MATERIALS AND METHODS

Preparation of Plant Extracts
Dry fruits of pepper fruit, Dennettia tripetala, black pepper, Piper guineense and African nut-meg, Monodora myristica and dry buds of clove, Eugenia aromatica were purchased in herbal stores at Erekesan Market in Akure, Nigeria. Pulverized parts were obtained according to the method described by Adedire and Lajide (2000). The plant materials were dried in an electric oven to a constant weight at 40°C for 8 h, ground thoroughly in an electric 5.0 HP electric grinder and sieved through a 40 holes mm^-2 mesh screen. To obtain the extract, ten grams of each of ground plant materials was put in a round bottomed flask and 100 mL of absolute ethanol was added and soaked for 24 h. The mixture was boiled at 60°C for 30 min in UNISCOPE SM801A Laboratory Water Bath and the solution was filtered using Whatman No.1 filter paper. The resulting filtrate was kept in a tightly covered dark brown sterile bottle prior to use.

Preparation of Fish Samples and Insect Culture
Samples of African mud catfish, C. gariepinus each weighing 100 g were obtained from dried fish markets in Akure, Nigeria. All fish samples and bioassay jars were disinfested by heat treatment in the Gallenkamp oven at 60°C for 1 h and allowed to cool at room temperature. The initial source of D. maculatus was obtained from naturally infested smoked catfish and was cultured in a Kilner jar covered with muslin cloth and maintained at tropical storage conditions of temperature (30±2°C) and relative humidity (65±5%). A new generation was obtained by placing adult insects from the stock culture on fresh disinfested fish and then removing the parent adults after 2-3 weeks period. Pieces of water- soaked cotton wool were supplied in the jar to induce oviposition.

Investigation of Extracts on Beetle Mortality and Repellency
An aliquot of 5 mL of each of the plant extracts of 2.5, 5.0, 7.5 and 10% concentration was evenly rubbed onto the body of 100 g disinfested smoked fish sample. The treated fish sample was air-dried for 1-2 h in order to remove traces of the solvent and placed in a plastic jar measuring 80 mm depth and 100 mm in diameter. Six newly emerged adults and six third instar larvae of D. maculatus were introduced into separate plastic jars which were then covered with muslin cloth. Each experiment was replicated thrice. A control experiment without ethanol extract was also set up in triplicate. The number of dead insects was assessed and recorded daily up to 7 days and the percentage mortality was calculated. The procedure described by Don-pedro (1985) and Egwunyenga et al. (1998) was adapted for the repellency test. 5 mL of 2.5% concentration of each of the plant extracts was rubbed onto the body of disinfested 100 g smoked fish and placed on one side of a plastic chamber, measuring 25x12x10 cm. The same weight of untreated dried fish was then placed on the opposite side of the chamber (10 cm apart). Six adults (2-4 day old) of insect species starved for 48 h were introduced at the centre of each of the triplicate chambers. Daily observations were made for a period of 7 days by counting the number of insects found on or within a 1.0 cm radius of treated and untreated fish in each chamber. The repellency rate (RR) was calculated by expressing the difference between the number of insects around the untreated and treated fish as a percentage of total number of introduced insects. The RR was assigned a repellency class (RC) as follows:

Data obtained were subjected to analysis of variance (ANOVA) and where significant differences existed at 0.05 significance level, the treatment means were separated using Tukey’s Test.

RESULTS

Each of the plant extract dosages caused significant difference (p<0.05) in mortality of D. maculatus adults on smoked fish compared with the control (Table 1). The effect of D. tripetala extract dosages proved superior to E. aromatica, P. guineense and M. myristica in the control of adult insects at 1 and 3 Days After Treatment (DAT). However, there was a total kill of the adults by the extracts of each plant material at 7 DAT. Comparatively, E. aromatica was less toxic to adult beetles at all corresponding concentration levels. The result shows a greater devastating effect of plant extracts on larval mortality. Larvae were totally killed by the extracts of D. tripetala and P. guineense at 3 DAT while there was no survivor in any of the treatments at 7 DAT (Table 2).

The presence of the extracts on dried fish repelled D. maculatus adults from smoked fish. Significantly lower number (p<0.05) of D. maculatus was consistently obtained around treated fish compared with the proportion associated with the control at observation time (Table 3). The repellency test classified D. tripetala, E. aromatica and P. guineense to the Repellency Class (RC)_IV with Repellency Rate (RR) 45.65, 46.83 and 48.41%, respectively while M. myristica was categorized as RC_III with RR 35.32%.

Table 1:	Effect of ethanol extracts on percentage mortality of Dermestes maculatus adults
Values are means of triplicate samples followed by the standard error of means. Means in the same vertical column with different superscripts for each plant material are significantly different (p<0.05) by Tukey’s Test

Table 2:	Effect of ethanol extracts on percentage mortality of Dermestes maculatus larvae
Values are means of triplicate samples followed by the standard error of means. Means in the same vertical column with different superscripts for each plant material are significantly different (p<0.05) by Tukey’s Test

Table 3:	Effect of ethanol extracts on repellence of Dermestes maculatus adults on smoked fish
Values are means of triplicate samples followed by the standard error of means. Means in the same column with different superscripts for each plant material are significantly different (p<0.05) by Tukey’s test RR: Repellency Rate, RC: Repellency Class

DISCUSSION

In this study, ethanol extracts of D. tripetala, P. guineense, M. myristica and E. aromatica significantly increased adult and larval mortality and were repellent to the main pest of the highly relished smoked catfish. The results obtained in this study are similar to the findings of Ofuya and Dawodu (2001) and Adedire and Lajide (2000) who reported the susceptibility of different ages of Callosobruchus maculatus and D. maculatus respectively to P. guineense powder at all rates of application. While Okorie et al. (1990) reported a 93% kill for D. maculatus larvae and total mortality of all adults when exposed to 2 g of neem seed powder / 25 g Tilapia species, Ofuya and Bamigbola (1991) and Fasakin (2003) reported the effectiveness of the crude extract of M. myristica against C. maculatus and D. maculatus, respectively.

In this study, the larvae were especially rapidly killed by the extracts. Lale (1995) reported that plant extracts are highly lipophilic and could penetrate the cuticle of insects. The result obtained in the study is in line with Aku et al. (1998) who reported that extract of Anonna senegalensis was more effective than the powder in the control of C. maculatus. Similarly, Okonkwo and Okoye (2001) reported 100% kill of larvae of D. maculatus when treated with extracts of D. tripetala and P. guineense at dosages lower than the powders. Odeyemi et al. (2000) observed that cases of high mortality occur in larvae partly because of their inability to detoxify plant toxins when feeding actively, especially at the 1st-4th instar larval stage. According to the authors, larvae are voracious eaters because of growth requirements, in contrast to the adult insects, which tend to have a reduced feeding habit.

Stoll (2000) reported repellence as a major mechanism by which plant products control insect damage to stored produce. This view has been largely supported by the significant repellent effect of the ethanol extracts of the four plant materials investigated in this study. The rating of the extracts as promising repellents in this study is partly in agreement with Egwunyenga et al. (1998) who also attributed the repellence of D. maculatus and N. rufipes from admixed fish to olfactory and gustory sensations.

The toxicity properties of P. guineense and D. tripetala have been attributed to their pungent and pepperish taste which could asphyxiate insects by blocking the spiracles (Amusan and Okorie, 2002) and the presence of bioactive ingredients, such as, alpha-pinene, limonene and linalool in P. guineense (Golob et al., 1999), 2, terpenes and linoleic acid in M. myristica and eugenol, tannic acid and asarone in E. aromatica (Golob et al., 1999).

The use of the studied plant materials could be desirable in protecting cured fish in the tropics, especially as organoleptic assessment has shown that treated fish do not exhibit adverse evidence of taint, smell or change in taste, texture or flavour (Akinwumi et al., 2006).

ACKNOWLEDGMENTS

The authors are grateful to Vice-Chancellor, Adekunle Ajasin University, Akungba-Akoko, Nigeria for supporting this study with a research grant.