Background
Protein malnutrition is a major cause of morbidity and mortality in developing countries where the cost and availability of animal protein remain prohibitive (Adesina, 2013). Cowpea (Vigna unguiculata (L) Walp) has the potential to substitute expensive animal protein. Its production is however low in Nigeria. Insect pests are key limiting elements in cowpea production as they cause considerable crop loss both on the field and in storage. The magnitude of competition between Callosobruchus maculatus (Coleoptera: Chrysomelidae) and human beings for this important crop necessitates its control to avoid food shortage and promote self-sufficiency. The rate of damage increases with the duration of storage under normal condition (Gujar and Yadav, 1978). To combat the problem of protein deficiency prevalent in developing countries, apart from mass production, there is a need to reduce qualitative as well as quantitative losses of pulses during storage (Babu et al. 1989).

Management of C. maculatus on stored cowpea in Nigeria has over the years been primarily through the use of synthetic insecticides. The use of synthetic insecticides led to numerous problems unforeseen at the time of their introduction: acute and chronic poisoning of applicators, farmworkers, and even consumers; destruction of fish, birds, and other wildlife; disruption of natural biological control and pollination; extensive groundwater contamination, potentially threatening human and environmental health; and the evolution of resistance to pesticides in pest populations (Perry et al 1998; National Research Council, 2000).

These serious limitations posed by the use of chemical pesticides as preservatives during storage called for the search of new alternative methods of controlling the stored product insect pests, such as the use of promising plants’ extracts and plant products that are less hazardous and environmentally safer. Many indigenous plant materials have been screened for bioactivity against pests and interest in the use as alternative to synthetic chemicals is increasing because it is readily available, cheaper and ecologically tolerable (Ivbijaro and Agbaje, 1985; Lale, 1992; Adedire and Lajide, 2003).

Heliotropium indicum of the family Boraginaceae is an annual plant found in tropical and non-tropical countries; which grows in all parts of Nigeria and is given different names by the local communities (Shoge et al., 2011). It bears different names, such as indicum Cock’s comb (Gambia), Indian Heliotrope, herb a verrues (France), Karkashen-koorama (Hausas-Nigeria), Ogbe Akuko or Agogo Igun (Yorubas-Nigeria) (Burkhill, 1985). It is used locally in Nigeria to treat ailments such as ulcer and fever, It’s most important local application is for skin lesions, wounds, abscesses, gastric and varicose ulcerations, rashes and warts (Sofowora, 1993). While Lawsonia inermis also known as Henna (English), Jalousie (French), ‘Laali’ (Yoruba-Nigeria) is of family Lythraceae commonly found in savanna and deciduous forest. Henna leaves are very popular natural dye to color hand, finger, nails and hair. The dye molecule, Lawson is the chief constituents of the plant. In folk medicines, henna has been used as astringent, antihemorrhagic, intestinal antineoplastic, cardio-inhibitory, hypotensive, sedative and also as therapeutic against amoebiasis, headache, gonorrhea, jaundice and leprosy (Raja sekaran, 2001, Olowokudejo et al 2008). In northern Nigeria L. inermis leaves have been used traditionally as a remedy against diarrhea, dysentery an  d other related diseases, (Aliyu, 2006).

The objective of the research reported herein was to: (i) to evaluate the oviposition and progeny deterrent activity; damage and infestation assessment of C. maculatus on cowpea seeds exposed to H. indicum and L. inermis leaves powders and (ii) determine the qualitative phytochemical constituents of the tested plants that might be responsible for its toxic activities.

1 Results and Discussion
The contact toxicity on the survival of adult beetles after treatment with the plant powders is presented in Table 1. The results revealed that in each treatment, the mortality of C. maculatus increased gradually with time of exposure and concentration of plant powders. Although none of the tested plant powder were able to cause 100% adult mortality; however, at 2.5 g/20 g of cowpea seeds L. inermis caused 81.14% adult mortality and H. indicum exerted 75% adult mortality at 120 h after exposure time, closely followed by L. inermis applied at 2.0 g and 1.0 g which resulted in 75% adult mortality. The results clearly indicated that plant powders tested as contact phtyo-insecticides significantly (P<0.05) reduced number of tested insect. In general, L. inermis powders were more toxic than H. indicum powders.

Table 1 Mean percentage mortality of C. maculatus treated with powders of H. indicum and L. Inermis

 
The ability of the plant powders to cause adult mortality can be attributed to contact toxicity effects of the plants. Insects breathe by means of trachea which usually opens at the surface of the body through spiracles (Adedire et al., 2011). These spiracles might have been blocked by the powders thereby preventing respiration via trachea leading to suffocation. Furthermore, significantly very high mortality indicates the probable presence of insecticidal properties in the plants. This confirm the findings that plants being rich source of bioactive chemicals with insecticidal properties (Rajkumar and Jebanesan, 2004).

The data shown in Table 2 revealed the effect of plant powders in suppressing oviposition of C. maculatus on cowpea seeds and reduction in adult emergence on treated cowpea seeds. The reduction in oviposition was increased with the increase in dosage of each treatment. Higher dosage rate of 2.5 g/20 g of cowpea seeds were found to be effective in suppressing egg laying as compared to lower dosage rate. Maximum oviposition deterrence activity was observed with L. inermis powder. Earlier, Olaifa and Erhun (1998) found that higher concentration of the powder of Piper guineense significantly reduced the oviposition. Oviposition detergency may be due to the changes induced in physiology and behaviour in the adult of C. maculatus due semiochemical nature of the powder which alters the behaviour and physiology of the insects affecting adversely the egg laying capacity (Shukla et al., 2007). Besides, plant powders can reduce insect movement, sexual communication and disrupt mating activities (Akinkurolere et al., 2009; Ileke et al., 2012) and as well as deterring females from laying eggs. This study was in support of Dolui et al (2010; 2012) who reported considerable reduction in the number of eggs laid per female Helopeltis theivora after the treatment with H. indicum extract.

Table 2 Mean percentage of oviposition deterrent and percentage reduction in adult emergence by C. maculatus on treated cowpea seeds

 
A significant reduction in adult emergence was recorded among the treatments (Table 2). In the present study, maximum reduction in the adult emergence was observed in the seeds treated with highest dosage rate of plant powder. The results indicated that adult emergence reduction is based on plant powder dosage rate. This corroborates the findings of Annie Bright et al (2001) and Raja et al. (2001) who reported that botanicals inhibited adult emergence in C. maculatus in cowpea. Jayakumar et al. (2003) reported that plant extracts have obvious effects on postembryonic survival of the insect and resulting reduction in adult emergence in all the concentrations of different plants. The reduction in adult emergence could either be due to egg mortality or larval mortality or even reduction in the hatching of the eggs. It has been reported that the larvae which hatch from the eggs of Callosobruchus species must penetrate the seeds to survive (FAO, 1999). The leaf powders might thus have inhibited the larval penetration into the seed and thus showed maximum adult emergence reduction (Khalequzzman and Goni, 2009). Further Enslee and Riddiford (1997) suggested that the failure of eggs to hatch could be attributed to incomplete blastokinesis and abnormal breakage of extra embryonic membranes in the embryo. The ability of the evaluated plant powders to significantly suppress adult emergence indicated that the plant might possess ovicidal and larvicidal properties. Several hundred plants have been reported as ovicides and oviposition deterrents (Ewete et al. 1996). Shukla et al (2007) reported that ovicidal activity of plant powders were observed to decrease progeny production of C. chinensis on stored green gram seeds.

Percentage infestation and weight loss was presented in Table 3. Result shows that infestation and weight loss was highest in untreated (control) seeds and lowest at 2.5 g/20 g of treated cowpea seeds. Meanwhile, cowpea seeds treated with H. indicum powder was significantly different (P<0.05) across treatments, while cowpea seeds treated with L. inermis was not significantly different (P>0.05) in reducing infestation and weight loss, respectively.

Table 3 Mean percentage seed damage by C. maculatus and percentage weight loss recorded on treated cowpea seeds

 
The low infestation and weight loss recorded in this study could be attributed to the reduction in F1 progeny as a result of low egg hatchability (ovicidal properties) and adult emergence in the treated seeds resulting from the toxicity effect exhibited by the plant powder. Plant products control the pests at various stages, which kill or extend the life stages (larvae, pupae, eggs) (Baskar and Ignacimuthu, 2012). The higher percentage of infestation and weight loss recorded in untreated cowpea seeds confirmed that the larval stage of the beetle is the destructive stage as the adults do not feed (Ileke et al., 2012).

Results of phytochemical constituents of both plant powders were presented in Table 4. The phytochemical screening showed the presence of Tannins, Cardiac glycosides, Saponins, Flavonoid, Steroids, and Terpenoids in both plant powders. Phlobatanins and Anthraquinone was observed to be absent in both plants. While alkaloid was present L. inermis and absent in H. indicum (Table 4). The findings from this study aligned with the previous findings of Dolui et al. 2010; 2012; Shoge et al. 2012; Arun et al. 2010; Kannahi and Vinotha, 2013; Kawo and Kwa, 2011.

Table 4 Phytochemical constituents of H. indicum and L. inermis leaves powder

 
Results of the phytochemical composition of H. indicum and L. inermis provide an insight into the mechanism of actions of H. indicum and L. inermis. Phytochemicals, such as tannins, have been reported to possess strong activities against several plant pathogens and insect pests (Mila et al., 1996). Karamanoli et al. (2011) reported that tannins exert their action by a combination of mechanisms that include iron chelation and enzyme inhibition. Though the exact mechanism behind the observed actions of H. indicum and L. inermis is not yet known, the preponderance of tannins in its powder may suggest a role for phytochemical. Chaieb (2010) extensively reviewed insecticidal effects of saponins, linking their insecticidal activity their interaction with cholesterol, which results in impaired ecdysteroid synthesis. Dolui et al. (2006) on the other hand reported that tannin combine with protein inhibit the enzyme activity and reduce the availability of protein in haemolymph insect and alkaloid responsible for toxic effect against Tribolium castaneum.

The insecticidal activity exhibited by the evaluated plants in this study correlates with the findings of Dolui et al. (2010 and 2012) who reported the insecticidal activity (antifeedant activity) of H. indicum against Helopeltis theivora, antimicrobial (Shoge et al 2011; Kannahi and Vinotha, 2013) and antibacterial activity (Arun et al 2010; Kawo and Kwa, 2011) thus suggesting the insecticidal and medicinal potentials of the plants.

In conclusion, the findings of the present study confirm the toxic effect of leaf powders of tested plants in suppressing oviposition and adult emergence of C. maculatus, thus are efficacious in protecting cowpea seeds from insect infestation and damage at farmer level at low volume of seeds and since adult C. maculatus do not feed on stored cowpea seeds but only deposit their eggs; admixing the plant leaves powder is recommended as ecofriendly and non-toxic methods in management of C. maculatus and recommended for short time storage.

2 Materials and Methods
2.1 Experimental location and insect culture
The experiment was conducted in the Biology Laboratory, Department of Science Laboratory Technology, Rufus Giwa Polytechnic, Owo, Ondo State, Nigeria. C. maculatus was obtained from an infested stock of cowpea bought from Oja-oba market, Akure, Ondo, Nigeria and sub cultured in 2 L kilner jar, covered with muslin cloth to allow for air circulation. Insect culture and experiments were carried out under ambient temperature of 28± 2℃ and 75± 5% relative humidity. The jars were kept for the insect to breed and multiply.

2.2 Plant collection and preparation
Leaves of H. indicum and L. inermis were collected from Uso, Owo, Ondo State, Nigeria and authenticated by the Forestry and Wood Technology Department of Rufus Giwa Polytechnic, Owo, Ondo State, Nigeria. These plant leaves were rinsed in clean water to removed dirt and other impurities and air dried in a well-ventilated laboratory for three weeks. Thereafter, the dried leaves were made into powder using mortar and pestle. The powders were further sieved and were packed in plastic containers with tight lids and stored in a laboratory cupboard prior to use.

2.3 Collection of cowpea seeds
Cowpea (Drum variety) seeds used for this study were obtained from newly stocked seeds free of insecticides at the Polytechnic Teaching, Commercial and Research Farms. Damaged seeds were sorted out by handpicking leaving a healthy seed for the conduct of the experiment. The clean seeds were oven dried at 105℃ for 90 minutes, and cooled at room temperature overnight in a desiccator to eliminate any possible C. maculatus infestation coming from field. Disinfested cowpea seeds were weighed using digital weigh balance model TS400D (Precision standard) into 20 g of 125 mL plastic container in triplicates for each concentration and stored in cool dry place.

2.4 Contact toxicity of plants powders on adult mortality
Fine leaves powder H. indicum and were evaluated at different concentrations of 0 g, 1.0 g, 1.5 g, 2.0 g and 2.5 g) admixed separately with 20 g of uninfested cowpea seeds in 125 mL plastic containers. Each plastic container was tumbled several times to ensure homogenous mixing of powder with grains. Ten unsexed adult C. maculatus (2~3 days old) were released into each plastic container; these were covered to prevent entry and exit of insects. Adult mortality was observed at 24 h interval (24, 48, 72, 96 and 120 h) after infestation for both plant samples. Adults were considered dead when they did not respond to gentle pressure using a fingertip. To avoid the possibility of death mimicry, the beetles were watched for 2 min again subjected to gentle pressure. Three replicates of the treated and untreated controls were laid out in Complete Randomized Block Design arranged on the laboratory work bench. Percentage adult mortality was calculated by using the method by Omotosho and Oso (2004).

Percentage mortality = no of dead insect/total no of introduced insect × 100/1

2.5 Oviposition deterrent activities
Oviposition deterrent activities were assessed by admixing 1.0 g, 1.5 g, 2.0 g and 2.5 g of the different plant powders with 20 g of uninfested cowpea seed in 125 mL plastic containers. Ten unsexed adults of C. maculatus were released in each plastic container and covered with a lid. They were allowed to remain in the container for 7 days till they laid eggs. One week after oviposition (i.e. 14 days after infestation) the number of eggs laid on treated seeds and control seeds were counted using hand lens prior to the demise of the female insects (14 days after treatment) and the percentage of oviposition deterrence activity was calculated using the formula adopted by Arivoli and Tennyson (2013).

%oviposition deterrent activity= (A-B)/(A+B) × 100/1 (A: no of eggs laid in control dish; B: no of eggs laid in treated dish)

2.6 Adult emergence deterrence activity
To determine the F1 progeny detergence efficacy of plant powders, 20 g of cowpea seeds were placed in 125 mL plastic container and admixed with different dosage rate of the plant powders as stated above. After the eggs were counted the experimental set up was kept undisturbed until the emergence of Fl adults. The number of F1 adults that emerged from each replicates in control seeds and treated seeds was counted with an aspirator and recorded at 30 days after infestation. The number of emerged adult from treated seeds and control seeds was used to calculate percentage reduction in adult emergence.

Percentage reduction in adult emergence = (C-D)/C × 100/1 (C: no of emerged adult from control dish; D: no of emerged adult from treated dish)

2.7 Damage and infestation assessment
Total number of adult emergence holes in each treatment was counted after 30 days of release and used to assess damage and subsequently percentage infestation.

Percentage infestation = no of seeds with adult exit hole/ total no of seeds observed in sample × 100/1

The final weight of each of the treatments was taken and this was used to calculate the percentage weigh loss due to infestation.

Percentage weight loss = (E-F)/E × 100/1 (E: initial cowpea seeds weight; F: final cowpea seed weight)

2.8 Phytochemical screening of plant material
Phytochemical analysis of the leaf of H. indicum and L. inermis was carried out by screening for the presence tannins, phlobatanin, cardiac glycosides, anthraquinones, saponins, steroids, terpenoids and flavonoids. For tannins, 5 g of each portion of plant extract was stirred with 10 ml of distilled water and filtered as described by Trease and Evans (1998). Blue black, green, or blue-green precipitate formed following the addition of few drops of 5% ferric chloride was taken as evidence for the presence of tannins. Deposition of a red precipitate when aqueous solutions of leaf extract was boiled with 1% (v/v) HCl was taken as evidence for the presence of phlobatannin (Trease and Evans, 1998). Salkowski’s test, as described by Sofowora (1993), was used to test for cardiac glycosides. Leaf extract (0.5 g) was dissolved in 2 ml of chloroform prior to the careful addition of 1% (v/v) H₂SO₄ to form a lower layer. A reddish-brown colour at the interface was taken as evidence for the cardiac glycoside. Borntrager’s test was used for the detection of anthraquinones (Heyde et al., 1984). Plant material (5 g) was shaken with 10 ml benzene and filtered. Ammonia solution (5 ml, 10%) was added to the filtrate and a pink, red, or violet colour formed in the ammoniacal (lower) phase was recorded as an indication of the presence of free anthraquinones.

2.9 Statistical analysis
Data collected were subjected to analysis of variance (ANOVA) using Statistical Package for Social Sciences (SPSS) for windows version 14. While egg count, adult emergence, damaged and undamaged seeds, were subjected to square roots formation and percentage were arc sine transformed before analysis, where significant differences existed, treatment means were separated using Least Significant Difference (LSD) at 0.05% probability level (Gomez and Gomez, 1984).

Acknowledgement
The authors are grateful to Joseph Sylvanus Nwankwo of the Department of Science Laboratory Technology, Rufus Giwa Polytechnic, Owo, Ondo State, Nigeria for his assistance in collecting data used in this study.

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