Antioviposition and Reduction of Callosobruchus chinensis Pic. 1902 (Coleoptera: Bruchidae) Emergence on Phaseolus vulgaris by Dioscorea sansibarensis Powder and Its Chemical Composition

Callosobruchus chinensis causes damage to the Phaseolus vulgaris seeds. Traditionally, Dioscorea sansibarensis serves as a medicinal plant. Naturally, D. sansibarensis has toxins that protect against herbivores and the surrounding invasive plants in its natural habitat. Phytochemical analysis by thin-layer chromatography (TLC) and laboratory experiments was carried out to determine the activity of D. sansibarensis leaves, bulbils, and yams powders on antioviposition and inhibition of the F 1 emergence of C. chinensis . Bioassay data were subjected to nonparametric (Kolmogorov–Smirnov) statistical analysis and a generalized linear model at P ≤ 0 . 05. Statistically, the powders had an antioviposition activity of 34.3% ( R 2 � 0.343). A recommendable activity on antioviposition was displayed by the yams powder; treatment by 0.8 g of yams powder had a Wald Chi-Square value of 1.291, P � 0 . 26. Inhibition of F 1 emergence was signiﬁcantly attained by the yams powder; the treatment by 0.6g of yams powder had a Wald Chi-Square value of 7.72, P � 0 . 01. Statistically, the bulbils powder displayed low antioviposition and inhibition of F 1 emergence. Observations on the TLC exposed compounds with similar R f values; saponin with an R f value of 0.72 was portrayed in the leaves, bulbils, and yams. A terpenoid and a ﬂavonoid with R f values of 0.37 and 0.71, respectively, were observed in bulbils and yams but absent in leaves. A terpenoid with an R f value of 0.49 was visualized in leaves and bulbils but not in the yams powder. The study concluded that the D. sansibarensis yams and leaves powders are viable for application by the farmers in the protection of stored legumes against attack by C. chinensis . However, there may be other diverse interests in other storage insects and other methods of phytochemical analysis that have


Introduction
e common bean, Phaseolus vulgaris (Fabaceae), is one of the principle staple foods and cash crops in Tanzania and serves as an important crop for food security in many countries in sub-Sahara Africa [1]. It is important for many low-income populations and significantly improves their nutrition, aimed at the prevention and treatment of diseases [2]. Several studies have demonstrated Callosobruchus chinensis attacks on P. vulgaris in silos, stacks, and barns throughout the world causing qualitative and quantitative damage by both larvae and the adults [3]. C. chinensis accounts for 60% loss of weight and 66.3% loss of protein content of P. vulgaris [4].
Callosobruchus chinensis Pic. 1902, also termed Bruchus chinensis Linnaeus 1758, of the family Bruchidae, has an average lifetime of 22-25 days from egg to the adult stage [5]. e infestation begins in the field during harvest; the bruchid is transported to store with the legumes where it lays eggs on the seed surface at the right atmospheric conditions and its life is completed inside the bean seed as it feeds. Damage by C. chinensis occurs after larvae hatch from the eggs as the first instars penetrate the bean seeds. e bruchids develop within the seeds and emerge as adults leaving damage in the form of the empty feeding chambers [6,7]. More than one larva can develop in a single grain [8].
Studies are being conducted on strategies to reduce common bean damage and losses caused by C. chinensis [4]. Farmers use banned and highly toxic synthetic insecticides in their quest to protect agricultural products, including stored bean seeds. Other setbacks associated with using synthetic pesticides include high costs, health hazards, residue problems, and the development of resistance by treated insects [9].
Farmers are opting for exploiting the readily available natural resources such as plants of medicinal importance and have contributed to the suppression of pests. Pretreatment of Vigna radiata seeds with inert clay by farmers led to adult mortality and reduced the oviposition of Callosobruchus chinensis [10]. In recent years, researchers have shifted attention to plant powders for control of insect pests in stored bean seeds, due to their biodegradability and being ecofriendly to the environment, thus becoming an alternative to synthetic pesticides in improving food security [11]. Leaves powders of Ocimum basilica (basil), Azadirachta indica (neem), Dennittia tripetala (pepper fruit), Curcuma longa (black pepper), Anacardium occidentale (nutshell), Alpinia galangal (Siamese ginger), Clusia palmicida (Clusia), and Curcuma zedoaria (zedoary) have been reported to induce antioviposition and reduce the number of emerging F 1 progeny of C. chinensis on chickpea, cowpea, and green gram seeds [11][12][13].
Plants store the active compounds in the form of phenols, alkaloids, flavonoids, tannins, and saponins in different parts of their organs for protection against herbivores and pathogens [8]. e genus Dioscorea is the largest genus of the monocotyledonous family Dioscoreaceae. e wild Dioscorea sansibarensis, also called (m)chochoni in Swahili, is a sun-loving ornamental climber that forms a canopy occurring in riverine forests of tropical sub-Sahara Africa, specifically in Madagascar, Zanzibar, and Tanganyika. D. sansibarensis documented history in traditional medicine goes to at least 2000 BC [14,15]. D. sansibarensis bulbils, yams, stem, and leaves are found to be toxic to pathogens, herbivores, and monkeys; hunters use its extract on arrows to hunt wild pigs [14,16].
is study investigated the antioviposition and ability to reduce the F 1 emergence of the C. chinensis from stored P. vulgaris using D. sansibarensis leaves, bulbils, and yams powders, in addition to determining the chemicals present in the plant parts.

Materials and Methods
2.1. Materials. Undamaged P. vulgaris seeds were brought directly from Kisarawe farm (7°10′0′ S, 38°50′0″ E and altitude of 252 m above sea level) after harvest. e seeds were sundried and stored with 10 ± 2% moisture content. e commercial synthetic pesticide Actellic 50 EC (100 g/90 kg Syngenta, Switzerland) was purchased from agricultural input suppliers in Dar es Salaam, Tanzania, for use as the check (positive control).
in-layer chromatography (TLC) plates and analytical grade solvents were purchased from Chem Equip Laboratory supplier in Dar es Salaam, Tanzania.
Fresh leaves, bulbils, and yams of D. sansibarensis were identified and harvested from the Zoology-Botany forest thickets at the University of Dar es Salaam (6°45′0″ S by 39°1 5′0″ E, altitude 80 m above sea level). Morphological identification was done at the Herbarium in the Department of Botany, University of Dar es Salaam, where voucher specimens bearing reference numbers FMM 3910a, 3910b, and 3910c for leaf, bulbil, and yam, respectively, are deposited. e plant materials were brought to the Entomology Laboratory of the Department of Zoology and Wildlife Conservation at the University of Dar es Salaam where they were dried under shade in a well-ventilated area for 5 days. After drying, they were pulverized and sieved using a 60 μm mesh sieve to obtain fine powders.

Qualitative Analysis by in-Layer
. Determination of Alkaloids. Weigh 2.5 g of plant powder into a 100 mL beaker to which 80 mL of 1% acetic acid (1 : 10 w/v) in ethanol at a ratio of 1 : 1 was added and allowed to stand for 5 hours, followed by filtration. e resulting filtrate concentrated on a water bath to one-third of the original volume. Concentrated ammonium hydroxide was added dropwise to precipitation. e precipitate was collected, washed with dilute ammonium hydroxide, and filtered. e dry residue was weighed and calculated as a percentage alkaloid. in-layer chromatography (TLC) of isolated alkaloids was performed by wetting the powdered test samples with a solvent mixture of chloroform and methanol at a ratio of 15 : 1 which was also used as the mobile phase for running thin-layer chromatography at room temperature. e TLC plate was visualized under UV light and further detection of alkaloid was aided by spraying with Dragendorff's reagent [17].

Determination of Flavonoids.
Plant powder (2.5 g) was soaked repeatedly with 50 mL of 75% aqueous methanol and filtered through Whatman filter paper at room temperature. e filtrate was dried in vacuo and weighed to calculate the percentage flavanoid. e filtrate was mixed with methanol in the ratio of 1 : 8 (g/mL) and heated on a water bath at 60°C for 5 minutes. Chloroform and methanol at a ratio of 18 : 2 were used as the mobile phase [18].

Determination of Saponins.
Weigh 2.5 g of plant powder into a conical flask and 50 mL of 20% aqueous ethanol added. e mixture was subjected to heating over a water bath at a temperature of 57°C for 5 minutes. On cooling, the mixture was filtered and the residue reextracted by 50 mL of 20% ethanol. e alcohol extract was reduced to half the original volume by heating on a water bath at 60°C. On cooling, 25 mL of diethyl ether was added and shaken for 5 minutes. e aqueous layer was recovered and washed with 15 mL of 5% aqueous sodium chloride. e aqueous layer was dried, weighed, and calculated for percentage saponin. TLC analysis was performed by eluting with chloroform, glacial acetic acid, methanol, and water solvent system in a

Determination of
Phenolics. Weigh 2.5 g of plant powder and transfer it into a conical flask to which 40 mL of 80% acetone (acetone to water at a ratio of 8 : 2) was added. e mixture was heated at 65°C in a water bath for 20 min. e supernatant was decanted and mixed with 80% acetone (acetone to water at a ratio of 8 : 2). e aqueous residue was dried, weighed, and calculated for percentage phenolic compounds. TLC analysis was done by eluting with butan-2-ol/acetic acid/water solvent system in a ratio of 14 : 1 : 5. en FeCl 3 -K 3 Fe(CN) 6 was sprayed on the TLC plate for the detection of the phenolic constituents [20]. e separating funnel was vigorously shaken for 5 min and then left to settle overnight. e hexane layer was decanted to a beaker for air drying. e hexane extract was treated as total terpenoids. TLC analysis was done using nhexane : ethyl acetate eluting solvent system in a ratio of 6.2 : 2.4. e TLC plate was visualized under the UV and light then heated to 105°C on a hot plate after spraying with freshly prepared p-anisaldehyde reagent [21].

Calculation of Retention Factor Values.
e retention factor (Rf ) values of different spots were calculated as follows: Rf value � distance traveled by solute distance traveled by the solvent . (1)

Preparation of Working Concentrations of the Powders.
Fine powders of the leaves, bulbils, and yams at 0.2, 0.4, 0.6, and 0.8 g/50 g of bean seed were measured for bioassays. e standard 0.05 g/50 g of the commercial pesticide, Actellic 50, EC was used in the bioassay as the positive control. Clean, undamaged, and sun-dried P. vulgaris seeds were used as the negative control (untreated seeds).

Stock Culture of Callosobruchus chinensis.
In a glass jar (10.3 cm wide and 15 cm deep) containing 350 g of dry and undamaged P. vulgaris beans, approximately 200 females of C. chinensis were placed and the mouth was covered with a muslin cloth. e jar was incubated for 1 week in darkness at 28 ± 2°C and relative humidity of 65 ± 5% to facilitate egglaying by the C. chinensis. e bruchids were removed and the beans were incubated for mass culturing of the F 1 progeny of C. chinensis. e pure culture of the F 1 breed was used in experimentation.

Antioviposition on P. vulgaris.
Twenty females of 3-dayold C. chinensis were selected from the culturing jar and added into different jars measuring 6.5 cm wide and 10 cm deep, with 50 g of P. vulgaris seeds treated with the different doses of the leaves, bulbils, and yams powders. Actellic 50 EC was used as the positive control and the untreated seeds were used as a negative control. e experiment was replicated in quadruples. e jars were incubated for 5 days in the dark at 28 ± 2°C with a relative humidity of 65 ± 5% for egg-laying. On the 5 th day of incubation, all bruchids were carefully isolated and the eggs on the P. vulgaris seed coat were counted with the aid of a dissecting microscope.

Prevention of the Emergence of F 1 Progeny.
Twenty females of 3-day-old C. chinensis were selected from the culturing jar and added into jars of 6.5 cm width and 10 cm depth with 50 g of undamaged and dry P. vulgaris seeds. Under incubation at 28 ± 2°C with a relative humidity of 65 ± 5% in darkness, the weevils were left to lay eggs for 5 days and the weevils were carefully removed from the jars. Different doses of the leaves, bulbils, yams powders, and the positive control were added to their respective jars and then incubated at 28 ± 2°C with a relative humidity of 65 ± 5%. e experiment was monitored daily until the first adults emerged from the negative control. Emerging C. chinensis were isolated and counted daily for 5 weeks to avoid overlap with their F 1 generation.

Statistical Analyses.
Antioviposition and reduction of F 1 emergence analyses were performed using the SPSS software (version 20). e analysis involved determination of Poisson distribution by nonparametric (Kolmogorov-Smirnov) test and descriptive statistics of the mean variance. e Poisson distributed data from the antioviposition and F 1 emergence were subjected separately to generalized linear model analysis at P ≤ 0.05. In the model of analyzing the antioviposition, the concentrations of the doses were the independent variables as data of antioviposition was the dependent variable. In the analysis of F 1 emergence, the concentration was the independent variable while the data obtained from the emerged F 1 progeny was utilized as the dependent variable.

Phytochemical Profiling.
e leaves, bulbils, and yams contained different compounds with different Rf values (Table 1). Alkaloids were absent on the plant parts. Saponins and terpenoids were spotted in all the plant parts. Two spots of phenolic compounds and three spots of saponins were observed in bulbils. Terpenoids had high intensity in the leaves with an observation of six spots. Five spots of flavonoids were visualized in bulbils (Table 1). e results also showed that the powders had compounds with similar Rf values; saponin with an Rf value of 0.72 was depicted in the leaves, bulbils, and yams. Powders of bulbils and yams had terpenoids with an Rf value of 0.37 and flavonoid of Rf value of 0.71, but were absent in leaves. e bulbils and leaves powders showed terpenoid with an Rf value of 0.49 which was absent in yams powder (Table 1).

Antioviposition.
Tabulated results showed counts of 423, 459, and 479 eggs on the seeds treated by 0.2 g of leaves powder, 0.2 g of yams powder, and 0.4 g of bulbils powders, respectively ( Table 2). When the eggs laid on treated seeds are counted against the eggs in the negative control (untreated seeds), 0.

Inhibition of F 1 Emergence.
e first C. chinensis adult emerged from the negative control (untreated seeds) on the 28th day of incubation from the untreated bean seeds. Graphical results showed that 362, 368, and 387 weevils had emerged from the treatments by 0.2 g of yams and the leaves and 0.4 g of the bulbils powder, respectively (Figure 1). When emerged weevils in treatments are compared to the negative control, 0.2 g of yams powder, 0.2 g of leaves powder, and 0.4 g of bulbils powder exhibited inhibition of weevil emergence from the seeds by 37.02%, 35.98%, and 32.58%, respectively (Figure 1). Kolmogorov-Smirnov test recorded 2.90 and descriptive statistics recorded a mean of 307.71 with a variance of 13727.66. e Poisson distributed data subjected to a generalized linear model reported a Wald Chi-Square value of 2.37, P � 0.12 on the treatment by 0.8 g of leaves powder and 7.72, P � 0.01 by 0.6 g of yams powder. Statistically, reduction of the F 1 emergence was highly displayed in the treatment by 0 g of yams powder where 188.5 weevils emerged from the seeds. Significant activity was displayed in the treatment by 0.2 g of leaves and 0.4 g of bulbils powders recorded 367.75 and 387.25 at P � 0.07. Treatments by 0.6 g of yams and 0.8 of leaves powders showed a significance in reduction of the emerging F 1 progeny at P � 0.332. Statistically, the seeds treated by the bulbils powder recorded a higher number of the emerged weevils than the leaves and the yams powders.

Discussion
In the family Dioscoreaceae, there is limited literature review concerning studies on wild D. sansibarensis especially on chemical analysis and insecticidal potency. Research on D. sansibarensis has surveyed its therapeutic application [22]. In this study, the D. sansibarensis leaves, yams, and bulbils powders were found to possess terpenoids, saponins, and phenolic compounds; these compounds might have been attributed to their biological activities on mortality, antioviposition, and reduction of the emergence of the C. chinensis from the P. vulgaris seeds. Dhandapani and Sabna reported that alkaloids, tannins, and phenolic compounds that are present in Aegle marmelos, Cynodon dactylon, Eclipta prostrata, Moringa pterygosperma, Pongamia pinnata, Sida acuta, and Tridax procumbens protected the plants against pathogens and herbivores [23]. In this study, terpenoid with Rf value of 0.37 and flavonoid of 0.71 were identified. According to Lazarević et al. [24] and Omotoso et al. [25], terpenoids located at Rf values of 0.37 ± 0.03 are associated with the prevention of the egg-laying process of bruchids. Chiluwal et al. [26] state that terpenoids with Rf values of 0.49 ± 0.05 are involved in the larvicidal activity of bruchids, thus preventing the emergence of F 1 progeny. Flavonoids with Rf values of 0.71 and 0.72 are associated with antioviposition of C. chinensis [27]. e results in this study showed that D. sansibarensis powders have significant control of C. chinensis in the reduction of oviposition activity and adult emergence. In this study, antioviposition recorded after 5 days on P. vulgaris beans treated with 0.6 g of D. sansibarensis leaves powder was relatively higher as compared to the treatment by 0.8 g of Jatropha curcas leaves powder on cowpea seeds which resulted in a 42% reduction in oviposition after 5 days of infestation by C. maculatus and 50.67% in reduction of emerging F 1 progeny [22]. e antioviposition and reduction of C. chinensis emergence might have been influenced by the presence of flavonoids in the leaves, bulbils, and yams. Flavonoids of plant sources are highly involved in the defence mechanisms by exerting toxic effects on the bruchids [28,29]. Upasani et al. reported that flavonoids extracted from the aqueous shade-dried leaves of Ricinus communis (Euphorbiaceae) showed excellent ovicidal and oviposition deterrent activity and reduced the number of bean seeds damaged and the C. chinensis emerging from cowpeas [30]. A considerable amount of saponins present in the D. sansibarensis might have influenced antioviposition and the reduction of the emerging C. chinensis. Saponin in form of diosgenin identified in Trigonella foenum-graecum prevents Tribolium castaneum and Acanthoscelides obtectus from laying eggs on the Zea mays seed coat and interrupts their larval development that led to the reduction of the emerging F 1 progeny from seeds [24]. Saponic compounds estimated to appear at Rf values of 0.71 to 0.80 have been highly associated with mortality, antioviposition, larvicidal activity, and emergence of F 1 progeny of the family Bruchidae [31]. Saponins from Nicotiana tabacum, Tephrosia vogelii, and Securidaca longepedunculata were found to be toxic to the larvae of C. chinensis and C. maculatus by preventing larval growth and reduction of adult emergence [32]. Saponins isolated from Vigna unguiculata seeds reduced the C. maculatus F 1 emergence from P. vulgaris seeds [33].

Conclusion
Based on the findings in this study, D. sansibarensis powders possess chemicals and have shown potency on C. chinensis in antioviposition and reduction of F 1 emergence. D. sansibarensis yams powder is viable for application by the farmers in the protection of P. vulgaris against attack by C. chinensis.

Data Availability
e datasets generated and analyzed in this study are available from the corresponding author upon reasonable request.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this article.  Psyche: A Journal of Entomology 5