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Contact Toxicity Effects of Selected Organic Leaf Extracts of Tithonia diversifolia (Hemsl.) A. Gray and Vernonia lasiopus (O. Hoffman) against Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae)

Contact Toxicity Effects of Selected Organic Leaf Extracts of Tithonia diversifolia (Hemsl.) A.... Hindawi International Journal of Zoology Volume 2021, Article ID 8814504, 14 pages https://doi.org/10.1155/2021/8814504 Research Article Contact Toxicity Effects of Selected Organic Leaf Extracts of Tithonia diversifolia (Hemsl.) A. Gray and Vernonia lasiopus (O. Hoffman) against Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) 1 1 1 Stephen Maina Gitahi , Mathew Piero Ngugi , David Nganga Mburu , and Alex Kingori Machocho Department of Biochemistry, Microbiology and Biotechnology, Kenyatta University, P. O Box 43844-00100, Nairobi, Kenya Department of Chemistry, Kenyatta University, P. O Box 43844-00100, Nairobi, Kenya Correspondence should be addressed to Stephen Maina Gitahi; gitahism@gmail.com Received 21 October 2020; Revised 22 February 2021; Accepted 4 September 2021; Published 16 September 2021 Academic Editor: Edson Gandiwa Copyright © 2021 Stephen Maina Gitahi et al. 'is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Maize weevil (Sitophilus zeamais) infestation results in a substantial reduction in the quantity and deterioration of the quality of stored maize. Most farmers control weevils using conventional pesticides which are usually associated with several human health risks as well as intoxication of the fauna and flora. However, bioinsecticides form an alternative intervention since they possess fewer side effects on human health, are ecofriendly, and are readily available. 'is study sought to validate and document, in a systematic way, the pesticidal properties of the species Tithonia diversifolia and Vernonia lasiopus used for many years by many people of the world on S. zeamais. 'e plant leaf samples were obtained from Embu County, Kenya. Dichloromethane and ethyl acetate solvents were used to extract active phytochemicals from the dried plant sample powder. 'e GC-MS analysis of the obtained extracts was performed at ICIPE laboratories to identify their phytochemical compositions. Twenty grams of maize grains were put in 50 ml plastic vials and admixed with different treatments. 'e positive control group was treated with Actellic Super , while the negative control was treated with the respective extracting solvent only. 'e experimental groups were separately treated with the plant leaf extracts at doses of 25, 50, 75, and 100%. After treatment, each of the six groups was infested with 10 male S. zeamais and weevil mortality as a result of contact toxicity of the treatments was assessed at 6, 24, 48, 72, and 96 hours after the insects were exposed to the extracts. Results of the study indicate that the selected organic leaf extracts of T. diversifolia and V. lasiopus possessed significant contact insecticidal effects that ranged between 1.67 to 99.98%. Furthermore, the GC-MS analysis revealed several active biocompounds in T. diversifolia and V. lasiopus extracts, which are known for their considerable insecticidal effects. Our data suggest that the organic leaf extracts of T. diversifolia and V. lasiopus have considerable insecticidal properties and would, therefore, be a valuable bioprotective agent for stored maize grains against S. zeamais. insect pest in the beetle family Curculionidae (snout beetles). It 1. Introduction is the single most important primary pest that attacks stored maize grains, among others insect pests such as the larger grain Postharvest losses due to storage pests have been recognized as borer Prostephanus truncatus Horn (Coleoptera: Bostrychidae) an increasingly important constraint to maize production. In [1, 2]. Weevil infestation causes an estimated annual loss of spite of the great value of maize, its availability and utilization 30–50% of stored maize grains in tropical Africa [3]. have been impaired due to grain damage caused by notorious Farmers normally control insect pests using synthetic postharvest insect pests in developing countries. Maize weevil, insecticides. However, these chemicals are expensive and are S. zeamais (Coleoptera: Curculionidae), is a small agricultural 2 International Journal of Zoology arguably associated with deleterious side effects on the Siakago division, Mbeere North subcounty, Embu County, environment. 'e synthetic pesticides invoke resistance over Kenya. 'e GPS locations for T. diversifolia and V. lasiopus ° ° ° time. Insecticide resistance among strains of maize weevil specimens were 0 35′39″S, 37 38′10″E and 0 35′39.51″S, has been found to different registered synthetic grain pro- 37 38′23.62″E, respectively. 'e fresh leaves were identified tectants such as deltamethrin, Primiphos-methyl, per- and collected from mature plants with the help of local methrin, and Lindane and may become a much more serious herbalists. 'e folklore information obtained included the problem in the future [4, 5]. local name of the plants, part of plants harvested, season of 'e toxicity effects of pesticides on insects such as harvesting, method of preparation, and other medicinal weevils have different possible physiological end points importance of the plants. Samples were properly sorted out, which practically impose toxic effects that kill the targeted cleaned, and transported in bags to Kenyatta University, in pests [6–8]. Pesticides can be inhaled (fumigants), ingested, the Biochemistry, Microbiology, and Biotechnology de- or absorbed via the insect body surface due to the abrasive partmental laboratories. 'e plant samples were provided to effect on the pest cuticle as contact toxicants [9]. Although an acknowledged taxonomist for botanical authentication with some exceptions, an ingested insecticide will induce a and voucher specimens deposited at the Kenyatta University more severe response than the same amount of insecticide Pharmacy and Complementary/Alternative Medicine re- encountered by an insect through direct contact or fumi- search herbaria. gation [10]. 'e nonconventional methods employed to control weevils 2.2. Sample Preparation and Extraction. 'e leaves of on stored maize include the use of inert dusts, sanitary pest T. diversifolia and V. lasiopus were air-dried separately control methods involving consistent cleaning and disinfes- under shade and at room temperature for a period of two tation of storage structures, freezing, and use of ionizing ra- weeks. 'e leaves were separately ground into fine powder diation, as well as the use of hermetic techniques [11–13]. using a grinding electric mill and sieved using a 300 µm Medicinal plants are also importantly used in the control mesh. 'e powder was used for extraction following the of weevils in stored grains. 'ey include Azadirachta indica, guideline used by Singh [25]. Extraction was separately Tanaecium nocturnum, Pogostemon cablin, T. diversifolia, conducted with dichloromethane (DCM) and ethyl acetate and V. lasiopus, among others [14]. Tithonia diversifolia and to ensure maximum extraction of a wide variety of com- Vernonia lasiopus plants have numerous medicinal values pounds [26]. among various societies. Tithonia diversifolia is reported to Two hundred grams of each plant leaf powder was exhibit antifungal, anti-inflammatory, analgesic, antima- separately soaked in 200 ml of the respective solvents for 12 larial, antiviral, antidiabetic, antidiarrhoeal, antimicrobial, hours. 'e extracts were decanted and 200 ml of solvent was antispasmodic, vasorelaxant and cancer chemopreventive, added and set for 24 hours. After 24 hours, filtration was and antibacterial activities [15, 16]. 'ere are also scientific carried out again and 200 ml of the respective solvent was reports on the biological properties of T. diversifolia against added for the final extraction until 48 hours when the last arthropods. For instance, T. diversifolia is reported to elicit filtrate was obtained. Occasional swirling was performed to significant toxic impacts against dog fleas (Ctenocephalides ensure thorough extraction. Aluminum foil and cotton wool canis (Curtis) and jigger (Tunga penetrans), as well as were always used to cover the flasks to prevent escape of Callosobruchus maculatus Fab. and Sitophilus zeamais solvent. Muslin cloth and Whatman No. 1 papers were used [17–22]. for the filtration of the extracts. 'e extract filtrates were Different plant materials of Vernonia lasiopus have also then concentrated in vacuum using the Heidolph rotary exhibited antifungal and antimalarial activities among evaporator, and the solvent was recovered. 'e concentrates others [1]. It is applied on cattle bodies to control livestock were further allowed to dry to remove traces of the solvents ticks (Boophilus microplus) in pastoralist communities in and yield dry extracts. All extracts were later kept in sample Kenya [23]. 'e leaves and stem decoction of V. lasiopus is bottles and refrigerated (4 C) awaiting use in pesticidal used by herbalists in Eastern Province, Kenya, to treat bioassays. 'e percent extraction yields of the DCM and malaria, helminths, and nonbacterial infections [24]. EtOAc leaf extracts of the two plants were determined using Farmers in Embu County, Kenya, use the leaves of the following formula: T. diversifolia and V. lasiopus on stored maize cobs to protect them against weevil infestation. However, no scientific re- mass of extract obtained (g) Percent (%) extract yield � × 100. search has been conducted to evaluate this described effect. It mass of sample(g) is against this background that this study was designed to (1) explore and provide information on the contact toxicity effects of the selected organic extracts from T. diversifolia and V. lasiopus against adult S. zeamais. 2.3. Preparation of Extract Concentrations. 'e plant extract concentrates were diluted with the respective solvents at a 2. Materials and Methods −1 concentration of 1 gml , and this was termed as the stock 2.1. Plant Sample Collection. 'e plants used in this study, solution (100% v/v concentration) as described by Desh- T. diversifolia and V. lasiopus, were collected from their mukh and Borie [27] with limited modifications. 'e natural habitat in Makunguru Village, Nthawa Location, concentrations used were 25% (v/v), 50% (v/v), 75% (v/v), International Journal of Zoology 3 and 100% (v/v). 'ese extract concentrations were prepared T. diversifolia and V. lasiopus was performed using the as follows: the 25% (v/v) concentration was prepared by procedure previously used by Dar et al. [30]. Analysis of the diluting 1 ml of the stock solution with 3 ml of solvent to sample was carried out using GC-MS (7890/5975, Agilent make up 4 ml. 'e 50% (v/v) concentration was prepared by Technologies, Inc., Beijing, China) consisting of a gas diluting 2 ml of the stock solution with 2 ml of the solvent to chromatograph interfaced to a mass spectrometer. make up 4 ml, while for the 75% (v/v) concentration, 1 ml of 'e GC-MS was equipped with an HP-5 MS (5% phenyl the solvent was added to 3 ml of stock solution to make up methyl siloxane) low-bleed capillary column of 30 m length, 4 ml. 0.25 mm diameter, and 0.25 µm film thickness. For GC-MS detection, an electron ionization system with an ionization energy of 70 Ev was used. 'e carrier gas used was helium 2.4. Preparation of Maize Grains. To eliminate the effect of (99.99%) at a constant flow rate of 1.25 ml/min in the split varietal resistance to S. zeamais infestation, a susceptible mode. 'e injector and mass transfer line temperature were maize variety was obtained as a test variety for use in this ° ° set at 250 C and 200 C, respectively, and an injection volume study [21]. 'e experimental grains were cleaned and of 1 µl was employed. 'e oven temperature was pro- standardized using the method of Sulherie et al. [28]. 'e ° ° grammed from 35 C for 5 minutes, with an increase of 10 C/ damaged kernels were sorted out and the clean ones were ° ° minute to 280 C for 10.5 minutes and then 50 C/minute to put in a deep freezer at −20 C for three days to eliminate any 285 C for 29.9 minutes with a run time of 70 minutes. 'e eggs, larvae, pupae, or adult weevils. 'e dead weevils were MS operating parameters were ionization energy: 70 eV; ion sieved out and grain aired for 72 hours prior to use. 'is source temperature: 230 C, solvent cut time: 3.3 minutes, acclimatization of the maize grains stabilized the moisture scan speed: 1666 µ/second; scan range: 40–550 m/z; and content at 12-13%, thereby ensuring its suitability for interface temperature: 250 C. Interpretation of mass spec- feeding by weevils [16]. trum from GC-MS analysis was performed using the central database of the National Institute of Standard and Tech- nology (NIST), which contains more than 62,000 patterns. 2.5. Rearing and Sexing of Maize Weevil (Sitophilus zeamais). As for the unknown components, their spectrum was A stock culture of the maize weevil, S. zeamais, was initiated compared with those which are known from the NIST li- by collecting adult weevils from infested maize grains and brary [30]. cultured in their food media (susceptible whole maize grains) under fluctuating ambient temperature and relative humidity. Two hundred unsexed adult weevils were intro- 2.7. Experimental Design. 'e determination of contact duced into five two 2 l bottles with 500 g of maize. 'e insects toxicity experiments adopted a randomized controlled study were allowed to oviposit for seven days after which they were design (RCD) with four replications. 'e experiments were sieved out and subsequently used for the bioassay experi- set up into six independent treatment groups (Table 1) with ments. 'e insect stock culture was further maintained in four replicates (n � 4) per treatment group. In the negative glass bottles of 2 l capacity containing maize grains. 'e control, maize grains were treated with the respective solvent weevils were reared subsequently by replacing devoured and only, while for the positive control group, the grains were infested grains with fresh, clean, uninfected grains in con- treated with Actellic Super at a recommended dose of 50 g/ tainers covered with muslin cloth to allow for air circulation 90 kg grains. and prevent escape of insects. 'e muslin cloths covering the Determination of contact toxicity tests was carried out as containers were held in place with rubber bands. 'e maize follows: twenty grams of maize grains were weighed and put dust was periodically sieved to prevent the growth of mould, into 50 ml plastic vials. 1.0 ml of each plant extract at pre- which may lead to the caking of grains and ultimate death of determined concentrations of 25, 50, 75, and 100% was the insects. Sitophilus zeamais breeding and experiments added. 'e mixture was shaken gently to ensure uniform were conducted at an ambient temperature of 27± 2 C, coating of grains. After the grains and extracts were thor- relative humidity of 75± 5.5%, and suitable photoperiod (LD oughly mixed, the setups were air-dried for 2 hours to 12 :12). 'e culture maintained was used throughout the evaporate all traces of solvents. Twenty male adult S. zeamais period of this study. were introduced into each plastic vial and then tightly 'e weevils were sexed morphologically under a dis- covered with a lid. Several tiny openings were made on the secting microscope using the methods of Ojo and Owoloye lid of the plastic vials to ensure ventilation. [29] by examining the weevil’s rostrum and abdominal shape Weevil mortality as a result of fumigant and contact of the insects. Male S. zeamais were identified with a rough, toxicity was assessed 6, 24, 48, 72, and 96 hours after the distinctly shorter, and wider rostrum, while the female was insects were exposed to the extracts. After these test ob- identified with a smooth, shiny, distinctly longer, and servation periods, the plastic vials were opened and the narrower rostrum than that of the male. 'e male and female weevils transferred into an open recovery tray for five weevils were, hence, separated into different insect stock jars. minutes before mortality was assessed. 'e insects were confirmed dead if they could not move their appendages 2.6. Gas Chromatography-Mass Spectrometry (GC-MS) when probed with a sharp pin on the abdomen [31]. Cor- rected mortality percentages were then computed using Analysis. Gas Chromatography-Mass Spectrometry (GC- MS) analysis of the DCM and ethyl acetate leaf extracts of Abbort formula [32]: 4 International Journal of Zoology Table 1: Treatment protocol for determination of contact toxicity effects. Group Treatment I (negative control) Solvents only II (positive control) Actellic Super III (experimental group A) 25% (v/v) plant leaf extract IV (experimental group B) 50% (v/v) plant leaf extract V (experimental group C) 75% (v/v) plant leaf extract VI (experimental group D) 100% (v/v) plant leaf extract Actellic Super � 50 g/90 kg grain; solvents � 1 ml DCM/EtOAc. V. lasiopus. 'ese compounds include lipids (fatty acid esters Pt − Pc P � × 100, (2) and phytosterols), terpenoids (monoterpenes, diterpenes, 100 − Pc triterpenes, and sesquiterpenoids), and phenolic compounds where P � corrected mortality. (Tables 2 and 3). P � % mortality in various extracts treatments ((Dn/Tn)(Dn/Tn)(Dn/Tn) × 100/1) 2.10. Contact Toxicity Effects of DCM Leaf Extracts of P � % mortality in solvent control T. diversifolia and V. lasiopus against S. zeamais. As Tables 3 ((Dn/Tn)(Dn/Tn) × 100/1) and 5 show, the DCM leaf extracts of T. diversifolia and D � number of dead insects V. lasiopus generally caused remarkable weevil mortality T � total number of insects upon contact. It was observed that the two plants manifested an increase in weevil mortality with an increase in exposure time to weevils throughout the experimental period. In this 2.8. Data Management and Statistical Analysis. 'e number study, the solvent (dichloromethane) was reported to have of dead weevils (D ) was obtained from the different groups no weevil-killing potential throughout the experimental for each of the two plant extracts at the five test durations. period (Tables 5 and 6). 'e data obtained were recorded and later tabulated on a 'e T. diversifolia extract caused dose-dependent effects, broad sheet. Corrected percent weevil mortalities (Pr) were except in the first and second observation times of 6 and 24 formulated and computed using Ms Excel program. 'e data hours after exposure to weevils. Six hours after exposure to were checked for normality using the Kolmogorov–Smirnov weevils, the T. diversifolia extract dose of 75% evoked the test and then analyzed through descriptive statistics and highest weevil mortality of 36.25%. However, this was not presented as mean± SEM. 'e data from different treatment significantly different from the effects caused by the rest of groups were also subjected to inferential statistics using one- the extract concentrations (p> 0.005; Table 5). way ANOVA followed by Tukey’s post hoc test for sepa- Twenty-four hours after exposure to weevils, the ration and pairwise comparisons of means. 'e significant T. diversifolia extract dose of 75% caused the least weevil difference between the treatment groups was reported at mortality (72.47%), which was significantly lower than the p≤ 0.005. 'e resulting data of this study were presented in effects caused by the other dosages (p< 0.005; Table 5). At the form of tables and bar graphs. Unpaired Student’s t-test the same time, the T. diversifolia extract doses of 25, 50, and was used for pairwise separation and comparison of means 100% evoked statistically similar weevil-killing abilities, between different treatment groups for the two plants. 'e which also matched the effects caused by Actellic Super analyses were conducted using Minitab version 17 software (p> 0.005; Table 5). as the statistical tool. Following 48, 72, and 96 hours of exposure to weevils, the contact toxicity effect of the T. diversifolia extract at all concentrations was comparable to each other (p> 0.005; 2.9. Quantitative Phytochemical Analysis of the Selected Table 5). Furthermore, the effects caused by the Organic Leaf Extract of T. diversifolia and V. lasiopus. T. diversifolia extract concentrations were found to be 'e GC-MS analysis of the organic leaf extract of statistically comparable to the effects of the standard pes- T. diversifolia and V. lasiopus revealed the presence of several ticide, Actellic Super , after these long durations of ex- phytocompounds as presented in Tables 2 and 3. Results of posure to S. zeamais (p> 0.005; Table 5). percentage (%) extract yields applied in determining the On the other hand, the DCM leaf extracts of V. lasiopus concentration (ng/g) of the phytochemical revealed in the also demonstrated considerable dose-dependent contact- organic leaf extracts of the two are tabulated in Table 4. 'e induced weevil mortality. However, it was apparent that dichloromethanolic extracts of T. diversifolia had the highest none of the V. lasiopus extracts caused weevil mortality in a yield of 1.69% followed by ethyl acetate extracts of comparable fashion to that caused by the standard pesticide, T. diversifolia (1.44%) (Table 4). 'e dichloromethanolic and Actellic Super (p< 0.005; Table 6). ethyl acetate extracts of V. lasiopus had the lowest yield of 'e V. lasiopus extract concentrations of 25 and 50% 0.56 and 0.54%, respectively (Table 4). 'e GC-MS results of caused comparable weevil mortality throughout the ex- the present study showed the presence of active insecticidal perimental period (p> 0.005; Table 6). 'e two highest compounds in the organic leaf extracts of T. diversifolia and International Journal of Zoology 5 Table 2: Phytochemical analysis of insecticidal compounds in the DCM leaf extract of T. diversifolia and V. lasiopus. T. diversifolia V. lasiopus Compound name Chemical class Molecular formula RT Conc. (ng/g) RT Conc. (ng/g) Squalene TD C H 30.85 392.50 30.85 149.90 30 50 Phytol acetate DT C H O 22.29 254.59 22.29 170.93 22 42 2 Methyl linoleate DT C H O 25.15 164.40 25.39 164.40 19 34 2 Phytol DT C H O 24.96 159.82 24.96 051.17 20 40 Isophytol DT C H O 23.38 007.05 — — 20 40 Eugenol P C H O 16.68 009.53 16.71 005.30 10 12 2 Linalool MT C H O 12.80 007.34 — — 10 18 Chondrillasterol ST C H O — — 37.63 067.52 29 48 Methyl linoleate FAD C H O 25.15 164.40 25.39 043.03 19 34 2 1-Nonadecene FAD C H — — 21.12 003.18 19 38 P stands for phenolic, STfor sesquiterpenoid, TTfor triterpenoid, DTfor diterpenoid, MTformonoterpenoid, S for phytosterol, AD for aldehyde, and FAD for fatty acid derivatives. Figures in parenthesis indicate retention time (RT). Table 3: Phytochemical analysis of insecticidal compounds in the ethyl acetate leaf extracts of T. diversifolia and V. lasiopus. T. diversifolia V. lasiopus Compound name Chemical class Molecular formula RT Conc. (ng/g) RT Conc. (ng/g) Nonanoic acid FAD C H O 15.34 008.05 — — 9 18 2 Squalene TT C H — — 30.84 122.41 30 50 α-Pinene MT C H — — 09.59 004.53 10 16 Methyl linoleate FAD C H O 25.18 370.28 25.13 121.30 19 34 2 Phytol DT C H O 24.97 321.37 24.95 060.40 20 40 Phytol acetate DT C H O 25.90 087.95 22.28 233.70 22 42 2 Sabinene MT C H O — — 12.25 004.57 10 18 Eugenol P C H O 16.68 016.89 — — 10 12 2 Coryophyllene ST C H O 19.65 019.33 19.65 011.87 15 24 Linalool MT C H O — — 12.79 007.58 10 18 Limonene MT C H — — 11.55 005.06 10 16 Citronellel FAD C H O 22.54 116.29 — — 14 26 2 Terpinen-4-ol MT C H O 14.08 006.58 14.08 005.84 10 18 0 L-α-terpineol MT C H 14.29 003.47 — — 14 22 P stands for phenolic, ST for sesquiterpenoid, TT for triterpenoid, DT for diterpenoid, MT for monoterpenoid, S for phytosterol, AD for aldehyde, and FAD for fatty acid derivatives. Figures in parenthesis indicate retention time (RT). Table 4: 'e percentage (%) of crude extraction yields. Plant under study Type of extract Mass of crude extract (g) Yield of crude extract (%) DCM extract 3.38 1.69 T. diversifolia EtOAc extract 2.88 1.44 DCM extract 1.12 0.56 V. lasiopus EtOAc extract 1.07 0.54 DCM and EtOAc used were of analytical grade (95.5%). Table 5: Contact toxicity effects of DCM leaf extracts of T. diversifolia against S. zeamais. Mean percent corrected mortality (pr) with exposure period (hours) Group Treatment (% extract) 6 hr 24 hr 48 hr 72 hr 96 hr c c b b b Negative control Solvent only 00.00± 0.00 00.00± 0.00 00.00± 0.00 00.00± 0.00 00.00± 0.00 a a a a a Positive control Actellic Super 95.00± 2.04 97.47± 2.49 99.98± 0.25 99.95± 0.29 99.93± 0.03 b a a a a DCM leaf extract 25% (v/v) 25.00± 0.00 92.50± 2.50 92.50± 2.50 94.95± 0.05 97.40± 2.50 b a a a a 50% (v/v) 33.75± 2.39 89.97± 4.56 94.97± 2.87 97.45± 1.41 98.68± 1.23 b b a a a 75% (v/v) 36.25± 3.15 72.47± 3.23 94.97± 2.06 98.70± 1.27 99.93± 0.03 b a a a a 100% (v/v) 33.75± 4.27 98.72± 1.24 99.98± 0.03 99.95± 0.02 99.93± 0.03 Values are expressed as mean± SEM for four replicates per group (n � 4). Statistical comparisons were made within a column, and values followed by the same superscripts along the column are not significantly different by one-way ANOVA (p≥ 0.005) followed by Tukey’s post hoc test. Actellic Super � 50 g/90 kg of maize grains; solvents � 1 ml DCM/EtOAc. 6 International Journal of Zoology Table 6: Contact toxicity effects of DCM leaf extracts of V. lasiopus against S. zeamais. Mean percent corrected mortality (pr) with exposure period (hours) Group Treatment (% extract) 6 hr 24 hr 48 hr 72 hr 96 hr d d e d e Negative control Solvent only 00.00± 0.00 00.00± 0.00 00.00± 0.00 00.00± 0.00 00.00± 0.00 a a a a a Positive control Actellic Super 95.00± 2.04 97.47± 2.49 99.98± 0.25 99.95± 0.29 99.93± 0.03 d d d c d DCM leaf extract 25% (v/v) 01.67± 1.67 06.63± 1.63 18.30± 3.30 24.93± 4.98 31.57± 1.67 d d d c d 50% (v/v) 02.50± 2.50 11.23± 2.38 18.73± 2.39 26.20± 3.15 32.43± 1.43 c c c b c 75% (v/v) 25.00± 2.04 29.98± 2.06 31.23± 2.42 43.70± 3.14 54.93± 2.06 b b b b b 100% (v/v) 36.25± 1.25 46.23± 1.23 47.48± 1.43 57.45± 3.20 66.18± 1.24 Values are expressed as mean± SEM for four replicates per group (n � 4). Statistical comparisons were made within a column, and values followed by the same superscripts along the column are not significantly different by one-way ANOVA (p≥ 0.005) followed by Tukey’s post hoc test. Actellic Super � 50 g/90 kg of maize grains; solvents � 1 ml DCM/EtOAc. extract concentrations (75 and 100%) caused significantly 50% was found to be equally effective as the extract doses of different weevil mortalities upon contact (p< 0.005; Table 6). 75 and 100% as well as the reference pesticide, Actellic Super 'is was only in exception of the observation noted after 72 (p> 0.005; Table 7). hours of exposure to weevils, when the two dosages caused Similarly, the ethyl acetate leaf extract of V. lasiopus also statistically similar mortalities of 43.7 and 57.45%, respec- caused dose-dependent weevil mortality, which increased tively (p> 0.005; Table 6). However, these effects were sig- with an increase in exposure time. Six hours after exposure nificantly lower than the effects of the reference pesticide, to weevils, the V. lasiopus extract evoked weevil mortalities Actellic Super (p< 0.005; Table 6). of 12.50, 23.75, 32.50, and 41.25% at doses of 25, 50, 75, and In comparison, it was observed that the DCM leaf extract 100%, respectively. 'ese effects were significantly different of T. diversifolia generally evoked a more effective weevil- from those reported by both positive and unprotected grain killing potential as compared to the V. lasiopus extract samples (p< 0.005; Table 8). (Figure 1). Nevertheless, following 6 hours of exposure to It was apparent that the effectiveness of the V. lasiopus weevils, the extracts of both T. diversifolia and V. lasiopus, at extract at low doses of 25 and 50% was not significantly the doses of 75 and 100%, showed no significant difference in different from each other (p> 0.005) except after 48 hours of their effectiveness (p> 0.005; Figure 1). However, during the exposure. Likewise, the high extract doses (75 and 100%) rest of the experimental period, the T. diversifolia extract evoked statistically similar weevil mortality except after 24 remained significantly the most potent plant against hours of the V. lasiopus extract exposure to weevils S. zeamais (p< 0.005; Figure 1). (p> 0.005; Table 8). 'is study further demonstrated that the effect of the V. lasiopus extract did not match the effectiveness of the 2.11. Contact Toxicity Effects of the Ethyl Acetate Leaf Extract synthetic pesticides after a short duration of exposure to of T. diversifolia and V. lasiopus against S. zeamais. weevils (<48 hours). However, the higher extract dosages of Overall, the ethyl acetate leaf extracts of T. diversifolia and V. lasiopus (75 and 100%) evoked mortalities which were V. lasiopus demonstrated weevil-killing ability, which in- comparable to each other as well to the effect caused by creased with the increase in extract dosage (Tables 7 and 8). Actellic Super following a long duration of exposure to 'e effectiveness of these plant extracts was also found to weevils (72 and 96 hours) (p> 0.005; Table 8). It was also increase with an increase in exposure time to maize weevils. evident that, after long durations of exposure to weevils (72 'e weevils in the negative control group were found alive, and 96 hours), the V. lasiopus extract doses of 25 and 50% showing zero percent mortality (Tables 7 and 8). remained equally effective (p> 0.005) but significantly lower 'is study demonstrated that, apart from the than other treatments (p≤ 0.005; Table 8). T. diversifolia extract dose of 100%, none of the other Upon comparison, the ethyl acetate leaf extract of dosages showed weevil mortality comparable to that caused T. diversifolia generally demonstrated more potent weevil- by Actellic Super following a short duration (six hours) of killing ability as compared to the V. lasiopus extract (Fig- exposure to weevils (p≤ 0.005; Table 7). In the rest of the ure 2). All concentrations of the T. diversifolia extract experimental period, it was found that the T. diversifolia showed significantly higher weevil mortality after 6, 24, and extract doses of 75 and 100% were as effective as the standard 48 hours of exposure to the weevils than the effects of the pesticide, Actellic Super (p> 0.005; Table 7). V. lasiopus extract (p≤ 0.005; Figure 2). After 48 and 72 hours of exposure to weevils, the ef- However, following 72 hours of exposure to weevils, the fectiveness of the T. diversifolia extract doses of 25 and 50% 100% dose of the two plants showed no significant statistical was found to be comparable to each other (p> 0.005) al- difference in their effectiveness against S. zeamais (p> 0.005; though significantly lower than that of the rest of the Figure 2). At the observation time of 96 hours, the two treatments (p≤ 0.005; Table 7). At the last observation time extracts, at extract concentrations of 25, 75, and 100%, were (96 hours after exposure), the T. diversifolia extract dose of equally effective against S. zeamais (p> 0.005; Figure 2). International Journal of Zoology 7 a a aa a a aa aa 100 a 40 a a b b b b bb -20 6hr 24hr 48hr 72hr 96hr Extract Concentration (w/v) and Exposure Time (hours) T.diversifolia V.lasiopus Figure 1: Comparison of the contact toxicity effects (corrected percent mortality) of the DCM leaf extracts of T. diversifolia and V. lasiopus against S. zeamais. Bar graphs with different superscripts within the same concentration are significantly different at the respective test period (p< 0.005). Table 7: Contact toxicity effects of the ethyl acetate leaf extract of T. diversifolia against S. zeamais. Mean percent corrected mortality (pr) with exposure period (hours) Group Treatment (% extract) 6 hr 24 hr 48 hr 72 hr 96 hr e d c c c Negative control Solvent only 00.00± 0.00 00.00± 0.00 00.00± 0.00 00.00± 0.00 00.00± 0.00 a a a a a Positive control Actellic Super 95.00± 2.04 97.47± 2.49 99.98± 0.25 99.95± 0.29 99.93± 0.03 d c b b b Ethyl acetate extract 25% (w/v) 31.67± 1.67 33.30± 1.65 35.30± 1.65 61.60± 1.65 74.93± 2.86 c b b b a 50% (w/v) 47.50± 1.44 54.98± 2.04 62.48± 5.22 66.20± 1.27 98.65± 1.25 b a a a a 75% (w/v) 66.25± 1.25 87.47± 1.43 92.47± 1.43 97.45± 2.52 98.65± 1.25 a a a a a 100% (w/v) 86.25± 3.15 92.47± 2.52 96.22± 2.42 98.70± 1.23 99.90± 0.04 Values are expressed as mean± SEM for four replicates per group (n � 4). Statistical comparisons were made within a column, and values followed by the same superscripts along the column are not significantly different by one-way ANOVA (p≥ 0.005) followed by Tukey’s post hoc test. Actellic Super � 50 g/90 kg of maize grains; solvents � 1 ml DCM/EtOAc. Table 8: Contact toxicity effects of ethyl acetate leaf extracts of V. lasiopus against S. zeamais. Mean percent corrected mortality (pr) with exposure period (hours) Group Treatment (% extract) 6 hr 24 hr 48 hr 72 hr 96 hr e e e c c Negative control Solvent only 00.00± 0.00 00.00± 0.00 00.00± 0.00 00.00± 0.00 00.00± 0.00 a a a a a Positive control Actellic Super 95.00± 2.04 97.47± 2.49 99.98± 0.25 99.95± 0.29 99.93± 0.03 d de d b b Ethyl acetate extract 25% (v/v) 12.50± 1.44 13.73± 2.41 19.98± 2.04 28.70± 2.42 63.65± 4.28 cd cd c b b 50% (v/v) 23.75± 2.39 26.23± 4.29 37.46± 1.43 43.70± 3.76 68.65± 3.17 bc c b a a 75% (v/v) 32.50± 2.50 38.73± 1.24 71.22± 3.12 82.45± 3.21 93.65± 2.36 b b b a a 100%(v/v) 41.25± 2.39 54.98± 2.04 77.47± 4.80 84.95± 7.34 97.40± 1.44 Values are expressed as mean± SEM for four replicates per group (n � 4). Statistical comparisons were made within a column, and values followed by the same superscripts along the column are not significantly different by one-way ANOVA (p≥ 0.005) followed by Tukey’s post hoc test. Actellic Super � 50 g/90 kg of maize grains; solvents � 1 ml DCM/EtOAc. Probit analysis was performed to obtain the 50% lethal 3. Discussion dose of the four selected organic leaf extracts. 'e con- 'e present study was designed to assess the toxicity effects centration of the selected organic leaf extracts of of organic leaf extracts of T. diversifolia and V. lasiopus T. diversifolia and V. lasopus that killed 50% of the 20 weevils against S. zeamais. It was apparent that the two plants exposed to extracts every 24 hours for a period of 96 hours possess contact toxicity properties on adult weevils. 'e was recorded. 'e LD revealed DCM leaf extract of extracts showed toxicity ratings that ranged between a T. diversifolia to be the most effective biopesticide, while moderately low toxicity of 31.57% and a very high toxicity of DCM leaf extract of V. lasopus was reported as the least 99.93% after 96 hours of exposure to weevils. effective of the tested extracts (Table 9). Weevil Mortality (%) 25% 50% 75% 100% 25% 50% 75% 100% 25% 50% 75% 100% 25% 50% 75% 100% 25% 50% 75% 100% 8 International Journal of Zoology a aa a a a 100 a a b a 80 b a a a a a b 40 b 6hr 24hr 48hr 72hr 96hr Extract Concentration (w/v) and Exposure Time (hours) T.diversifolia V.lasiopus Figure 2: Comparison of the contact toxicity effects (corrected percent mortality) of ethyl acetate leaf extracts of T. diversifolia and V. lasiopus against S. zeamais. Bar graphs with different superscripts within the same concentration are significantly different at the respective test period (p≤ 0.005). Table 9: Lethal dose (LD ) of selected organic leaf extracts of T. diversifolia and V. lasopus against S. zeamais in stored maize grains. Lethal dose (LD ) Plant Extract 24 h 48 h 72h 96 h d d d d DCM 23.17± 1.92 19.92± 1.09 12.96± 1.15 07.29± 1.32 T. diversifolia c c c c EtOAc 36.96± 0.73 29.55± 0.83 24.71± 0.63 14.30± 0.21 a a a a DCM 84.19± 1.68 57.57± 1.08 37.00± 1.21 29.48± 1.68 V. lasiopus b b b b EtOAc 57.21± 0.33 37.40± 0.88 30.62± 1.07 23.84± 0.93 Values are expressed as mean± SEM for four replicates per group (n � 4). Statistical comparisons were made within column, and values followed by different superscripts. 'e insecticidal findings of the present study are in To evaluate the contact toxicity effects of the alcoholic leaf extracts of T. diversifolia on termites, Oyedokun et al. accordance with many other studies that reported botanicals as effective controls against major stored grain pest species. [37] used similar test dosages used in this study and dem- 'ere is immense scientific literature on both crude extracts onstrated equally high toxicity effects of T. diversifolia on of plants and isolated phytochemicals with insecticidal ef- termites. Similar laboratory-based tests were carried out to fects against storage pests [33]. For instance, according to determine the toxicities of methanolic, hexane, and meth- Ikbal and Pavela [34], O. basilicum, M. piperita, P. anisum, anolic: hexane blend extracts of Allium sativum on maize M. pulegium, A. indica, and F. vulgare, among others plant weevils using four concentration levels of 25, 50, 75, and species, have shown outstanding effectiveness against insects 100% by Ouko et al. [39]. [34]. 'e results of this study showed a direct relationship between the level at which the plant extract treatments were 'e essential oils of cinnamon, clove, rosemary, ber- gamot, and Japanese mint also showed effective fumigant applied and their effectiveness on S. zeamais . 'e effects of the treatments at different extract concentration levels on the toxicity against pulse beetle [35]. Similarly, the present findings support the results of Stoll [36], who reported that S. zeamais were notably different from each other. In organic leaf extracts of various plants have effective toxicity general, the bioactivities of DCM and EtOAc leaf extracts of against insect pests of various crops in the field and in stores. T. diversifolia and V. lasiopus were directly proportional to 'e insecticidal nature of essential oils of P. angolensis and the extract concentrations. 'e higher the plant extract P. quadrifolia was also manifested by contact action on adult concentration, the more potent the extract. insects of S. cerealella [36]. 'at the number of dead weevils increased with the In consonance with the findings of the present study, the increasing concentration could be due to the increase in extracts of T. diversifolia, P. amarus, and A. albida also bioactive components as the concentration of the extract increases such that it is likely that, at the lower dose, there demonstrated 42–88%, 40–56%, and 24–60% insect mor- tality, respectively [37]. Trivedi et al. [38] demonstrated 96% was no sufficient concentration of the active principle(s). fumigant toxicity against the stored grain pest Calloso- 'is variability can also be explained by the fact that the bruchus chinensis. Ouko et al. [39] reported up to 100% probability of feeding on the botanical insecticidal fumigant toxicity effect of hexane, a methanolic blend ex- compounds along with the extract particles increases with tract of O. basilicum, on adult S. zeamais. the increase in concentration. 'is correlation suggests Weevil Mortality (%) 25% 50% 75% 100% 25% 50% 75% 100% 25% 50% 75% 100% 25% 50% 75% 100% 25% 50% 75% 100% International Journal of Zoology 9 'e GC-MS analysis revealed that the organic leaf ex- that the organic extracts of the two plants can best be applied at 100% v/v concentration to have a better kill of tracts of T. diversifolia and V. lasiopus contain phyto- chemical compounds which are toxic to insect pests and maize weevils. According to Ouko et al. [39], this may be due to the fact that the combination of the active phy- parasites. 'ese compounds include phytosterols, fatty acids, tocompounds was in the best proportional mixture for α-pinene, citronellol, 1,8-cineole, limonene, linalool, optimum insecticidal activity at 100% v/v extract con- α-terpineol, caryophyllene oxide, sabinene, and eugenol centration. 'at the higher dose such as that of 100% was among others [56, 57]. When absorbed through the insect not as effective as at the lower dose level of 75% may be body surface, these compounds interfere with the basic due to the fact that the high dose takes longer to be metabolic, biochemical, physiological, and behavioral functions of the target insects. Insecticidal properties may be absorbed across the insect cuticle to the targeted site. 'at the insecticidal effectiveness increased with extract linked to the main phytochemicals extracted, reportedly acting alone or in synergy with others including minor concentration and exposure time in this study is consistent with previous findings on the effects of organic plant extracts constituents, thus potentiating its contact toxicity effects on weevils [58, 59]. on various pest insects including S. zeamais, A. obtectus, B. brassicae, and T. castaneum [39–47]. 'e extracts man- Fatty acids identified by GC-MS analysis of the selected ifested a higher mortality with an increased exposure time of organic leaf extracts of T. diversifolia and V. lasiopus have the weevils to the treated maize grains. 'is observation previously been demonstrated to have insecticidal effec- could be explained by the fact that an increase in exposure tiveness against S. zeamais among other insect pests [60, 61]. time allows for more contact time with the target pest and, It is, therefore, not strange that the extracts killed the adult hence, permits an increase in uptake of active constituents, weevils in the present assay. Furthermore, the insecticidal effects of these fatty acids have been suggested to enhance the hence the observed higher mortality with longer exposure span. efficacy of microbial insecticides such as Bacillus thur- ingiensis [62]. 'is is consistent with other previous studies carried out using Citrullus colocynthis, Cannabis indica, and Artemisia 'e observed contact toxicity on weevils may be due to nonanoic acid, which is a naturally occurring saturated fatty argyi extracts against insect pests. 'ese extracts exerted adverse effects against insect pests such as Brevicoryne acid also found in the studied extracts. Usually, ammonium brassicae L. at increasing concentrations and prolonged salt, which is a form of nonanoic acid, is used as a herbicide exposure periods [48]. 'e trend was also consistent with the that works by stripping the waxy cuticle of the plant, thereby findings in [49, 50] that showed a positive concentration- causing cell disruption, cell leakage, and death of plants by dependent correlation of A. sativum versus mortality in desiccation. Similarly, the chemical might have stripped off pulse beetle and maize weevils, respectively. the insects’ cuticles causing cell leakage and eventual mortality of the weevils [63]. 'e toxicity activities of the two plant extracts on the weevils varied relatively according to the solvent used during Major phytosterols also revealed in the GC-MS analysis of the organic leaf extracts of T. diversifolia and V. lasiopus extraction. 'e content and activity of the extracted phy- tochemicals depend on the polarity of the solvent and the can be associated with the extract toxicity effects on the solubility of the bioactive compounds in the mother solvent. weevils. Stigmasterol is among the phytosterol compounds 'erefore, the extracting solvent plays an important role in whose accumulation in the body leads to cardiac injury and, the biocidal potency of plant crude extracts [48, 51, 52], and hence, promotes mortality [64]. this was evident in the present study. Para-xylene may cause death of organisms through Previous studies have shown that nonpolar organic affecting the central nervous system if swallowed or causing solvents such as ethyl acetate extract, pesticidal nonpolar chemical pneumonitis when breathed into the lungs [65]. compounds such as terpenoids and phytosterols [53], and 'is suggests that p-xylene found in the organic leaf extracts of T. diversifolia and V. lasiopus could be responsible for medium-polar solvents such as dichloromethane effectively extract flavonoids, terpenoids, phytosterols, fatty acids, al- toxicity effects of the extracts on weevils in the present study. Furthermore, previous studies have suggested that p-xylene kaloids, and phenols [53, 54] which also exhibit pesticidal properties. Polar organic solvents such as methanol usually could cause damage to development and reproductive extract polar compounds such as amino acids, sugars, and systems [65]. glycosides, which are not particularly associated with pes- 'e pesticidal effects of the selected organic leaf extracts ticidal activities [54, 55]. of T. diversifolia and V. lasiopus could also be due to the 'e variation in the toxicity effect of the extracts in this presence of α-pinene in these extracts. Benelli et al. [66] study can, therefore, be attributed to the varying phyto- reported that α-pinene contained in the organic leaf extract of C. sativa contributed to 98.20% insect mortality. Insec- chemical composition of the extracts. 'e higher mortality exhibited by the ethyl acetate leaf extracts indicated that this ticidal properties of α-pinene have been demonstrated against Tribolium confusum, Tribolium castaneum, Sitophi- organic solvent extracted more active compounds with in- secticidal activity than the DCM leaf extracts. 'erefore, in lus zeamais, Callosobruchus maculatus, and Rhyzopertha dominica. Furthermore, the results of Benelli et al. [67] also this study, the contact insecticide activities of EtOAc and DCM leaf extracts exhibited no significant difference, which indicated a similarly high mortality of M. persicae, also suggests that using either of the two extraction solvents associated with the presence of α-pinene in the organic renders no difference. extracts of Aulacorthum solani. 10 International Journal of Zoology previously reported to have insecticidal effects on insects Uptake of eugenol from the plant extracts may also have contributed to the high mortality of adult insects in this [80–84]. As much as the extracts of V. lasiopus showed the study. Eugenol has been reported to have effective toxicity effects against insects such as aphids, houseflies, and presence of several insecticidal phytochemicals (terpenoids, cockroaches [68, 69]. 'e insecticidal activities of various phenolics, phytosterols, fatty acids, and alkaloids), their plant species such as C. cinnamomum and C. cymbopogon concentration levels were notably low to induce an effective against houseflies (M. domestica) have also been largely weevil mortality in comparison to the effectiveness of associated with the predominance of eugenol phytochemi- T. diversifolia extracts. 'erefore, that the organic leaf ex- cals in plant essential oils [69]. tracts of V. lasiopus had lower toxicity against maize weevils could be attributed to the presence of these potent com- According to the GC-MS analysis, the organic leaf ex- tracts of T. diversifolia and V. lasiopus contained limonene pounds in lower concentrations than the concentrations in the organic leaf extracts of T. diversifolia. whose insecticidal activities have been extensively reported against various insects such as C. maculatus, C. subinnotatus, 'e standard insecticide used in this study, Actellic, is a persistent broad-spectrum insecticide. Actellic has fumigant, B. brassicae, fleas, and ticks among others [70–72]. Similarly, it is probable that the death of weevils in this study was as a stomach, and contact activity against insect pests. It is result of their exposure to limonene. conventionally used for the control of storage pests in bulk 'e toxicity effects of these extracts could also be as a stored grains, bagged grains, and storage surfaces. It effec- result of the presence of linalool. Insecticidal properties of tively controls weevils, large grain borers, and other insects linalool have been demonstrated against Sitophilus zeamais, and mites on stored grains and pulses. It contains per- Tribolium confusum, Tribolium castaneum, Callosobruchus methrin (3 g/kg) and pirimiphos-methyl (16 g/kg) as its active ingredients, which gives Actellic an effective control maculatus, and Rhyzopertha dominica. Few reports have been published regarding the mode of action of linalool in against storage pests. Pirimiphos-methyl is taken by the insect through its respiratory system and affects the pests insects. However, similar to limonene, linalool is thought to cause the death of insects by affecting the activity of nerves in through its fumigant and repellence effects. On the other hand, permethrin is able to penetrate the insect cuticle and, insects [73–75]. 'e insecticidal activity of the organic leaf extracts of hence, effect its contact and stomach actions on pests [85]. M. lucida has previously been attributed to sabinene among It is worth noting that, during the 48 hours of the test other known major components of oxygenated monoter- observation time, the fumigant activity of EtOAc leaf extract penes in the extract [76]. It is, therefore, likely that the of T. diversifolia at the highest tested extract concentration of toxicity effect of organic leaf extracts of T. diversifolia and 100% was marginally more effective (97.47% and 91.22%, V. lasiopus on adult weevils in this study was contributed by respectively) than Actellic (82.47%). 'is suggests a possibly better insecticidal mechanism of the extracts or mimicry of sabinene, which was also identified during phytochemical analysis of these extracts. the Actellic mode of action by the active phytochemicals in the studied extracts. It is also possible that the EtOAc and 'e toxicity of the extracts on adult weevils in the present study could also be linked to the presence of α-bulnesene in DCM leaf extract of T. diversifolia was efficiently inhibiting the organic leaf extracts of T. diversifolia and V. lasiopus. alternative mechanisms for killing weevils. According to Albuquerque et al. [77], α-bulnesene extracted 'e possible cause of toxicity of these extracts on weevils from Pogostemon cablin exhibits insecticidal activities is through inhibition of acetylcholinesterase enzyme. Many against various urban ant species. phytochemicals affect neurotransmission and signal trans- Additionally present in the studied extracts is car- duction in organisms [86]. Binding of these antagonists to yophyllene oxide, which is an insecticidal sesquiterpene and, acetylcholinesterase receptors causes physiological and hence, probably responsible for the toxicity of the organic biochemical disturbances and blockage. 'e subsequently observed effects include restlessness, lack of coordination, leaf extracts of T. diversifolia and V. lasiopus on weevils. According to the work in [78], sesquiterpenes such as (E)- unconsciousness, and eventual death of the insect as simi- larly observed in the present study [86]. 'ese observed caryophyllene oxide are naturally pesticidal. Caryophyllene oxide identified in the organic extracts of Melaleuca sty- effects as well as the rapid action of the extracts against phelioides was found to exhibit strong insecticidal properties weevils were suggested as indicative of a neurotoxic mode of against Aphis spiraecola, Aphis gossypii, and M. persicae [79]. action and interference of the neuromodulator (acetylcho- Caryophyllene in the root bark of Chinese bittersweet, line or octopamine) or the GABA-gated chloride channels Celastrus angulatus Max., was largely associated with the [87]. toxicity effects of the plant extract against insects such as Njoroge et al. [88] suggested that the aqueous and DCM leaf extracts of G. glauca leaves possess acetylcholinesterase Mythimna separate [78]. Furthermore, a closely similar (E)- caryophyllene, myrecene, that was extracted from C. sativa, enzyme inhibitory activity in Chilo partellus larvae at con- centrations of 0.25, 5, and 7 mg/mL due to the presence of was reported to exhibit effective insect-killing potential on A. solani and M. persicae [66, 67]. 'e insecticidal activity of insecticidal phenols, terpenoids, alkaloids, and phytosteroids in the extracts. Furthermore, a study by Friedman and organic leaf extracts of T. diversifolia and V. lasiopuscould be as a result of other terpenoids such as 1,8-cineole ?-- McDonad [89] greatly associated glycoalkaloids to the in- terpineol, terpinen-4-ol, and linalool that were found to be hibition of acetylcholinesterase enzyme in beetles. Terpe- highly present in the studied extracts. 'ese are also noids such as 1,8-cineole, eugenol ?-pinene, ?-terpineol, International Journal of Zoology 11 terpinen-4-ol, camphor, and linalool are also reported to Acknowledgments have demonstrated inhibitory effects on insects [80–84]. 'e authors are grateful to the members of the animal Nevertheless, the observed toxicity of the studied ex- breeding and experimentation laboratory in the Department tracts on weevils could also be due to that the active con- of Biochemistry, Microbiology, and Biotechnology, Kenyatta stituents in these extracts targeted voltage-gated sodium University. channels. 'ese channels are vital for electrical signaling in most excitable cells. Pesticidal alkaloids from sabadilla, pyrethrins, and Tanacetum cinerariae folium target these References channels and bind to the specific receptors on them, hence altering their gating functions [90]. Likewise, the pesticidal [1] C. O. Adedire, R. O. Akinkurolere, and O. O. 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Contact Toxicity Effects of Selected Organic Leaf Extracts of Tithonia diversifolia (Hemsl.) A. Gray and Vernonia lasiopus (O. Hoffman) against Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae)

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Copyright © 2021 Stephen Maina Gitahi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Abstract

Hindawi International Journal of Zoology Volume 2021, Article ID 8814504, 14 pages https://doi.org/10.1155/2021/8814504 Research Article Contact Toxicity Effects of Selected Organic Leaf Extracts of Tithonia diversifolia (Hemsl.) A. Gray and Vernonia lasiopus (O. Hoffman) against Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) 1 1 1 Stephen Maina Gitahi , Mathew Piero Ngugi , David Nganga Mburu , and Alex Kingori Machocho Department of Biochemistry, Microbiology and Biotechnology, Kenyatta University, P. O Box 43844-00100, Nairobi, Kenya Department of Chemistry, Kenyatta University, P. O Box 43844-00100, Nairobi, Kenya Correspondence should be addressed to Stephen Maina Gitahi; gitahism@gmail.com Received 21 October 2020; Revised 22 February 2021; Accepted 4 September 2021; Published 16 September 2021 Academic Editor: Edson Gandiwa Copyright © 2021 Stephen Maina Gitahi et al. 'is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Maize weevil (Sitophilus zeamais) infestation results in a substantial reduction in the quantity and deterioration of the quality of stored maize. Most farmers control weevils using conventional pesticides which are usually associated with several human health risks as well as intoxication of the fauna and flora. However, bioinsecticides form an alternative intervention since they possess fewer side effects on human health, are ecofriendly, and are readily available. 'is study sought to validate and document, in a systematic way, the pesticidal properties of the species Tithonia diversifolia and Vernonia lasiopus used for many years by many people of the world on S. zeamais. 'e plant leaf samples were obtained from Embu County, Kenya. Dichloromethane and ethyl acetate solvents were used to extract active phytochemicals from the dried plant sample powder. 'e GC-MS analysis of the obtained extracts was performed at ICIPE laboratories to identify their phytochemical compositions. Twenty grams of maize grains were put in 50 ml plastic vials and admixed with different treatments. 'e positive control group was treated with Actellic Super , while the negative control was treated with the respective extracting solvent only. 'e experimental groups were separately treated with the plant leaf extracts at doses of 25, 50, 75, and 100%. After treatment, each of the six groups was infested with 10 male S. zeamais and weevil mortality as a result of contact toxicity of the treatments was assessed at 6, 24, 48, 72, and 96 hours after the insects were exposed to the extracts. Results of the study indicate that the selected organic leaf extracts of T. diversifolia and V. lasiopus possessed significant contact insecticidal effects that ranged between 1.67 to 99.98%. Furthermore, the GC-MS analysis revealed several active biocompounds in T. diversifolia and V. lasiopus extracts, which are known for their considerable insecticidal effects. Our data suggest that the organic leaf extracts of T. diversifolia and V. lasiopus have considerable insecticidal properties and would, therefore, be a valuable bioprotective agent for stored maize grains against S. zeamais. insect pest in the beetle family Curculionidae (snout beetles). It 1. Introduction is the single most important primary pest that attacks stored maize grains, among others insect pests such as the larger grain Postharvest losses due to storage pests have been recognized as borer Prostephanus truncatus Horn (Coleoptera: Bostrychidae) an increasingly important constraint to maize production. In [1, 2]. Weevil infestation causes an estimated annual loss of spite of the great value of maize, its availability and utilization 30–50% of stored maize grains in tropical Africa [3]. have been impaired due to grain damage caused by notorious Farmers normally control insect pests using synthetic postharvest insect pests in developing countries. Maize weevil, insecticides. However, these chemicals are expensive and are S. zeamais (Coleoptera: Curculionidae), is a small agricultural 2 International Journal of Zoology arguably associated with deleterious side effects on the Siakago division, Mbeere North subcounty, Embu County, environment. 'e synthetic pesticides invoke resistance over Kenya. 'e GPS locations for T. diversifolia and V. lasiopus ° ° ° time. Insecticide resistance among strains of maize weevil specimens were 0 35′39″S, 37 38′10″E and 0 35′39.51″S, has been found to different registered synthetic grain pro- 37 38′23.62″E, respectively. 'e fresh leaves were identified tectants such as deltamethrin, Primiphos-methyl, per- and collected from mature plants with the help of local methrin, and Lindane and may become a much more serious herbalists. 'e folklore information obtained included the problem in the future [4, 5]. local name of the plants, part of plants harvested, season of 'e toxicity effects of pesticides on insects such as harvesting, method of preparation, and other medicinal weevils have different possible physiological end points importance of the plants. Samples were properly sorted out, which practically impose toxic effects that kill the targeted cleaned, and transported in bags to Kenyatta University, in pests [6–8]. Pesticides can be inhaled (fumigants), ingested, the Biochemistry, Microbiology, and Biotechnology de- or absorbed via the insect body surface due to the abrasive partmental laboratories. 'e plant samples were provided to effect on the pest cuticle as contact toxicants [9]. Although an acknowledged taxonomist for botanical authentication with some exceptions, an ingested insecticide will induce a and voucher specimens deposited at the Kenyatta University more severe response than the same amount of insecticide Pharmacy and Complementary/Alternative Medicine re- encountered by an insect through direct contact or fumi- search herbaria. gation [10]. 'e nonconventional methods employed to control weevils 2.2. Sample Preparation and Extraction. 'e leaves of on stored maize include the use of inert dusts, sanitary pest T. diversifolia and V. lasiopus were air-dried separately control methods involving consistent cleaning and disinfes- under shade and at room temperature for a period of two tation of storage structures, freezing, and use of ionizing ra- weeks. 'e leaves were separately ground into fine powder diation, as well as the use of hermetic techniques [11–13]. using a grinding electric mill and sieved using a 300 µm Medicinal plants are also importantly used in the control mesh. 'e powder was used for extraction following the of weevils in stored grains. 'ey include Azadirachta indica, guideline used by Singh [25]. Extraction was separately Tanaecium nocturnum, Pogostemon cablin, T. diversifolia, conducted with dichloromethane (DCM) and ethyl acetate and V. lasiopus, among others [14]. Tithonia diversifolia and to ensure maximum extraction of a wide variety of com- Vernonia lasiopus plants have numerous medicinal values pounds [26]. among various societies. Tithonia diversifolia is reported to Two hundred grams of each plant leaf powder was exhibit antifungal, anti-inflammatory, analgesic, antima- separately soaked in 200 ml of the respective solvents for 12 larial, antiviral, antidiabetic, antidiarrhoeal, antimicrobial, hours. 'e extracts were decanted and 200 ml of solvent was antispasmodic, vasorelaxant and cancer chemopreventive, added and set for 24 hours. After 24 hours, filtration was and antibacterial activities [15, 16]. 'ere are also scientific carried out again and 200 ml of the respective solvent was reports on the biological properties of T. diversifolia against added for the final extraction until 48 hours when the last arthropods. For instance, T. diversifolia is reported to elicit filtrate was obtained. Occasional swirling was performed to significant toxic impacts against dog fleas (Ctenocephalides ensure thorough extraction. Aluminum foil and cotton wool canis (Curtis) and jigger (Tunga penetrans), as well as were always used to cover the flasks to prevent escape of Callosobruchus maculatus Fab. and Sitophilus zeamais solvent. Muslin cloth and Whatman No. 1 papers were used [17–22]. for the filtration of the extracts. 'e extract filtrates were Different plant materials of Vernonia lasiopus have also then concentrated in vacuum using the Heidolph rotary exhibited antifungal and antimalarial activities among evaporator, and the solvent was recovered. 'e concentrates others [1]. It is applied on cattle bodies to control livestock were further allowed to dry to remove traces of the solvents ticks (Boophilus microplus) in pastoralist communities in and yield dry extracts. All extracts were later kept in sample Kenya [23]. 'e leaves and stem decoction of V. lasiopus is bottles and refrigerated (4 C) awaiting use in pesticidal used by herbalists in Eastern Province, Kenya, to treat bioassays. 'e percent extraction yields of the DCM and malaria, helminths, and nonbacterial infections [24]. EtOAc leaf extracts of the two plants were determined using Farmers in Embu County, Kenya, use the leaves of the following formula: T. diversifolia and V. lasiopus on stored maize cobs to protect them against weevil infestation. However, no scientific re- mass of extract obtained (g) Percent (%) extract yield � × 100. search has been conducted to evaluate this described effect. It mass of sample(g) is against this background that this study was designed to (1) explore and provide information on the contact toxicity effects of the selected organic extracts from T. diversifolia and V. lasiopus against adult S. zeamais. 2.3. Preparation of Extract Concentrations. 'e plant extract concentrates were diluted with the respective solvents at a 2. Materials and Methods −1 concentration of 1 gml , and this was termed as the stock 2.1. Plant Sample Collection. 'e plants used in this study, solution (100% v/v concentration) as described by Desh- T. diversifolia and V. lasiopus, were collected from their mukh and Borie [27] with limited modifications. 'e natural habitat in Makunguru Village, Nthawa Location, concentrations used were 25% (v/v), 50% (v/v), 75% (v/v), International Journal of Zoology 3 and 100% (v/v). 'ese extract concentrations were prepared T. diversifolia and V. lasiopus was performed using the as follows: the 25% (v/v) concentration was prepared by procedure previously used by Dar et al. [30]. Analysis of the diluting 1 ml of the stock solution with 3 ml of solvent to sample was carried out using GC-MS (7890/5975, Agilent make up 4 ml. 'e 50% (v/v) concentration was prepared by Technologies, Inc., Beijing, China) consisting of a gas diluting 2 ml of the stock solution with 2 ml of the solvent to chromatograph interfaced to a mass spectrometer. make up 4 ml, while for the 75% (v/v) concentration, 1 ml of 'e GC-MS was equipped with an HP-5 MS (5% phenyl the solvent was added to 3 ml of stock solution to make up methyl siloxane) low-bleed capillary column of 30 m length, 4 ml. 0.25 mm diameter, and 0.25 µm film thickness. For GC-MS detection, an electron ionization system with an ionization energy of 70 Ev was used. 'e carrier gas used was helium 2.4. Preparation of Maize Grains. To eliminate the effect of (99.99%) at a constant flow rate of 1.25 ml/min in the split varietal resistance to S. zeamais infestation, a susceptible mode. 'e injector and mass transfer line temperature were maize variety was obtained as a test variety for use in this ° ° set at 250 C and 200 C, respectively, and an injection volume study [21]. 'e experimental grains were cleaned and of 1 µl was employed. 'e oven temperature was pro- standardized using the method of Sulherie et al. [28]. 'e ° ° grammed from 35 C for 5 minutes, with an increase of 10 C/ damaged kernels were sorted out and the clean ones were ° ° minute to 280 C for 10.5 minutes and then 50 C/minute to put in a deep freezer at −20 C for three days to eliminate any 285 C for 29.9 minutes with a run time of 70 minutes. 'e eggs, larvae, pupae, or adult weevils. 'e dead weevils were MS operating parameters were ionization energy: 70 eV; ion sieved out and grain aired for 72 hours prior to use. 'is source temperature: 230 C, solvent cut time: 3.3 minutes, acclimatization of the maize grains stabilized the moisture scan speed: 1666 µ/second; scan range: 40–550 m/z; and content at 12-13%, thereby ensuring its suitability for interface temperature: 250 C. Interpretation of mass spec- feeding by weevils [16]. trum from GC-MS analysis was performed using the central database of the National Institute of Standard and Tech- nology (NIST), which contains more than 62,000 patterns. 2.5. Rearing and Sexing of Maize Weevil (Sitophilus zeamais). As for the unknown components, their spectrum was A stock culture of the maize weevil, S. zeamais, was initiated compared with those which are known from the NIST li- by collecting adult weevils from infested maize grains and brary [30]. cultured in their food media (susceptible whole maize grains) under fluctuating ambient temperature and relative humidity. Two hundred unsexed adult weevils were intro- 2.7. Experimental Design. 'e determination of contact duced into five two 2 l bottles with 500 g of maize. 'e insects toxicity experiments adopted a randomized controlled study were allowed to oviposit for seven days after which they were design (RCD) with four replications. 'e experiments were sieved out and subsequently used for the bioassay experi- set up into six independent treatment groups (Table 1) with ments. 'e insect stock culture was further maintained in four replicates (n � 4) per treatment group. In the negative glass bottles of 2 l capacity containing maize grains. 'e control, maize grains were treated with the respective solvent weevils were reared subsequently by replacing devoured and only, while for the positive control group, the grains were infested grains with fresh, clean, uninfected grains in con- treated with Actellic Super at a recommended dose of 50 g/ tainers covered with muslin cloth to allow for air circulation 90 kg grains. and prevent escape of insects. 'e muslin cloths covering the Determination of contact toxicity tests was carried out as containers were held in place with rubber bands. 'e maize follows: twenty grams of maize grains were weighed and put dust was periodically sieved to prevent the growth of mould, into 50 ml plastic vials. 1.0 ml of each plant extract at pre- which may lead to the caking of grains and ultimate death of determined concentrations of 25, 50, 75, and 100% was the insects. Sitophilus zeamais breeding and experiments added. 'e mixture was shaken gently to ensure uniform were conducted at an ambient temperature of 27± 2 C, coating of grains. After the grains and extracts were thor- relative humidity of 75± 5.5%, and suitable photoperiod (LD oughly mixed, the setups were air-dried for 2 hours to 12 :12). 'e culture maintained was used throughout the evaporate all traces of solvents. Twenty male adult S. zeamais period of this study. were introduced into each plastic vial and then tightly 'e weevils were sexed morphologically under a dis- covered with a lid. Several tiny openings were made on the secting microscope using the methods of Ojo and Owoloye lid of the plastic vials to ensure ventilation. [29] by examining the weevil’s rostrum and abdominal shape Weevil mortality as a result of fumigant and contact of the insects. Male S. zeamais were identified with a rough, toxicity was assessed 6, 24, 48, 72, and 96 hours after the distinctly shorter, and wider rostrum, while the female was insects were exposed to the extracts. After these test ob- identified with a smooth, shiny, distinctly longer, and servation periods, the plastic vials were opened and the narrower rostrum than that of the male. 'e male and female weevils transferred into an open recovery tray for five weevils were, hence, separated into different insect stock jars. minutes before mortality was assessed. 'e insects were confirmed dead if they could not move their appendages 2.6. Gas Chromatography-Mass Spectrometry (GC-MS) when probed with a sharp pin on the abdomen [31]. Cor- rected mortality percentages were then computed using Analysis. Gas Chromatography-Mass Spectrometry (GC- MS) analysis of the DCM and ethyl acetate leaf extracts of Abbort formula [32]: 4 International Journal of Zoology Table 1: Treatment protocol for determination of contact toxicity effects. Group Treatment I (negative control) Solvents only II (positive control) Actellic Super III (experimental group A) 25% (v/v) plant leaf extract IV (experimental group B) 50% (v/v) plant leaf extract V (experimental group C) 75% (v/v) plant leaf extract VI (experimental group D) 100% (v/v) plant leaf extract Actellic Super � 50 g/90 kg grain; solvents � 1 ml DCM/EtOAc. V. lasiopus. 'ese compounds include lipids (fatty acid esters Pt − Pc P � × 100, (2) and phytosterols), terpenoids (monoterpenes, diterpenes, 100 − Pc triterpenes, and sesquiterpenoids), and phenolic compounds where P � corrected mortality. (Tables 2 and 3). P � % mortality in various extracts treatments ((Dn/Tn)(Dn/Tn)(Dn/Tn) × 100/1) 2.10. Contact Toxicity Effects of DCM Leaf Extracts of P � % mortality in solvent control T. diversifolia and V. lasiopus against S. zeamais. As Tables 3 ((Dn/Tn)(Dn/Tn) × 100/1) and 5 show, the DCM leaf extracts of T. diversifolia and D � number of dead insects V. lasiopus generally caused remarkable weevil mortality T � total number of insects upon contact. It was observed that the two plants manifested an increase in weevil mortality with an increase in exposure time to weevils throughout the experimental period. In this 2.8. Data Management and Statistical Analysis. 'e number study, the solvent (dichloromethane) was reported to have of dead weevils (D ) was obtained from the different groups no weevil-killing potential throughout the experimental for each of the two plant extracts at the five test durations. period (Tables 5 and 6). 'e data obtained were recorded and later tabulated on a 'e T. diversifolia extract caused dose-dependent effects, broad sheet. Corrected percent weevil mortalities (Pr) were except in the first and second observation times of 6 and 24 formulated and computed using Ms Excel program. 'e data hours after exposure to weevils. Six hours after exposure to were checked for normality using the Kolmogorov–Smirnov weevils, the T. diversifolia extract dose of 75% evoked the test and then analyzed through descriptive statistics and highest weevil mortality of 36.25%. However, this was not presented as mean± SEM. 'e data from different treatment significantly different from the effects caused by the rest of groups were also subjected to inferential statistics using one- the extract concentrations (p> 0.005; Table 5). way ANOVA followed by Tukey’s post hoc test for sepa- Twenty-four hours after exposure to weevils, the ration and pairwise comparisons of means. 'e significant T. diversifolia extract dose of 75% caused the least weevil difference between the treatment groups was reported at mortality (72.47%), which was significantly lower than the p≤ 0.005. 'e resulting data of this study were presented in effects caused by the other dosages (p< 0.005; Table 5). At the form of tables and bar graphs. Unpaired Student’s t-test the same time, the T. diversifolia extract doses of 25, 50, and was used for pairwise separation and comparison of means 100% evoked statistically similar weevil-killing abilities, between different treatment groups for the two plants. 'e which also matched the effects caused by Actellic Super analyses were conducted using Minitab version 17 software (p> 0.005; Table 5). as the statistical tool. Following 48, 72, and 96 hours of exposure to weevils, the contact toxicity effect of the T. diversifolia extract at all concentrations was comparable to each other (p> 0.005; 2.9. Quantitative Phytochemical Analysis of the Selected Table 5). Furthermore, the effects caused by the Organic Leaf Extract of T. diversifolia and V. lasiopus. T. diversifolia extract concentrations were found to be 'e GC-MS analysis of the organic leaf extract of statistically comparable to the effects of the standard pes- T. diversifolia and V. lasiopus revealed the presence of several ticide, Actellic Super , after these long durations of ex- phytocompounds as presented in Tables 2 and 3. Results of posure to S. zeamais (p> 0.005; Table 5). percentage (%) extract yields applied in determining the On the other hand, the DCM leaf extracts of V. lasiopus concentration (ng/g) of the phytochemical revealed in the also demonstrated considerable dose-dependent contact- organic leaf extracts of the two are tabulated in Table 4. 'e induced weevil mortality. However, it was apparent that dichloromethanolic extracts of T. diversifolia had the highest none of the V. lasiopus extracts caused weevil mortality in a yield of 1.69% followed by ethyl acetate extracts of comparable fashion to that caused by the standard pesticide, T. diversifolia (1.44%) (Table 4). 'e dichloromethanolic and Actellic Super (p< 0.005; Table 6). ethyl acetate extracts of V. lasiopus had the lowest yield of 'e V. lasiopus extract concentrations of 25 and 50% 0.56 and 0.54%, respectively (Table 4). 'e GC-MS results of caused comparable weevil mortality throughout the ex- the present study showed the presence of active insecticidal perimental period (p> 0.005; Table 6). 'e two highest compounds in the organic leaf extracts of T. diversifolia and International Journal of Zoology 5 Table 2: Phytochemical analysis of insecticidal compounds in the DCM leaf extract of T. diversifolia and V. lasiopus. T. diversifolia V. lasiopus Compound name Chemical class Molecular formula RT Conc. (ng/g) RT Conc. (ng/g) Squalene TD C H 30.85 392.50 30.85 149.90 30 50 Phytol acetate DT C H O 22.29 254.59 22.29 170.93 22 42 2 Methyl linoleate DT C H O 25.15 164.40 25.39 164.40 19 34 2 Phytol DT C H O 24.96 159.82 24.96 051.17 20 40 Isophytol DT C H O 23.38 007.05 — — 20 40 Eugenol P C H O 16.68 009.53 16.71 005.30 10 12 2 Linalool MT C H O 12.80 007.34 — — 10 18 Chondrillasterol ST C H O — — 37.63 067.52 29 48 Methyl linoleate FAD C H O 25.15 164.40 25.39 043.03 19 34 2 1-Nonadecene FAD C H — — 21.12 003.18 19 38 P stands for phenolic, STfor sesquiterpenoid, TTfor triterpenoid, DTfor diterpenoid, MTformonoterpenoid, S for phytosterol, AD for aldehyde, and FAD for fatty acid derivatives. Figures in parenthesis indicate retention time (RT). Table 3: Phytochemical analysis of insecticidal compounds in the ethyl acetate leaf extracts of T. diversifolia and V. lasiopus. T. diversifolia V. lasiopus Compound name Chemical class Molecular formula RT Conc. (ng/g) RT Conc. (ng/g) Nonanoic acid FAD C H O 15.34 008.05 — — 9 18 2 Squalene TT C H — — 30.84 122.41 30 50 α-Pinene MT C H — — 09.59 004.53 10 16 Methyl linoleate FAD C H O 25.18 370.28 25.13 121.30 19 34 2 Phytol DT C H O 24.97 321.37 24.95 060.40 20 40 Phytol acetate DT C H O 25.90 087.95 22.28 233.70 22 42 2 Sabinene MT C H O — — 12.25 004.57 10 18 Eugenol P C H O 16.68 016.89 — — 10 12 2 Coryophyllene ST C H O 19.65 019.33 19.65 011.87 15 24 Linalool MT C H O — — 12.79 007.58 10 18 Limonene MT C H — — 11.55 005.06 10 16 Citronellel FAD C H O 22.54 116.29 — — 14 26 2 Terpinen-4-ol MT C H O 14.08 006.58 14.08 005.84 10 18 0 L-α-terpineol MT C H 14.29 003.47 — — 14 22 P stands for phenolic, ST for sesquiterpenoid, TT for triterpenoid, DT for diterpenoid, MT for monoterpenoid, S for phytosterol, AD for aldehyde, and FAD for fatty acid derivatives. Figures in parenthesis indicate retention time (RT). Table 4: 'e percentage (%) of crude extraction yields. Plant under study Type of extract Mass of crude extract (g) Yield of crude extract (%) DCM extract 3.38 1.69 T. diversifolia EtOAc extract 2.88 1.44 DCM extract 1.12 0.56 V. lasiopus EtOAc extract 1.07 0.54 DCM and EtOAc used were of analytical grade (95.5%). Table 5: Contact toxicity effects of DCM leaf extracts of T. diversifolia against S. zeamais. Mean percent corrected mortality (pr) with exposure period (hours) Group Treatment (% extract) 6 hr 24 hr 48 hr 72 hr 96 hr c c b b b Negative control Solvent only 00.00± 0.00 00.00± 0.00 00.00± 0.00 00.00± 0.00 00.00± 0.00 a a a a a Positive control Actellic Super 95.00± 2.04 97.47± 2.49 99.98± 0.25 99.95± 0.29 99.93± 0.03 b a a a a DCM leaf extract 25% (v/v) 25.00± 0.00 92.50± 2.50 92.50± 2.50 94.95± 0.05 97.40± 2.50 b a a a a 50% (v/v) 33.75± 2.39 89.97± 4.56 94.97± 2.87 97.45± 1.41 98.68± 1.23 b b a a a 75% (v/v) 36.25± 3.15 72.47± 3.23 94.97± 2.06 98.70± 1.27 99.93± 0.03 b a a a a 100% (v/v) 33.75± 4.27 98.72± 1.24 99.98± 0.03 99.95± 0.02 99.93± 0.03 Values are expressed as mean± SEM for four replicates per group (n � 4). Statistical comparisons were made within a column, and values followed by the same superscripts along the column are not significantly different by one-way ANOVA (p≥ 0.005) followed by Tukey’s post hoc test. Actellic Super � 50 g/90 kg of maize grains; solvents � 1 ml DCM/EtOAc. 6 International Journal of Zoology Table 6: Contact toxicity effects of DCM leaf extracts of V. lasiopus against S. zeamais. Mean percent corrected mortality (pr) with exposure period (hours) Group Treatment (% extract) 6 hr 24 hr 48 hr 72 hr 96 hr d d e d e Negative control Solvent only 00.00± 0.00 00.00± 0.00 00.00± 0.00 00.00± 0.00 00.00± 0.00 a a a a a Positive control Actellic Super 95.00± 2.04 97.47± 2.49 99.98± 0.25 99.95± 0.29 99.93± 0.03 d d d c d DCM leaf extract 25% (v/v) 01.67± 1.67 06.63± 1.63 18.30± 3.30 24.93± 4.98 31.57± 1.67 d d d c d 50% (v/v) 02.50± 2.50 11.23± 2.38 18.73± 2.39 26.20± 3.15 32.43± 1.43 c c c b c 75% (v/v) 25.00± 2.04 29.98± 2.06 31.23± 2.42 43.70± 3.14 54.93± 2.06 b b b b b 100% (v/v) 36.25± 1.25 46.23± 1.23 47.48± 1.43 57.45± 3.20 66.18± 1.24 Values are expressed as mean± SEM for four replicates per group (n � 4). Statistical comparisons were made within a column, and values followed by the same superscripts along the column are not significantly different by one-way ANOVA (p≥ 0.005) followed by Tukey’s post hoc test. Actellic Super � 50 g/90 kg of maize grains; solvents � 1 ml DCM/EtOAc. extract concentrations (75 and 100%) caused significantly 50% was found to be equally effective as the extract doses of different weevil mortalities upon contact (p< 0.005; Table 6). 75 and 100% as well as the reference pesticide, Actellic Super 'is was only in exception of the observation noted after 72 (p> 0.005; Table 7). hours of exposure to weevils, when the two dosages caused Similarly, the ethyl acetate leaf extract of V. lasiopus also statistically similar mortalities of 43.7 and 57.45%, respec- caused dose-dependent weevil mortality, which increased tively (p> 0.005; Table 6). However, these effects were sig- with an increase in exposure time. Six hours after exposure nificantly lower than the effects of the reference pesticide, to weevils, the V. lasiopus extract evoked weevil mortalities Actellic Super (p< 0.005; Table 6). of 12.50, 23.75, 32.50, and 41.25% at doses of 25, 50, 75, and In comparison, it was observed that the DCM leaf extract 100%, respectively. 'ese effects were significantly different of T. diversifolia generally evoked a more effective weevil- from those reported by both positive and unprotected grain killing potential as compared to the V. lasiopus extract samples (p< 0.005; Table 8). (Figure 1). Nevertheless, following 6 hours of exposure to It was apparent that the effectiveness of the V. lasiopus weevils, the extracts of both T. diversifolia and V. lasiopus, at extract at low doses of 25 and 50% was not significantly the doses of 75 and 100%, showed no significant difference in different from each other (p> 0.005) except after 48 hours of their effectiveness (p> 0.005; Figure 1). However, during the exposure. Likewise, the high extract doses (75 and 100%) rest of the experimental period, the T. diversifolia extract evoked statistically similar weevil mortality except after 24 remained significantly the most potent plant against hours of the V. lasiopus extract exposure to weevils S. zeamais (p< 0.005; Figure 1). (p> 0.005; Table 8). 'is study further demonstrated that the effect of the V. lasiopus extract did not match the effectiveness of the 2.11. Contact Toxicity Effects of the Ethyl Acetate Leaf Extract synthetic pesticides after a short duration of exposure to of T. diversifolia and V. lasiopus against S. zeamais. weevils (<48 hours). However, the higher extract dosages of Overall, the ethyl acetate leaf extracts of T. diversifolia and V. lasiopus (75 and 100%) evoked mortalities which were V. lasiopus demonstrated weevil-killing ability, which in- comparable to each other as well to the effect caused by creased with the increase in extract dosage (Tables 7 and 8). Actellic Super following a long duration of exposure to 'e effectiveness of these plant extracts was also found to weevils (72 and 96 hours) (p> 0.005; Table 8). It was also increase with an increase in exposure time to maize weevils. evident that, after long durations of exposure to weevils (72 'e weevils in the negative control group were found alive, and 96 hours), the V. lasiopus extract doses of 25 and 50% showing zero percent mortality (Tables 7 and 8). remained equally effective (p> 0.005) but significantly lower 'is study demonstrated that, apart from the than other treatments (p≤ 0.005; Table 8). T. diversifolia extract dose of 100%, none of the other Upon comparison, the ethyl acetate leaf extract of dosages showed weevil mortality comparable to that caused T. diversifolia generally demonstrated more potent weevil- by Actellic Super following a short duration (six hours) of killing ability as compared to the V. lasiopus extract (Fig- exposure to weevils (p≤ 0.005; Table 7). In the rest of the ure 2). All concentrations of the T. diversifolia extract experimental period, it was found that the T. diversifolia showed significantly higher weevil mortality after 6, 24, and extract doses of 75 and 100% were as effective as the standard 48 hours of exposure to the weevils than the effects of the pesticide, Actellic Super (p> 0.005; Table 7). V. lasiopus extract (p≤ 0.005; Figure 2). After 48 and 72 hours of exposure to weevils, the ef- However, following 72 hours of exposure to weevils, the fectiveness of the T. diversifolia extract doses of 25 and 50% 100% dose of the two plants showed no significant statistical was found to be comparable to each other (p> 0.005) al- difference in their effectiveness against S. zeamais (p> 0.005; though significantly lower than that of the rest of the Figure 2). At the observation time of 96 hours, the two treatments (p≤ 0.005; Table 7). At the last observation time extracts, at extract concentrations of 25, 75, and 100%, were (96 hours after exposure), the T. diversifolia extract dose of equally effective against S. zeamais (p> 0.005; Figure 2). International Journal of Zoology 7 a a aa a a aa aa 100 a 40 a a b b b b bb -20 6hr 24hr 48hr 72hr 96hr Extract Concentration (w/v) and Exposure Time (hours) T.diversifolia V.lasiopus Figure 1: Comparison of the contact toxicity effects (corrected percent mortality) of the DCM leaf extracts of T. diversifolia and V. lasiopus against S. zeamais. Bar graphs with different superscripts within the same concentration are significantly different at the respective test period (p< 0.005). Table 7: Contact toxicity effects of the ethyl acetate leaf extract of T. diversifolia against S. zeamais. Mean percent corrected mortality (pr) with exposure period (hours) Group Treatment (% extract) 6 hr 24 hr 48 hr 72 hr 96 hr e d c c c Negative control Solvent only 00.00± 0.00 00.00± 0.00 00.00± 0.00 00.00± 0.00 00.00± 0.00 a a a a a Positive control Actellic Super 95.00± 2.04 97.47± 2.49 99.98± 0.25 99.95± 0.29 99.93± 0.03 d c b b b Ethyl acetate extract 25% (w/v) 31.67± 1.67 33.30± 1.65 35.30± 1.65 61.60± 1.65 74.93± 2.86 c b b b a 50% (w/v) 47.50± 1.44 54.98± 2.04 62.48± 5.22 66.20± 1.27 98.65± 1.25 b a a a a 75% (w/v) 66.25± 1.25 87.47± 1.43 92.47± 1.43 97.45± 2.52 98.65± 1.25 a a a a a 100% (w/v) 86.25± 3.15 92.47± 2.52 96.22± 2.42 98.70± 1.23 99.90± 0.04 Values are expressed as mean± SEM for four replicates per group (n � 4). Statistical comparisons were made within a column, and values followed by the same superscripts along the column are not significantly different by one-way ANOVA (p≥ 0.005) followed by Tukey’s post hoc test. Actellic Super � 50 g/90 kg of maize grains; solvents � 1 ml DCM/EtOAc. Table 8: Contact toxicity effects of ethyl acetate leaf extracts of V. lasiopus against S. zeamais. Mean percent corrected mortality (pr) with exposure period (hours) Group Treatment (% extract) 6 hr 24 hr 48 hr 72 hr 96 hr e e e c c Negative control Solvent only 00.00± 0.00 00.00± 0.00 00.00± 0.00 00.00± 0.00 00.00± 0.00 a a a a a Positive control Actellic Super 95.00± 2.04 97.47± 2.49 99.98± 0.25 99.95± 0.29 99.93± 0.03 d de d b b Ethyl acetate extract 25% (v/v) 12.50± 1.44 13.73± 2.41 19.98± 2.04 28.70± 2.42 63.65± 4.28 cd cd c b b 50% (v/v) 23.75± 2.39 26.23± 4.29 37.46± 1.43 43.70± 3.76 68.65± 3.17 bc c b a a 75% (v/v) 32.50± 2.50 38.73± 1.24 71.22± 3.12 82.45± 3.21 93.65± 2.36 b b b a a 100%(v/v) 41.25± 2.39 54.98± 2.04 77.47± 4.80 84.95± 7.34 97.40± 1.44 Values are expressed as mean± SEM for four replicates per group (n � 4). Statistical comparisons were made within a column, and values followed by the same superscripts along the column are not significantly different by one-way ANOVA (p≥ 0.005) followed by Tukey’s post hoc test. Actellic Super � 50 g/90 kg of maize grains; solvents � 1 ml DCM/EtOAc. Probit analysis was performed to obtain the 50% lethal 3. Discussion dose of the four selected organic leaf extracts. 'e con- 'e present study was designed to assess the toxicity effects centration of the selected organic leaf extracts of of organic leaf extracts of T. diversifolia and V. lasiopus T. diversifolia and V. lasopus that killed 50% of the 20 weevils against S. zeamais. It was apparent that the two plants exposed to extracts every 24 hours for a period of 96 hours possess contact toxicity properties on adult weevils. 'e was recorded. 'e LD revealed DCM leaf extract of extracts showed toxicity ratings that ranged between a T. diversifolia to be the most effective biopesticide, while moderately low toxicity of 31.57% and a very high toxicity of DCM leaf extract of V. lasopus was reported as the least 99.93% after 96 hours of exposure to weevils. effective of the tested extracts (Table 9). Weevil Mortality (%) 25% 50% 75% 100% 25% 50% 75% 100% 25% 50% 75% 100% 25% 50% 75% 100% 25% 50% 75% 100% 8 International Journal of Zoology a aa a a a 100 a a b a 80 b a a a a a b 40 b 6hr 24hr 48hr 72hr 96hr Extract Concentration (w/v) and Exposure Time (hours) T.diversifolia V.lasiopus Figure 2: Comparison of the contact toxicity effects (corrected percent mortality) of ethyl acetate leaf extracts of T. diversifolia and V. lasiopus against S. zeamais. Bar graphs with different superscripts within the same concentration are significantly different at the respective test period (p≤ 0.005). Table 9: Lethal dose (LD ) of selected organic leaf extracts of T. diversifolia and V. lasopus against S. zeamais in stored maize grains. Lethal dose (LD ) Plant Extract 24 h 48 h 72h 96 h d d d d DCM 23.17± 1.92 19.92± 1.09 12.96± 1.15 07.29± 1.32 T. diversifolia c c c c EtOAc 36.96± 0.73 29.55± 0.83 24.71± 0.63 14.30± 0.21 a a a a DCM 84.19± 1.68 57.57± 1.08 37.00± 1.21 29.48± 1.68 V. lasiopus b b b b EtOAc 57.21± 0.33 37.40± 0.88 30.62± 1.07 23.84± 0.93 Values are expressed as mean± SEM for four replicates per group (n � 4). Statistical comparisons were made within column, and values followed by different superscripts. 'e insecticidal findings of the present study are in To evaluate the contact toxicity effects of the alcoholic leaf extracts of T. diversifolia on termites, Oyedokun et al. accordance with many other studies that reported botanicals as effective controls against major stored grain pest species. [37] used similar test dosages used in this study and dem- 'ere is immense scientific literature on both crude extracts onstrated equally high toxicity effects of T. diversifolia on of plants and isolated phytochemicals with insecticidal ef- termites. Similar laboratory-based tests were carried out to fects against storage pests [33]. For instance, according to determine the toxicities of methanolic, hexane, and meth- Ikbal and Pavela [34], O. basilicum, M. piperita, P. anisum, anolic: hexane blend extracts of Allium sativum on maize M. pulegium, A. indica, and F. vulgare, among others plant weevils using four concentration levels of 25, 50, 75, and species, have shown outstanding effectiveness against insects 100% by Ouko et al. [39]. [34]. 'e results of this study showed a direct relationship between the level at which the plant extract treatments were 'e essential oils of cinnamon, clove, rosemary, ber- gamot, and Japanese mint also showed effective fumigant applied and their effectiveness on S. zeamais . 'e effects of the treatments at different extract concentration levels on the toxicity against pulse beetle [35]. Similarly, the present findings support the results of Stoll [36], who reported that S. zeamais were notably different from each other. In organic leaf extracts of various plants have effective toxicity general, the bioactivities of DCM and EtOAc leaf extracts of against insect pests of various crops in the field and in stores. T. diversifolia and V. lasiopus were directly proportional to 'e insecticidal nature of essential oils of P. angolensis and the extract concentrations. 'e higher the plant extract P. quadrifolia was also manifested by contact action on adult concentration, the more potent the extract. insects of S. cerealella [36]. 'at the number of dead weevils increased with the In consonance with the findings of the present study, the increasing concentration could be due to the increase in extracts of T. diversifolia, P. amarus, and A. albida also bioactive components as the concentration of the extract increases such that it is likely that, at the lower dose, there demonstrated 42–88%, 40–56%, and 24–60% insect mor- tality, respectively [37]. Trivedi et al. [38] demonstrated 96% was no sufficient concentration of the active principle(s). fumigant toxicity against the stored grain pest Calloso- 'is variability can also be explained by the fact that the bruchus chinensis. Ouko et al. [39] reported up to 100% probability of feeding on the botanical insecticidal fumigant toxicity effect of hexane, a methanolic blend ex- compounds along with the extract particles increases with tract of O. basilicum, on adult S. zeamais. the increase in concentration. 'is correlation suggests Weevil Mortality (%) 25% 50% 75% 100% 25% 50% 75% 100% 25% 50% 75% 100% 25% 50% 75% 100% 25% 50% 75% 100% International Journal of Zoology 9 'e GC-MS analysis revealed that the organic leaf ex- that the organic extracts of the two plants can best be applied at 100% v/v concentration to have a better kill of tracts of T. diversifolia and V. lasiopus contain phyto- chemical compounds which are toxic to insect pests and maize weevils. According to Ouko et al. [39], this may be due to the fact that the combination of the active phy- parasites. 'ese compounds include phytosterols, fatty acids, tocompounds was in the best proportional mixture for α-pinene, citronellol, 1,8-cineole, limonene, linalool, optimum insecticidal activity at 100% v/v extract con- α-terpineol, caryophyllene oxide, sabinene, and eugenol centration. 'at the higher dose such as that of 100% was among others [56, 57]. When absorbed through the insect not as effective as at the lower dose level of 75% may be body surface, these compounds interfere with the basic due to the fact that the high dose takes longer to be metabolic, biochemical, physiological, and behavioral functions of the target insects. Insecticidal properties may be absorbed across the insect cuticle to the targeted site. 'at the insecticidal effectiveness increased with extract linked to the main phytochemicals extracted, reportedly acting alone or in synergy with others including minor concentration and exposure time in this study is consistent with previous findings on the effects of organic plant extracts constituents, thus potentiating its contact toxicity effects on weevils [58, 59]. on various pest insects including S. zeamais, A. obtectus, B. brassicae, and T. castaneum [39–47]. 'e extracts man- Fatty acids identified by GC-MS analysis of the selected ifested a higher mortality with an increased exposure time of organic leaf extracts of T. diversifolia and V. lasiopus have the weevils to the treated maize grains. 'is observation previously been demonstrated to have insecticidal effec- could be explained by the fact that an increase in exposure tiveness against S. zeamais among other insect pests [60, 61]. time allows for more contact time with the target pest and, It is, therefore, not strange that the extracts killed the adult hence, permits an increase in uptake of active constituents, weevils in the present assay. Furthermore, the insecticidal effects of these fatty acids have been suggested to enhance the hence the observed higher mortality with longer exposure span. efficacy of microbial insecticides such as Bacillus thur- ingiensis [62]. 'is is consistent with other previous studies carried out using Citrullus colocynthis, Cannabis indica, and Artemisia 'e observed contact toxicity on weevils may be due to nonanoic acid, which is a naturally occurring saturated fatty argyi extracts against insect pests. 'ese extracts exerted adverse effects against insect pests such as Brevicoryne acid also found in the studied extracts. Usually, ammonium brassicae L. at increasing concentrations and prolonged salt, which is a form of nonanoic acid, is used as a herbicide exposure periods [48]. 'e trend was also consistent with the that works by stripping the waxy cuticle of the plant, thereby findings in [49, 50] that showed a positive concentration- causing cell disruption, cell leakage, and death of plants by dependent correlation of A. sativum versus mortality in desiccation. Similarly, the chemical might have stripped off pulse beetle and maize weevils, respectively. the insects’ cuticles causing cell leakage and eventual mortality of the weevils [63]. 'e toxicity activities of the two plant extracts on the weevils varied relatively according to the solvent used during Major phytosterols also revealed in the GC-MS analysis of the organic leaf extracts of T. diversifolia and V. lasiopus extraction. 'e content and activity of the extracted phy- tochemicals depend on the polarity of the solvent and the can be associated with the extract toxicity effects on the solubility of the bioactive compounds in the mother solvent. weevils. Stigmasterol is among the phytosterol compounds 'erefore, the extracting solvent plays an important role in whose accumulation in the body leads to cardiac injury and, the biocidal potency of plant crude extracts [48, 51, 52], and hence, promotes mortality [64]. this was evident in the present study. Para-xylene may cause death of organisms through Previous studies have shown that nonpolar organic affecting the central nervous system if swallowed or causing solvents such as ethyl acetate extract, pesticidal nonpolar chemical pneumonitis when breathed into the lungs [65]. compounds such as terpenoids and phytosterols [53], and 'is suggests that p-xylene found in the organic leaf extracts of T. diversifolia and V. lasiopus could be responsible for medium-polar solvents such as dichloromethane effectively extract flavonoids, terpenoids, phytosterols, fatty acids, al- toxicity effects of the extracts on weevils in the present study. Furthermore, previous studies have suggested that p-xylene kaloids, and phenols [53, 54] which also exhibit pesticidal properties. Polar organic solvents such as methanol usually could cause damage to development and reproductive extract polar compounds such as amino acids, sugars, and systems [65]. glycosides, which are not particularly associated with pes- 'e pesticidal effects of the selected organic leaf extracts ticidal activities [54, 55]. of T. diversifolia and V. lasiopus could also be due to the 'e variation in the toxicity effect of the extracts in this presence of α-pinene in these extracts. Benelli et al. [66] study can, therefore, be attributed to the varying phyto- reported that α-pinene contained in the organic leaf extract of C. sativa contributed to 98.20% insect mortality. Insec- chemical composition of the extracts. 'e higher mortality exhibited by the ethyl acetate leaf extracts indicated that this ticidal properties of α-pinene have been demonstrated against Tribolium confusum, Tribolium castaneum, Sitophi- organic solvent extracted more active compounds with in- secticidal activity than the DCM leaf extracts. 'erefore, in lus zeamais, Callosobruchus maculatus, and Rhyzopertha dominica. Furthermore, the results of Benelli et al. [67] also this study, the contact insecticide activities of EtOAc and DCM leaf extracts exhibited no significant difference, which indicated a similarly high mortality of M. persicae, also suggests that using either of the two extraction solvents associated with the presence of α-pinene in the organic renders no difference. extracts of Aulacorthum solani. 10 International Journal of Zoology previously reported to have insecticidal effects on insects Uptake of eugenol from the plant extracts may also have contributed to the high mortality of adult insects in this [80–84]. As much as the extracts of V. lasiopus showed the study. Eugenol has been reported to have effective toxicity effects against insects such as aphids, houseflies, and presence of several insecticidal phytochemicals (terpenoids, cockroaches [68, 69]. 'e insecticidal activities of various phenolics, phytosterols, fatty acids, and alkaloids), their plant species such as C. cinnamomum and C. cymbopogon concentration levels were notably low to induce an effective against houseflies (M. domestica) have also been largely weevil mortality in comparison to the effectiveness of associated with the predominance of eugenol phytochemi- T. diversifolia extracts. 'erefore, that the organic leaf ex- cals in plant essential oils [69]. tracts of V. lasiopus had lower toxicity against maize weevils could be attributed to the presence of these potent com- According to the GC-MS analysis, the organic leaf ex- tracts of T. diversifolia and V. lasiopus contained limonene pounds in lower concentrations than the concentrations in the organic leaf extracts of T. diversifolia. whose insecticidal activities have been extensively reported against various insects such as C. maculatus, C. subinnotatus, 'e standard insecticide used in this study, Actellic, is a persistent broad-spectrum insecticide. Actellic has fumigant, B. brassicae, fleas, and ticks among others [70–72]. Similarly, it is probable that the death of weevils in this study was as a stomach, and contact activity against insect pests. It is result of their exposure to limonene. conventionally used for the control of storage pests in bulk 'e toxicity effects of these extracts could also be as a stored grains, bagged grains, and storage surfaces. It effec- result of the presence of linalool. Insecticidal properties of tively controls weevils, large grain borers, and other insects linalool have been demonstrated against Sitophilus zeamais, and mites on stored grains and pulses. It contains per- Tribolium confusum, Tribolium castaneum, Callosobruchus methrin (3 g/kg) and pirimiphos-methyl (16 g/kg) as its active ingredients, which gives Actellic an effective control maculatus, and Rhyzopertha dominica. Few reports have been published regarding the mode of action of linalool in against storage pests. Pirimiphos-methyl is taken by the insect through its respiratory system and affects the pests insects. However, similar to limonene, linalool is thought to cause the death of insects by affecting the activity of nerves in through its fumigant and repellence effects. On the other hand, permethrin is able to penetrate the insect cuticle and, insects [73–75]. 'e insecticidal activity of the organic leaf extracts of hence, effect its contact and stomach actions on pests [85]. M. lucida has previously been attributed to sabinene among It is worth noting that, during the 48 hours of the test other known major components of oxygenated monoter- observation time, the fumigant activity of EtOAc leaf extract penes in the extract [76]. It is, therefore, likely that the of T. diversifolia at the highest tested extract concentration of toxicity effect of organic leaf extracts of T. diversifolia and 100% was marginally more effective (97.47% and 91.22%, V. lasiopus on adult weevils in this study was contributed by respectively) than Actellic (82.47%). 'is suggests a possibly better insecticidal mechanism of the extracts or mimicry of sabinene, which was also identified during phytochemical analysis of these extracts. the Actellic mode of action by the active phytochemicals in the studied extracts. It is also possible that the EtOAc and 'e toxicity of the extracts on adult weevils in the present study could also be linked to the presence of α-bulnesene in DCM leaf extract of T. diversifolia was efficiently inhibiting the organic leaf extracts of T. diversifolia and V. lasiopus. alternative mechanisms for killing weevils. According to Albuquerque et al. [77], α-bulnesene extracted 'e possible cause of toxicity of these extracts on weevils from Pogostemon cablin exhibits insecticidal activities is through inhibition of acetylcholinesterase enzyme. Many against various urban ant species. phytochemicals affect neurotransmission and signal trans- Additionally present in the studied extracts is car- duction in organisms [86]. Binding of these antagonists to yophyllene oxide, which is an insecticidal sesquiterpene and, acetylcholinesterase receptors causes physiological and hence, probably responsible for the toxicity of the organic biochemical disturbances and blockage. 'e subsequently observed effects include restlessness, lack of coordination, leaf extracts of T. diversifolia and V. lasiopus on weevils. According to the work in [78], sesquiterpenes such as (E)- unconsciousness, and eventual death of the insect as simi- larly observed in the present study [86]. 'ese observed caryophyllene oxide are naturally pesticidal. Caryophyllene oxide identified in the organic extracts of Melaleuca sty- effects as well as the rapid action of the extracts against phelioides was found to exhibit strong insecticidal properties weevils were suggested as indicative of a neurotoxic mode of against Aphis spiraecola, Aphis gossypii, and M. persicae [79]. action and interference of the neuromodulator (acetylcho- Caryophyllene in the root bark of Chinese bittersweet, line or octopamine) or the GABA-gated chloride channels Celastrus angulatus Max., was largely associated with the [87]. toxicity effects of the plant extract against insects such as Njoroge et al. [88] suggested that the aqueous and DCM leaf extracts of G. glauca leaves possess acetylcholinesterase Mythimna separate [78]. Furthermore, a closely similar (E)- caryophyllene, myrecene, that was extracted from C. sativa, enzyme inhibitory activity in Chilo partellus larvae at con- centrations of 0.25, 5, and 7 mg/mL due to the presence of was reported to exhibit effective insect-killing potential on A. solani and M. persicae [66, 67]. 'e insecticidal activity of insecticidal phenols, terpenoids, alkaloids, and phytosteroids in the extracts. Furthermore, a study by Friedman and organic leaf extracts of T. diversifolia and V. lasiopuscould be as a result of other terpenoids such as 1,8-cineole ?-- McDonad [89] greatly associated glycoalkaloids to the in- terpineol, terpinen-4-ol, and linalool that were found to be hibition of acetylcholinesterase enzyme in beetles. Terpe- highly present in the studied extracts. 'ese are also noids such as 1,8-cineole, eugenol ?-pinene, ?-terpineol, International Journal of Zoology 11 terpinen-4-ol, camphor, and linalool are also reported to Acknowledgments have demonstrated inhibitory effects on insects [80–84]. 'e authors are grateful to the members of the animal Nevertheless, the observed toxicity of the studied ex- breeding and experimentation laboratory in the Department tracts on weevils could also be due to that the active con- of Biochemistry, Microbiology, and Biotechnology, Kenyatta stituents in these extracts targeted voltage-gated sodium University. channels. 'ese channels are vital for electrical signaling in most excitable cells. Pesticidal alkaloids from sabadilla, pyrethrins, and Tanacetum cinerariae folium target these References channels and bind to the specific receptors on them, hence altering their gating functions [90]. Likewise, the pesticidal [1] C. O. Adedire, R. O. Akinkurolere, and O. O. 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International Journal of ZoologyHindawi Publishing Corporation

Published: Sep 16, 2021

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