used to determine the LC₅₀ of the botanicals by using probit analysis with SPSS (version 16.0) software package [33].

Samples for protein assays were prepared following Askar, et al., [17]. Adult weevils exposed to LC₅₀ of ethanolic extracts of the botanicals for 7 days were freeze killed at - 20°C. A sample of 0.5g of the insects was taken from each bottle and homogenized in 10 times (5mL) volumes (w/v) of phosphate buffer (pH 7.2) using a glass homogenizer under ice. The total homogenates were centrifuged at 4000 rpm for 10min at 4°C using a centrifuge. The supernatant was transferred to new eppendof’s tubes and preserved at - 20°C for spectrophotometry.

The body protein was estimated following the method of Lowry, et al., in triplicates with Bovine Serum Albumin (BSA) as the standard [34]. The absorbance was measured spectrophotometrically after 30mins at 750nm against the blank for both test samples and the standard solutions. The protein concentration was calculated and expressed as µg/ml wet tissue using the formula:

S. zeamais adults were fumigated with LC₅₀ of ethanolic extracts of E. balsamifera, L. inermis, M. hirtus and E. balsamifera, L. inermis, M. hirtus as fumigant toxicity assay. After seven days of fumigation, the weevils were homogenized in phosphate buffer saline (50mM, pH 8) using a Teflon glass tissue homogenizer. The homogenate was centrifuged at 10,000 rpm for 20min at 4°C. The supernatant was filtered and used as the AChE preparation. Methods of Ellman, et al., and Chaubey were adopted for the assay and AChE activity was determined spectrophotometrically [28,35]. Absorbance in the treated samples was evaluated at 412nm for 6min and compared to that of the control. All measurements were made in triplicate [25,26,28].

AChE activity inhibition was calculated as follows [26]:

Data generated were tested for normality using Shapiro-Wilk and Jacque-Bera normality tests [36-38]. Data from adult mortality tests were subjected to two-way ANOVA using Graph Pad Prism (version 7.03). Probit analysis was employed to calculate LC₅₀ of the extracts with the significant differences determined by X² goodness of fit using SPSS (version 16.0). One-way ANOVA was used to test if there were significant differences in the body protein and AChE activity inhibition in adults of S. zeamais exposed to LC₅₀ of the extracts by using PAST (version 2.17). Significantly different means were separated by Bonferroni’s and Tukey’s multiple comparisons tests [38]. All analyses were performed as statistically significant p<0.05.

Ethanolic leaf extracts of the selected botanicals applied at different concentrations of 2.5, 5.0 and 10.0x10⁴ ppm exhibited varying percentage mortalities of S. zeamais within 1, 2 and 3 days after treatment (Table 1).

Treatments	Conc.(x 104ppm)	Mean Adult Mortality (% ± S. E.)
		Days After Treatment (DAT)
		1	2	3
	2.5	65.00 ± 2.89bc	90.00 ± 4.08ab	100.00 ± 0.00a
E. balsamifera	5	75.00 ± 2.89ab	95.00 ± 2.89a	100.00 ± 0.00a
	10	85.00 ± 2.89a	100.00 ± 0.00a	100.00 ± 0.00a
	2.5	55.00 ± 2.89c	77.50 ± 4.79b	97.50 ± 2.50a
L. inermis	5	72.50 ± 2.50b	92.50 ± 2.50ab	100.00 ± 0.00a
	10	77.50 ± 2.50ab	95.00 ± 2.89a	100.00 ± 0.00a
	2.5	67.50 ± 4.89bc	80.00 ± 4.08ab	95.00 ± 2.89a
M. hirtus	5	70.00 ± 4.08bc	92.50 ± 4.89ab	100.00 ± 0.00a
	10	75.00 ± 2.89ab	95.00 ± 2.89a	100.00 ± 0.00a
	2.5	55.00 ± 2.89c	72.50 ± 2.50b	82.50 ± 2.50b
E. balsamifera, L. inermis, M. hirtus	5	67.50 ± 4.79bc	85.00 ± 2.89ab	95.00 ± 2.89a
	10	70.00 ± 4.08bc	90.00 ± 4.08ab	100.00 ± 0.00a
Control	0	0.00 ± 0.00d	0.00 ± 0.00c	0.00 ± 0.00c

Analysis of variance indicated that the difference in adult mortalities of S. zeamais was highly significant among the botanicals (F (4, 45) = 2947.00, p<0.0001) as well as within the concentrations (F (2, 45) = 18.00, p<0.0001). Bonferroni’s multiple comparisons test revealed that at 2.5 x 10⁴ ppm, E. balsamifera, L. inermis and M. hirtus caused statistically similar mortality which was higher than that in E. balsamifera, L. inermis, M. hirtus at 3 DAT. At 5.0 and 10.0 x 10⁴ ppm, all the botanicals had the same effect on the weevils’ mortality within 3 DAT.

Table 2 shows that L. inermis had the highest LC₅₀ value as 1.72 x 10⁴ ppm, while E. balsamifera had the lowest as 1.12 x 10⁴ ppm at 24 HAT. The probit equations were y = 1.074x - 0.053, y = 1.065x - 0.251, y = 0.365x + 0.296 and y = 0.669x - 0.100 for E. balsamifera, L. inermis, M. hirtus and E. balsamifera, L. inermis, M. hirtus , respectively (Table 2). The slopes varied between 0.365 ± 0.723 and 1.074 ± 0.739. X²goodness of fit (0.005 - 0.092) reveals that the LC₅₀ of ethanolic extracts against the weevils was not significantly different (p>0.05) among concentrations of each of the botanicals.

Botanicals	LC50 (x 104 ppm)	95% CI	Slope ± S.E.	X2	p value
E. balsamifera	1.12	0.97 - 3.11	1.074 ± 0.739	0.005	0.946
L. inermis	1.72	0.88 - 3.01	1.065 ± 0.712	0.092	0.762
M. hirtus	1.15	1.07 - 2.30	0.365 ± 0.723	0.006	0.938
E. balsamifera, L. inermis, M. hirtus	1.41	1.21 - 2.55	0.669 ± 0.701	0.065	0.798

The protein in S. zeamais exposed to ethanolic extracts of the botanicals appeared to follow the order: E. balsamifera<L. inermis< M. hirtus<S. obtusifolia<control (Figure 1). Significant difference (F (4, 10) = 2861.00, p < 0.0001) existed in protein content among the weevils exposed to sorghum grains treated with LC₅₀ of ethanolic extracts of different test botanicals. Further, Tukey’s multiple comparisons test revealed that all treatments differed from each other with insects exposed to E. balsamifera having the least body protein content.

 

Figure 1: Concentration of body protein in S. zeamais exposed to LC₅₀ of ethanolic leaf extracts.

 

The inhibition of AChE activity in S. zeamais by ethanolic extracts was 79.61, 77.65, 74.90 and 70.98 for E. balsamifera, L. inermis, M. hirtus and E. balsamifera, L. inermis, M. hirtus , while none was recorded in weevils exposed to the untreated grains (Figure 2). One-way ANOVA revealed that inhibition of the AChE activity was significantly different (F (4, 10) = 4190.00, p<0.0001) among the botanical treatments. Tukey’s pairwise comparisons showed that the inhibition activity was statistically similar in weevils exposed to E. balsamifera and L. inermis but higher than the rest.

 

Figure 2: AChE activity inhibition in S. zeamais exposed to LC₅₀ of ethanolic leaf extracts.

Ethanolic extracts of the selected botanicals were found to be highly effective by resulting in total mortality of adults of S. zeamais in sorghum grains at all concentrations of E. balsamifera and higher rates of L. inermis, M. hirtus and E. balsamifera, L. inermis, M. hirtus . The efficacy of the test botanicals is in line with earlier findings in which ethanolic extracts of some plant species caused adult mortality of maize weevils [10,13]. Ajayi reported that ethanolic extract of D. regia seeds applied at 2.0mg/g maize grains resulted in 12.50% adult mortality of S. zeamais after five days of exposure [10]. Similarly, findings of Ibrahim, et al., showed that application of ethanolic leaf extract of C. odorata at 10ml / 50g maize caused 14.00% adult mortality of S. zeamais at 7 DAT [13]. The observed increase in mortality of S. zeamais caused by ethanolic extracts with increase in post treatment period in this study agrees with previous findings [10,12,13].

Ethanolic leaf extracts of E. balsamifera, L. inermis, M. hirtus and E. balsamifera, L. inermis, M. hirtus were generally found toxic to adult S. zeamais and suppressed their survival in sorghum grains by causing considerably high mortality. All the selected botanicals were effective against the weevil even at lower concentrations. This was possible because plant species contain secondary metabolites which are vast storehouse of compounds such as the steroids, phenolic compounds and tannins with wide range of biological activity reported to have great impact on insecticidal activities [39,40]. Other bioactive compounds such as terpenoids, flavonoids, alkaloids, saponins and glycosides were found in the leaf extracts of E. balsamifera, L. inermis and E. balsamifera, L. inermis, M. hirtus by others and reported to be pesticidal in nature [40-42]. Ileke, et al., reported the presence of alkaloids, flavonoids, saponins and tannins, in the powders and methanolic extracts of M. fragrans and A. melegueta and concluded their insecticidal activity against S. zeamais [14]. The characteristic smell of the study botanicals might have also contributed to their insecticidal activity by repelling the insects away from the grains as suggested by Ileke, et al., [14].

The present study investigated LC₅₀ of E. balsamifera, L. inermis, M. hirtus and E. balsamifera, L. inermis, M. hirtus against S. zeamais in sorghum grains due to lack of information on this aspect in previous studies. However, lethal concentrations of other botanicals used to control insect pests of storage were determined [43-45].

LC₅₀ of the plants showed that the ethanolic extracts had great efficiency by causing 50% adult mortality of S. zeamais in sorghum even at a concentration below the lowest amount applied within 24 HAE. E. balsamifera was more effective than the other botanicals followed by M. hirtus. The effectiveness the selected botanicals in killing adult weevils at lower concentration within short period is in conformity with Biswas, et al., who reported high efficiency of L. inermis against T. castaneum [39].

Low LC₅₀ values of ethanolic extracts of the test botanicals concur with Rani and Devanand, who reported that among methanolic extracts of the plants tested Momordica charantia L [43]. was the most effective with LC₅₀ of 2.82mg / 20g maize grains against S. zeamais.

There was a decrease in body protein of the maize weevils exposed to lethal concentrations of all the botanicals compared to the control. The reduction in body protein agrees with Askar, et al., who reported that both clove oil and diatomaceous earth reduced body protein of S. zeamais and S. oryzae from 0.10mg/ml in the control to 0.09 and 0.06mg/ ml, respectively [17]. It was earlier reported by Piri, et al., that sub-lethal concentrations of spinosad resulted in a significant decrease in protein content of bombyx mori Walker [20]. This decrease in body protein could be attributed to low feeding efficiency of the insects due to antifeedant effect of many other insecticides and hence a decrease in protein concentration [40,46]. Another reason might be as a result of protein break down into amino acids which help in energy supply to the insect [47].

It was found that application of E. balsamifera, L. inermis, M. hirtus and E. balsamifera, L. inermis, M. hirtusin the form of powders and extracts significantly affected body protein of S. zeamais which could probably affect the aforementioned biological aspects of the weevil. The reduction in concentration of the body protein might have contributed to decline in the insect’s population and hence decreased infestations in the sorghum grains. This could also be attributed to decrease in vitellogenin, a precursor yolk component, present of in the body protein of vitellogenic females, which its amount reflects the insect reproductive performance [48].

AChE is a hydrolytic enzyme in insect body that influences the nervous system [27]. In the present study the LC₅₀ of ethanolic extracts of E. balsamifera, L. inermis, M. hirtus and E. balsamifera, L. inermis, M. hirtus significantly inhibited the activity of AChE in S. zeamais agreeing with Li, et al., who reported that methyl alcohol, ethyl acetate and petroleum ether fruit extracts of Illicium verum hook. F [27]. inhibited the activity of AChE in S. zeamais. Similarly, the LC₅₀ of essential oils of Pimpinella anisum and Ocimum basilicum have demonstrated their interference with AChE activity in S. granarius [25]. Recently, methanolic extract of Urginea maritime at a concentration of 1000µg/ml was found to result in 73.37% inhibition of the activity of AChE in S. oryzae [26]. Also, another finding has confirmed the inhibitory effect of botanicals on the activity of AChE where Chaubey reported that LC₅₀ of Cuminum cyminum essential oil reduced AChE activity in S. zeamais to 66.90% compared to 31.59% of the control [28].

The inhibitory effect on AChE activity in S. zeamais exposed to lethal concentrations of all the botanicals has shown their possible potentiality as biopesticides. This is because it has been reported that inhibition of AChE in cholinergic synapses of the nervous system is the primary mechanism of acute toxicity of insecticides [25]. Therefore, the botanicals probably have neurotoxic effects on S. zeamais by interfering with the passage of impulses in the insect nervous system leading to inability of AChE to hydrolyze acetylcholine which builds up in the synapse and results in excessive neuro excitation [24]. The mortality effects of E. balsamifera, L. inermis, M. hirtus and E. balsamifera, L. inermis, M. hirtus against S. zeamais recorded in this study may also be attributed to their ability to inhibit the activity of AChE as suggested by Rajashekar, et al., [24]. Thus, the neurotransmitting system of the weevils represents a target for insect control.