MATERIALS AND METHODS

Compounds: The essential oil compounds used were trans-anethol [1-Methoxy-4-(1-proenyl) benzene], thymol (99.5% 5-methyl-2-isopropylphenol), eugenol [2-methoxy-4-(2-prophenyl) phenol] were purchased from Sigma, USA and cinnamaldehyde (trans-3-phenyl-2-propenal) was procured from Chem Industry, USA.

Test insect: Laboratory reared: Laboratory reared strain of T. castaneum (originally collected from the Slough Laboratory, UK and the culture are maintained in the Crop Protection and Toxicology Laboratory, Department of Zoology, University of Rajshahi since, 1991) were used. A standard mixture of whole-wheat flour with powdered dry yeast in a ratio of 19:1 was used as food medium. Adults used in the experiments were 3-7 day old and of mixed sex and larvae were 10 day and 18 day old used in contact and fumigant toxicity experiments. All stage were reared at 28±1°C without light and humidity control. The experiments were carried out during January to September 2008.

Contact toxicity: To examine the contact toxicity of the tested compounds to different days of the insect, 6 cm petridishes were used. A serial dilution of each compound of essential oils was prepared using acetone as a solvent. Aliquots of 0.5 mL of the dilutions were applied into the petridish. After evaporated the solvent for 5 min the adults (3-7 days old) and larvae (10 and 18 day old) were released in group of 10 for each replication of doses and control. Then the petridishes were kept at 28±1°C in the incubator. Each concentration and control was replicated three times. Mortality was determined after 6, 12, 18, 24 and 48 h from commencement of exposure.

Fumigant toxicity: Fumigant toxicity was done in 650 mL gastight jars with screwed caps as exposure chamber. A filter paper (Whatman No. 1), cut into 6 cm diameter was attached to the under surface of the cap to serve as a diffuser, on which varying doses of the tested compounds were applied while the control diffuser was left untreated. Insects were exposed to the 5 cm Petri dish that were placed at the bottom of each jar, ten individuals for each replication was placed and exposed to the various concentrations of vapour. The insects had no contact with the diffuser and stayed at the bottom of the chamber throughout the experiment. A 6, 12, 18, 24 and 48 h exposure time was considered adequate to assess mortality. After exposure, the insects were taken out of the jar and subsequently the dead insects were counted. Five replications were performed for each dose and control.

Statistical analysis: Data on percentage mortality were corrected for heterogeneity of treatment variances using arcsine-transformation (Papachristos and Stamopoulos, 2002a; Leatemia and Isman, 2004) before being subjected to ANOVA. Differences between treatment means were determined using Least Significant Difference (LSD) test (Gomez and Gomez, 1984).

RESULTS AND DISCUSSION

In contact toxicity for the tested monoterpene compounds, it can be seen from Table 1 and 2 that, at the lowest concentration of 0.036 mg cm^-2 and highest exposure time of 48 h, trans-anethol caused 70% mortality for adult, 76.66% mortality for 10 day old larvae and no mortality were observed for 18 day old larvae. Whereas, at the lowest concentration of 0.053 mg cm^-2 and highest exposure time of 48 h, thymol showed 43.33% mortality for adult, 76.66% mortality for 10 day old larvae and 23.33% mortality for 18 day old larvae. In case of eugenol, at the lowest concentration of 0.013 mg cm^-2 and highest exposure time of 48 h, 100% mortality were observed for adult, 96.66% mortality for 10 day old larvae and 46.66% mortality for 18 day old larvae. At the lowest concentration of 0.057 mg cm^-2 and highest exposure time of 48 h, cinnamaldehyde showed 93.33% mortality for adult, 100% mortality for 10 day old larvae and 30.0% mortality for 18 day old larvae. Complete mortality was obtained after 48 h at highest dose level above with all compounds for adult and 10 day old larvae, but not for 18 day old larvae. Here, the toxic order is Eugenol> cinnamaldehyde> trans-anethol> thymol in comparing the percent mortality at the lowest dose level and highest exposure time of 48 h.

In fumigation toxicity, at the lowest concentration of 1.08 mg^-1 and highest exposure time of 48 h, trans-anethol caused 3.33% mortality for adult, 60.0% mortality for 10 day old larvae and no mortality were observed for 18 day old larvae. Whereas, at the lowest concentration of 14.43 mg^-1 and highest exposure time of 48 h, thymol showed 56.66% mortality for adult, 90.0% mortality for 10 day old larvae and 6.66% mortality for 18 day old larvae. In case of eugenol, at the lowest concentration of 0.769 mg^-1 and highest exposure time of 48 h, 50.0% mortality were observed for adult, 46.66% mortality for 10 day old larvae and no mortality were observed for 18 day old larvae.

Table 1:	Percentage mortality of different stages of Tribolium castaneum in contact toxicity to trans-anethol and thymol
Means in each column and row followed by the same letters do not differ significantly using Least Significant Difference (LSD) test at **p<0.01

At the lowest concentration of 10.14 mg^-1 and highest exposure time of 48 h, cinnamaldehyde showed 100% mortality for adult, 83.33% mortality for 10 day old larvae and 56.66% mortality for 18 day old larvae. Here the toxic order is Eugenol> trans-anethol > cinnamaldehyde > thymol in comparing the percent mortality at the lowest dose and concentration level and highest exposure time of 48 h (Table 2-4).

The insecticidal constituents of many plant extracts and essential oils are monoterpenoids. Due to their high volatility they have fumigant activity that might be of importance for controlling stored-product insects (Regnault-Roger and Hamraoui, 1995; Ahn et al., 1998). The insecticidal activity varied with insect species, oil concentrations and exposure time. In the space fumigation studies the oil and its aromatic constituents showed strong species-specific toxicity that was highly dependent upon the dosage and time after treatment (Ogendo et al., 2008). Essential oil products are generally broad-spectrum, due to the presence of several active ingredients that operate via several modes of action (Chiasson et al., 2004).

Table 2:	Percentage mortality of different stages of Tribolium castaneum in contact toxicity to eugenol and cinnamaldehyde
Means in each column and row followed by the same letters do not differ significantly using Least Significant Difference (LSD) test at *p<0.05 and **p<0.01, NS: Not significant

Positive results for contact and fumigant activity of monoterpenes were obtained against T. castaneum. Monoterpenoids are typically volatile and rather lipophilic compounds, which can rapidly penetrate into insects and interfere with their physiological functions (Lee et al., 2002). Eugenol a monoterpenoid had fumigant toxicity to T. castaneum (Rozman et al., 2007). The essential oil from Vitex pseudonegundo, 1, 8-cineol was determined to be the major constituents has fumigant toxicity to T. castaneum (Sahaf et al., 2008). Of all the oils tested thus far, cinnamaldehyde is the only essential oil-derived compound that exerts contact toxicity to T. castaneum. The essential oil of garlic (Ho et al., 1996) as well as the n-hexane extract of star anise (Ho et al., 1995) and its main constituent, anethole (Ho et al., 1997) were more toxic to T. castaneum than to Sitophilus zeamais. On the other hand, the n-hexane extract of clove flower buds (Ho et al., 1994) and its main constituent, eugenol were more toxic to S. zeamais than to T. castaneum. Therefore, cinnamaldehyde is more advantageous as a contact poison as it is equally effective against stored product of insects.

Table 3:	Percentage mortality of different stages of Tribolium castaneum in fumigant toxicity to trans-anethol and thymol
Means in each column and row followed by the same letters do not differ significantly using Least Significant Difference (LSD) test at *p<0.05. and **p<0.01, NS: Not significant

The larvae of T. castaneum were progressively more tolerant to cinnamaldehyde with age, a trend similarly observed by other authors with hexane extracts of clove (Ho et al., 1994) and star anise (Ho et al., 1995) as well as the essential oil of garlic (Ho et al., 1996). Clemente et al. (2000) obtained positive results by treating Tribolium castaneum with the essential oil of Lavandula spica L. that contained 40% of 1,8-cineole. The laboratory studies of Lee et al. (2003) proved that T. castaneum could be controlled by 1,8-cineole, 1-fechone and pulegone, but only at the highest doses. The results of present study are mostly in the agreement with the results of other investigators.

Erler (2005) reported the fumigant activity of thymol against adults and eggs of T. confusum and larvae and eggs of Ephestia kuehniella Zeller. Concerning the larval stage, Stamopoulos et al. (2007) observed that susceptibility to vapours varied with age. In fact, as the larva develops it becomes less susceptible.

Table 4:	Percentage mortality of different stages of Tribolium castaneum in fumigant toxicity to eugenol and cinnamaldehyde
Means in each column and row followed by the same letters do not differ significantly using Least Significant Difference (LSD) test at *p<0.05. and **p<0.01, NS: Not significant

The adult of T. castaneum to all tested compounds shows that adults were much more susceptible than larvae. As the larvae grew older, they became less susceptible, except for the 16 day old larvae. There was a tendency for the larvae to become more tolerant to the tested compounds as they grew older provided they did not moult during exposure. As in the case of contact toxicity, T. castaneum larvae became progressively more tolerant with age (Huang and Ho, 1998). Furthermore, the essential oil evaluated in this study is used as a pharmaceutical agent and is thus considered to be less harmful to humans and the environment than the majority of conventional insecticides (Isman, 2005). Consequently, the possibility of employing these natural fumigants to control insects in stored products may warrant further investigation.

We extended the range of active oil substances of aromatic plants tested by four more compounds (trans-anethol, thymol, eugenol and cinnamaldehyde) in the control of T. castaneum all of which proved capable of achieving complete control within 48 h at the lowest doses. In the present study, cinnamaldehyde, eugenol, thymol and trans-anethol were demonstrated both contact and fumigant toxicity to T. castaneum adults and larvae. The observed contact and fumigant activity demonstrates that essential oils are a source of biologically active compounds which may potentially prove to be efficient insecticides.