Fumigant toxicity of volatile synthetic compounds and natural oils against red flour beetle Tribolium castaneum (Herbst) (Coleopetera: Tenebrionidae)

2.1 Insect

T. castaneum adults and larvae were obtained from a stock culture that was continuously maintained for several generations on wheat flour at the Entomology Research Institute insectary at laboratory conditions (29 ± 1 °C; 60–65% R.H.; 11 ± 0.5 h photoperiod). The adults used in the fumigation toxicity experiments were 5–7 day-old and the larvae were 12 day-old.

2.2 Volatile oils and volatile compounds

Camphor oil obtained from Cinnamomum camphora (also known as Laurus camphora) and eucalyptus oil obtained from Eucalyptus globulus Labill. were procured from the Central Institute of Medicinal and Aromatic plants, Lucknow, India. The two synthetic volatile compounds, benzaldehyde and propionic acid, were purchased from Ranbaxy Fine Chemicals Limited (RFCL), New Delhi, India.

2.3 Treatments and concentrations

The volatile oils and volatile compounds were tested individually as well as in different combinations. Besides a set of control, 12 different treatments were used as follows: (i) propionic acid (PA); (ii) benzaldehyde (B); (iii) eucalyptus oil (EO); (iv) camphor oil (CO); (v) PA + B (1:1 ratio); (vi) EO + CO (1:1 ratio); (vii) PA + EO (1:1 ratio); (viii) PA + CO (1:1 ratio); (ix) B + EO (1:1 ratio); (x) B + CO (1:1 ratio); (xi) PA + EO + CO (1:1:1 ratio) and (xii) B + EO + CO (1:1:1 ratio). Concentrations for fumigation studies were prepared based on the volume of the container used and expressed as μl/l of air. In the combination treatments the volatile oils and/or compounds were mixed in equal proportion and required concentrations were prepared. Five different concentrations viz., 20, 40, 80, 120 and 160 μl/l of air were screened in fumigation toxicity studies against larvae and adults. The ovicidal activity and adult emergence were studied at three different concentrations viz., 20, 40 and 80 μl/l of air. The fecundity studies were conducted using the sublethal concentrations viz., LC₁₀, LC₂₀ and LC_30.

2.4 Fumigation toxicity on T. castaneum adults and larvae

The toxicity of the volatile oils (eucalyptus and camphor) and the volatile compounds (propionic acid and benzaldehyde) on T. castaneum adults was tested by filter paper dip method at laboratory conditions (30 ± 1 °C; 60–65% R.H.). Whatman No. 1 filter paper discs (2 cm diameter) were impregnated with different concentrations of essential oils or compounds separately and were attached to the under surface of the screw caps of glass vials (volume 45 ml) separately as described by Negahban et al. (2007). The cap was screwed tightly on the vial after the release of 10 adult beetles (1–7 days old) or larvae along with little amount of wheat flour as food. For comparison a set of control, without volatile oils and compounds, was maintained. Each treatment and control was replicated five times. Mortality was recorded after 4, 8, 12 and 24 h from the commencement of exposure. When no leg or antennal movements were observed, insects were considered dead. Percentage insect mortality was calculated using the Abbott’s correction formula (Abbott, 1925).

2.5 Fecundity

Ten male and ten female T. castaneum beetles were taken in a 220 ml air tight petri dish and a little amount of wheat flour was added as food. Filter paper discs (Whatman No. 1; 2 cm diameter) impregnated with LC₁₀ concentration of benzaldehyde were placed inside the petri dish. After 24 h of treatment the adults were transferred to an untreated petri dish and number of eggs laid was counted up to 2 days under a stereo microscope. Fecundity per female was calculated. The other treatments and other sublethal concentrations (LC₂₀ and LC₃₀) were also tested by the same method. Five replications were maintained for control and treatment groups.

2.6 Egg hatchability, larval survival and adult emergence

Fresh eggs of T. castaneum were separated from insect culture vials and carefully transferred to 50 ml glass vials with the help of a smooth hair brush. About 15 eggs were separately treated with each concentration of each treatment by the same fumigation method described earlier. In the controls filter paper discs without volatiles were placed inside the vials. All treatments and control were replicated five times (n = 50). The egg hatchability was recorded daily and converted into percentage. Hatched larvae were maintained on wheat flour and the larval survival and percent adult emergence were recorded.

2.7 Fumigation toxicity inside 1 m³ wooden cage

The efficacy of effective treatments was tested inside a 1 m³ wooden cage. The wooden cage was used because it simulates a common storage structure. The wooden cage had a small opening with a sliding door. The cage was completely sealed with adhesive tape at all joints. Four treatments namely benzaldehyde, propionic acid, benzaldehyde + propionic acid and benzaldehyde + camphor oil were tested at their respective LC₉₉ concentrations. The LC₉₉ concentration of benzaldehyde solution required for 1 m³ volume was calculated and taken in a glass bottle. The bottle with benzaldehyde solution was kept at the centre inside the cage. One hundred adult beetles were released on 50 g wheat flour taken in a plastic bottle (3.5 l) and the mouth of the vial was closed with a muslin cloth. The plastic bottle with the beetles was kept inside the cage. Three replications were maintained for one treatment. The door of the cage was closed and the gaps were completely sealed with adhesive tape. The plastic vials were taken out after every 15 days; the mortality was recorded and the dead insects were replaced with new live insects. The experiment was continued for 90 days. Similarly other treatments were also tested. All treatments were replicated nine times. The percentage mortality was calculated and corrected using Abbott’s formula (Abbott, 1925).

2.8 Statistical analysis

The fumigation toxicity, fecundity, egg hatchability, larval survival and adult emergence were subjected to analysis of variance (ANOVA). Analysis of covariance (ANCOVA) was used to find treatment effects on egg hatchability and larval survival. Significant differences between treatments were determined by Tukey’s HSD tests (p ⩽ 0.05). The median lethal concentration of LC₅₀ and LC₉₉ values were calculated using probit analysis (Finney, 1971).

3.1 Adulticidal activity

Table 1 shows the lethal concentrations (LC₅₀ and LC₉₉) of all the volatile oil and volatile compound treatments against T. castaneum adults. Among the various treatments benzaldehyde + propionic acid combination killed more beetles in less period of exposure compared to the other treatments. The LC₅₀ and LC₉₉ concentrations of benzaldehyde + propionic acid combination were calculated as 30.6 and 99.28 μl/l, respectively. Individual treatments of benzaldehyde and propionic acid were also more effective than volatile oils. The LC₅₀ concentrations of propionic acid and benzaldehyde were 36.79 and 34.07 μl/l, respectively (Table 1).

The volatile oils were less effective than benzaldehyde and propionic acid treatments. However, camphor oil + benzaldehyde combination showed increased toxicity compared to individual camphor oil treatment and the increase was statistically significant (p > 0.05) after 24 h exposure (LC₅₀ = 40.98 μl/l).

3.2 Larvicidal activity

Benzaldehyde + propionic acid combination recorded the maximum larvicidal activity and the LC₅₀ value was 78.03 μl after 24 h exposure (Table 2). Benzaldehyde individual treatment was the second most effective treatment (LC₅₀ = 94.15 μl). The larvicidal activity of volatile oils, eucalyptus and camphor increased when they were mixed with benzaldehyde. The LC₅₀ values of benzaldehyde + camphor oil and benzaldehyde + eucalyptus oil were 110.38 and 119.17 μl, respectively, after 24 h of exposure.

3.3 Fecundity

The exposure of gravid female T. castaneum beetles to sublethal concentrations (LC₁₀, LC₂₀ and LC₃₀) of different treatments for 24 h resulted in reduced fecundity. The average fecundity of the beetle in control group was recorded as 4.7 eggs/female. In all the treatments the fecundity was reduced and it was directly proportional to the concentrations tested. Benzaldehyde + propionic acid combination recorded the lowest fecundity at LC₁₀ (2.44 eggs/female), LC₂₀ (1.32 eggs/female) and LC₃₀ (0.8 eggs/female) concentrations (Table 3). Benzaldehyde individual treatment was also found to be effective; this treatment recorded 2.76, 1.38 and 0.92 eggs/female insect at the LC₁₀, LC₂₀ and LC₃₀ concentrations, respectively, in 24 h.

3.4 Egg hatchability and larval survival

The egg hatchability was found to be much reduced by the treatment with benzaldehyde + propionic acid combination in a highly significant manner compared with propionic acid (F value in ANCOVA = 18.00; p < 0.001) and benzaldehyde individually (F value in ANCOVA = 12.73; p < 0.001). Benzaldehyde + propionic acid combination and benzaldehyde individual treatments recorded 100% ovicidal activity at 80 μl concentration (Table 4). In the control group the egg hatchability and survival of the hatched larvae were recorded as 86.7% and 84.6%, respectively. Benzaldehyde + propionic acid treatment showed the lowest egg hatchability of 27.8% and 12.5% at 20 and 40 μl concentration, respectively, and the larval survival was also the lowest in this treatment. The larval survival in benzaldehyde + propionic acid treatment was recorded as 10% and 6.6% at 20 and 40 μl concentrations, respectively, whereas these variations are less significant (Table 5). Propionic acid treatment at 80 μl concentration registered only 6.6% egg hatchability and all the hatched larvae died. Camphor and eucalyptus oil when treated individually affected the egg hatchability drastically at higher concentrations viz. 40 and 80 μl. The egg hatchability in eucalyptus oil treatment was recorded as 80%, 66.6% and 54% at 20, 40 and 80 μl concentrations, respectively. The oils were also found to be toxic to the hatched larvae and the survival of larvae at 20 μl concentration of camphor oil and eucalyptus oil was nearly 50% of total hatched larvae. When camphor oil was mixed with benzaldehyde only 27.3% egg hatching was recorded at 80 μl concentration, which was less than individual camphor and eucalyptus oil treatments. At 20 μl/l concentration, egg hatchability was statistically same in the treatments of propionic acid, benzaldehyde + eucalyptus oil, benzaldehyde + camphor oil and benzaldehyde + eucalyptus oil + camphor oil.

3.5 Emergence of F1 adults

All the treatments including volatile oils significantly affected the adult emergence compared to control. In control 80.2% adult emergence was recorded. In benzaldehyde + propionic acid treatment some larvae survived in 20 and 40 μl concentration treatments; the F1 adult emergence was noticed only in 20 μl concentration as 20% (Table 6). In benzaldehyde, propionic acid and benzaldehyde + camphor oil treatments adults did not emerge at 80 μl concentration.

3.6 Fumigation toxicity inside confined wooden cage

The efficiency of the effective treatments namely benzaldehyde, benzaldehyde + propionic acid, propionic acid and benzaldehyde + camphor oil was tested inside a confined wooden cage (1 m³) and the results are presented in Table 7. The LC₉₉ concentrations of these effective treatments presented good fumigation toxicity effects against the adult beetles. On the first observation period (15th day), 99.3% mortality was noticed in benzaldehyde + propionic acid treatment and the difference was highly significant (F value by ANCOVA = 7.646; p = 0.003) compared with other treatments. The same treatment was found to be significantly effective throughout the study period; it recorded 61.1% mortality on the 90th day (last observation). Benzaldehyde treatment recorded 91% mortality in the beginning (15th day). The toxic effect of all the treatments decreased gradually at every consecutive observation period.