Essential oil composition of Callistemon citrinus (Curtis) and its protective assessment towards Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae)

Essential oil (EO) was extracted from Callistemon citrinus leaves by hydro-distillation. The extracted oil was analysed by GC and Mass Spectroscopy. Analysis report showed that the major constituent of the essential oil was eucalyptol (40.44%). The EO of C. citrinus exhibited 100% fumigation toxicity (adult mortality) against adult and 95.8% larvicidal activity against Tribolium castaneum at 160 μL/L (12 hrs) and 320 μL/L (48 hrs), respectively. The effective concentration of 37.05 μL/L (adult) and 144.31 μL/L (larva) at 24 and 48 hrs respectively. A 100% repellent activity was observed at 20 μl for adult beetles and 93.3% for larvae of T. castaneum at 20 μl after 24 h. Exposure to C. citrinus EO significantly reduced beetle fecundity, ovicidal activity, egg hatching, larvae survival, and emergence of adult. The effect of EO on detoxification enzymes of T. castaneum adults was examined. Results indicated that the activity of detoxification enzymes drastically varied when compared with control. This EO had toxicant effects on all stages of the life of T. castaneum. Hence it may be used as fumigant instead of the use of using synthetic chemical fumigants.


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Oil Extraction and GC-MS analysis 75 Callistemon citrinus (Bottle brush) was collected from the campus of University of 76 Madras. The oil was extracted from freshly-collected plant leaves by hydro-distillation, then it 77 was subjected to GC-MS analysis. 78 Toxicity Study 79 Fumigation toxicity of C. citrinus EO was tested in the laboratory using filter paper 80 method on adult insect at 28 ± 2ºC and 60-70% RH [23]. Two different ranges of concentrations 81 such as 40, 80, 120, 160 and 200 μl/L, and 40, 80, 120, 160, 200, 240, 280, and 320 μl/L air were 82 evaluated for fumigation toxicity on adults and larvae, respectively. Ten freshly-emerged 3-7-83 day-old adult/10-12 days, old larvae were released in a bottle along with a small amount of flour 84 as feed. The EO was poured on filter paper and it was adhered inside the screw cap of the bottle 85 then closed tightly. Without treatment of essential oil to be consider control. Five replications 86 were made for all treatments and controls. Mortality of adults and larvae were recorded after 3, The repellency of EO was measured using the diet impregnation method on larvae.  Ten adults were released into Petri dish (100 ml) with known quantity of wheat flour.

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Filter paper (Whatman No. 1) discs measuring about 2 cm dia, were impregnated with different 106 concentrations (5, 10, 20 and 30 µL/L) of C. citrinus EO. At 24 hrs after treatment the adult were 107 transferred to new Petri dish with food. The Petri dishes were carefully examined and recorded 108 the number of eggs laid in control and treatments for a period of 2 days by using compound 109 microscope. Knock-down adults were counted separately and recorded as a knock-down effect.

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Five replications were used for each treatment and the control group.

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Growth inhibition effects 112 Fifty adult beetles released in a Petri dish containing known quantity of wheat flour.

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After 48 h, the adult beetles were removed and numbers of eggs laid in each Petri dish were 114 counted. Subsequently, the filter paper treated with different sub-lethal concentrations of EO was 115 placed inside a Petri dish. Filter paper discs devoid of any volatiles were used as a control. The  Adult insects were treated with sub-lethal concentrations of 5, 10 and 20 µL/L of EO.

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The live insects were used in the biochemical analysis which consisted of three replicates.

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Treated adults (10 individuals for each concentration) were transferred separately and 124 homogenized with 500 μl of ice-cold phosphate buffer (20 mM, pH 7.0) using a Teflon, hand 125 homogenizer to estimate the total protein, esterase, phosphatase, and Glutathione-S-Transferase  The total protein, acid & alkaline phosphatases and ß-carboxylesterase of adult were 137 examined by discontinuous PAGE gel using non-denatured conditions. The gel electrophoresis 138 was run by using 5% stacking gel (pH 6.8) and 8% separating gel (pH 8.8) in Tris-glycine buffer Student's 't' test was carried out to determine the significant differences between the 148 biochemical constituents and enzyme activity in the treatments and control. Differences between 149 means were considered as significant at p ≤ 0.05. All statistical analyses done original data (after 150 transformed also the data did not showed significant distribution Shapiro wilk test). The probit 151 analysis was done for fumigation toxicity. Significant different between the treatment group was 152 calculated Duncans test followed by F-Test. The SPSS software, version 25was used for 153 analysis.

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Oil yield 156 EO of C. citrinus was extracted from the leaves using a Clevenger apparatus at 65 ºC for 157 3 h. Initially the oil was whitish in colour but later turned a pale yellow. The maximum yield of 158 650 µL/100 g (fresh weight of leaves) was obtained.  (Table 2b). The fumigation toxicity of 180 C. citrinus EO was concentration and time dependent against T. castaneum larvae, however, the 181 larvae appeared to be more tolerance to the EO than adults.  (Table 3).
The larval repellency was conducted in Petri dishes using a choice-based method at 5, 10, 188 15 and 20 μL concentrations and different observation period of 2, 4, 6, 12, and 24 h. The 189 maximum repellency (93.3%) was observed at 20 μL concentrations at 24 h observation. Lowest 190 concentration showed 30% repellent activity against T. castaneum larvae at 24 h (Table 3). 191 Overall, the results indicate that the EO exhibited good repellence potential on both larvae and 192 adults. total protein content of T. castaneum adult was highly and significantly reduced relative to the 222 control value of 7.43 mg/mL of protein to 6.19 mg/mL, 5.72 mg/mL, 5.32 mg/mL in adults 223 exposed to different concentrations (5, 10 and 20 µL/L) of EO, respectively (Fig. 1a).

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Acetylcholinesterase activity dramatically increased in the 10 and 20 µL/L , treatments when 225 compared to the control; while, at 20 µL/L concentration treatment reduced Acetylcholinesterase 226 activity (Fig. 1b).

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The level of α-Carboxylesterase activity was significantly increased at three of the 228 selected sub-lethal concentrations of EO, compared to the control (Fig. 1c). β-carboxylesterase 229 activity level was also drastically elevated in the 5, 10 and 20 µL/L treatments compared to the 230 control, however, no significant difference was observed between the different sub-lethal 231 treatments (Fig. 1d).

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Exposure of T. castaneum adults to C. citrinus EO resulted in decreased level of acid 233 phosphatase at selected concentrations when compared to control group (Fig. 1e). Alkaline 234 phosphatase activity was significantly reduced in the 5 µL/L treatment and drastically elevated in 235 the 10 µL/L treatment. Significantly lower activity was recorded in the 20 treatment when 236 compared to the control group (Fig. 1f). Glutathione-S-Transferase levels significantly increased 237 in the 10 and 20 µL/L treatments, relative to the control group, but was significantly lower, 238 relative to the control, in the 10 µL/L treatment (Fig. 1g).

Qualitative analysis of T. castaneum adult biochemistry 240
A qualitative analysis of total proteins was analysed using the native PAGE method.

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Protein extracted from T. castaneum adults treated with sub-lethal concentrations of C. citrinus 242 EO showed a reduction in the number protein bands, relative to the control (Fig. 2a). The when beetles were exposed to higher concentration of EO (Fig. 2b). Electrophoretic analysis of 248 acid and alkaline phosphatase enzyme activity in adult beetles was not affected by exposure to 249 the range of sub-lethal concentrations of EO used in the experiment (Fig. 2c,d). Since they are generally volatile in nature, essential oils are very easy to use and kill the pests of 256 stored grains. In the present study, essential oil extracted from C. citrinus leaves was tested for 257 its potential against the larvae and adult beetles of T. castaneum.  indicated the decreased oviposition (28%) at 5.2 mg/cm 2 concentration when the adult beetles 302 were exposed to EO derived from leaves of C. loga. The reduction in oviposition was probably 303 due to physically weakened insects as well as lesser surviving insects [46]. 304 The inhibition of egg hatchability or ovicidal activity was rapidly exhibited by exposure 305 to the EO without any direct contact with the eggs due the volatiles released by the EO. The