Insect culture
Newly emerged adult C. maculatus were obtained from infested cowpea seeds in Entomology Research Laboratory. The insects were reared on 600 g of cowpea seeds. Eighty (80) pairs of C. maculatus were introduced into a glass Kilner jar holding V. unguiculata (variety Ife brown) obtained from Agricultural Development Programme Akure, Ondo State. The culture was placed in an insect rearing cage with an ambient temperature of 28 ± 2 °C and 75 ± 5% relative humidity.
Sample collection and preparation of agro-waste powder
Uninfected cowpea (variety Ife Brown) were collected from the seed unit of the Agricultural Development Project (ADP), Akure, and Sorted and kept in the freezer at – 10 °C for 4 weeks to remove any hidden infestation and then air-dried before usage.
Rice husk, cowpea pod, maize cob and groundnut pod were collected from uninfected plants and unpolluted areas within Akure metropolis, Ondo State and brought to Entomology Research Laboratory, Biology Department, Federal University of Technology Akure (FUTA), Ondo State, Nigeria for subsequent processing.
The agricultural wastes were thoroughly cleaned separately with water, air-dried in the laboratory for 21 days, pulverized into coarse powder using pestle and mortar before grinding in an electric blender, JTC Omni Blender V (Model TM-800). The fine powders were allowed to pass through a nylon mesh of 1mm2 dimension. The powders were then packed into an air tight containers and put in a refrigerator at 4 °C to retain its good quality before application.
Preparation of agricultural wastes extract
One hundred and fifty grams (150 g) of powdered agro wastes were weighed into separate glass jars and the ethanol was measured into the jars in ratio 1:3 (W/V). The mixture was stirred for about ten minutes until the solution becomes homogenous. The tip of the cover was taped to prevent evaporation and kept in a dark cupboard for 72 h. The dark cupboard was used because light penetration may denature active ingredients that are photosensitive and heating from sunlight may support evaporation. Filtration was carried out on the mixture using a double layer of Whatman no. 1 filter paper and the ethanol was evaporated using a rotary evaporator at 30–40 °C with rotary speed of 3–6 rpm for 8 h (Udo 2011). Extracts were air-dried to remove traces of solvent present in them and were separately kept in air-tight containers. All extracts in bottles with lid were kept in the refrigerator until needed.
Phytochemical test
Phytochemical constituents (saponin, alkaloids, phytate and oxalate) of ethanolic extracts of rice husk, maize cob, groundnut, and cowpea pods were quantified using the methods described by Harborne (1973); Trease and Evans (1985); Sofowora (1993).
Bioassay
Toxicity of agricultural wastes powder to C. maculatus
Twenty grams each of cowpea seeds were weighed into separate plastic containers and the powder of agricultural wastes at different doses of 0.1, 0.2, 0.3, 0.4, and 0.5 g were separately mixed with seeds, after which ten pairs of C. maculatus (0–24 h old) were introduced into the containers in three replicates. Untreated cowpea seed was used as control. They were arranged in a completely randomized design. Mortality was assessed every 24 h for 4 days, data on percentage adult mortality was calculated using Abbott’s (1925) formula thus:
$$P_{T} = \frac{{P_{o} - P_{c} }}{{100 - P_{o} }} \times 100$$
where PT = corrected mortality (%), PO = observed mortality (%), PC = control mortality (%).
Live and dead insects were removed and oviposition was counted and recorded after the removal of life and dead insects at day 5.
Treated and control treatments were kept in a protective cage for the emergence of the first filia (F1) generation. The number of the emerged adult was expressed in percentages (Odeyemi and Daramola 2000) as follows:
$$\mathrm{\%\,Adult\,emergence}= \frac{\mathrm{Total\,number\,of\,adult\,emergence}}{\mathrm{Total\,number\,of\,eggs\,laid}} \times 100$$
Weight loss of the cowpea seeds was expressed as percentage loss in weight as follows:
$$\mathrm{\%\,Weight\,loss}= \frac{\mathrm{Change\,in\,weight}}{\mathrm{Initial\,weight}} \times 100$$
The total numbers of seeds damaged in each treatment were estimated in Percentage as follows:
$$\mathrm{\%\,Seed\,damage}= \frac{\mathrm{Number\,of\,seeds\,damaged}}{\mathrm{Total\,number\,of\,seeds}} \times 100$$
Beetle Perforation Index (BPI) was evaluated using the method Fatope et al. (1995) as follows:
$$\mathrm{BPI}= \frac{\mathrm{\%\,treated\,cowpea\,seeds\,perforated}}{\mathrm{\%\,control\,cowpea\,seeds\,perforated}} \times 100$$
BPI value that exceeds 50 was viewed as an enhancement of beetle infestation.
Toxicity of agricultural wastes extract to C. maculatus
Twenty grams (20 g) each of cowpea seeds were weighed into separate plastic containers and the ethanolic extract of agricultural wastes at different concentrations of 0.1. 0.2, 0.3, 0.4, and 0.5 ml were separately mixed vigorously with the seeds inside the containers. The extracts were air-dried for 5–10 min to remove traces of solvents, after which ten pairs of C. maculatus (0–24 h old) were introduced into the containers in three replicates. They were arranged in a completely randomized design. Untreated cowpea seed was used as control. Mortality was assessed every 24 h for 4 days, data on percentage adult mortality was calculated using Abbott’s (1925) formula. Live and dead insects were removed and oviposition was counted and recorded. Treated and control treatments were kept in a protective cage for the emergence of the first filia (F1) generation. Adult emergence, weight loss and seed were calculated using the parameters described above.
Statistical analysis
Data were subjected to analysis of variance (ANOVA) and treatment means were separated using New Duncan’s Multiple Range Test. Log-Probit model analysis was carried out on percentage mortality of the adult C. maculatus to determine the 50% lethal dose (LD50/LC50) and 90% lethal concentration (LD90/LC90). The ANOVA and Probit analysis were done using Statistical Package for the Social Sciences (SPSS) version 20.
