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Vol. 11 No. 1 (2019)

The journal of Toxicology and pest control is one of the series issued twice by the Egyptian
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Citation: Egypt. Acad. J. Biolog. Sci. (F. Toxicology & Pest control) Vol. 11(1) pp. 27- 38 (2019)
 Egypt. Acad. J. Biolog. Sci., 11(1): 27– 38 (2019)
Egyptian Academic Journal of Biological Sciences
F. Toxicology & Pest control
ISSN: 2090 - 0791
http://eajbsf.journals.ekb.eg/

Toxicity of some Essential oils and its Biochemical Effect against Red Flour
Beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae)

El-Gizawy, K. KH.1; Halawa, S. M.1;Mehany, A. L.2and Mohamed, S.A. 2

1-Plant Protection Dept., Fac. of Agric. Moshtohor, Benha Uni., Egypt.
2-Plant Research Dept., Nuclear Research Center, Atomic Energy Authority,
Abozaabal, Egypt
E-mail: karam.elgizawy@fagr.bu.edu.eg

ARTICLE INFO ABSTRACT

Article History Fumigant activities for four essential oils; Garlic oil (Allium
Received:2/1/2019 sativumL); Basil oil(Ocimum basilicum); Pine (Pinus longifolia L.) and
Accepted:28/1/2019 Eucalyptus (Eucalyptus oblique L.) at different concentrations after 1, 2, 3, 5
_______________ and 7 days at 28±1° C were tested against 4th instar larvae and adults of the
Keywords:
red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae)
Toxicity, essential in the laboratory. The results showed that mortality increased with increasing
oils, biochemical oil concentration and time of exposure, also the higher essential oils toxicity
effect, Tribolium at the LC50 after 7 days post-treatment was Garlic oil. Effects of Garlic
castaneum essential oils on some enzymes activity in 4th instars’ larvae of T. castaneum
were investigated. From this study, we conclude that these essential oils have
a potential for applications in IPM programs for stored-grain pests because of
its high volatility and fumigant activity.
INTRODUCTION

Red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) is one

of the most destructive beetle species to several grains and flours. The progeny
production rate of T. castaneum is so high. The fourth instars larvae are highly active in
the rainy season and cause a very high infestation. This insect pest had a long association
with individual stored food and has been found in a relationship with an array of
commodities including grain, flour, peas, beans, cacao, nuts, dried vegetables, and spices,
but milled grain products such as flour seem to be their preferred food. Good (1936);
Campbelland Runnion (2003). Control of these insects depends on the use of synthetic
insecticides and fumigants, which has generated problems such as disturbances of the
environment, increasing costs of application, pest resurgence, pest amount of resistance
to pesticides and deadly effects on non-target organisms in addition to direct toxicity to
users Muniz et al., 2008; Jembere et al., 1995; Boyeret 's., 2012 and Simoniello et al.,
2008. Fumigation was being the most effective method for controlling stored grains pest pests.
Utilized of plants for infestations control on stored grain seems to offer desired
solutions, especially in the developing tropical countries where plants are found in
abundance everywhere throughout the 12 months. Moreover, there have been growing
interest in the use of both plant extracts and their essential oils since they exhibit low
mammalian toxicity and low persistence in environmental surroundings Fouad et al.,

Citation: Egypt. Acad. J. Biolog. Sci. (F. Toxicology & Pest control) Vol. 11(1) pp. 27- 38 (2019)
28 El-Gizawy, K. KH et al.

(2012); Papachristos and Stamopoulos(2002). Indicated that the essential herbal oils
produced in various external and internal glands of these plants are containing a very
complex combination of terpenes, sesquiterpenes, their oxygenated derivatives and other
aromatic compounds Ogendo et al., (2008). Many studies of the fumigant activity of such
natural substances have been undertaken to ascertain new control practices with lower
mammalian toxicity and low persistence in environmental surroundings Isikber et al.,
)2006); Erler (2005) and Mishra et al., )2014). Most natural plant compounds and
microorganisms employed in controlling pests are proven to affect the enzymatic profiles
(Nathan, et al., 2005). Cytochrome P450 monooxygenases (CYPs), glutathione-S-
transferases (GSTs) and esterase (ESTs) are 3 major detoxifying enzymes in most
organisms. At least one of them is involved with detoxification of insecticides in insects
(Bull, 1981). Insect GSTs have recently been implicated in resistance to insecticides,
organophosphorus and pyrethroid, through direct insecticide metabolism (Wei et al.,
2001) or by protecting against secondary harmful effects, such as raises in lipid
peroxidation, caused by insecticide exposure (Dou et al., 2009). A member of the esterase
cluster probably plays a role in the detoxification of xenobiotic esters. (Gacarand Tasksn,
2009). Alkaline phosphatase (ALP) is a brush border membrane gun enzyme and is
especially active in tissues with active membrane transport, such as intestinal epithelial
cellular material (Etebari and Matindoost, 2004a), Malpighian tubules (Etebari and
Matindoost, 2004b) and hemolymph(Etebari et al., 2007). This study investigates toxic
effectiveness of three essential herbal oils against the Red flour beetle, Tribolium
castaneum (Herbst) for protection of stored-grain from insect infestation also we had
investigated their biochemical effects against some nutrients process of the 4th stretcher larvae.
MATERIALS AND METHODS

The experiments were conducted at the stored product pests’ laboratory of the Plant
Protection Dept. Faculty of Agric., Moshtohor, Benha University
Insect Culture:
Tribolium castaneum was reared in a glass container (250ml) containing wheat
flour covered with a fine mesh cloth for ventilation. The cultures were maintained in the
dark in an incubator at 28 ±1 ºC and 60 ± 5% RH. Adults were obtained from laboratory
stock cultures maintained at the Plant Protection Dept. Faculty of Agric., Moshtohor,
Benha University, Egypt.
Essential Oils:
Four essential oils in Table (1) belonging to different families; Amaryllidaceae;
Lamiaceae; Myrtaceae; and Pinaceae were used during these investigations. All the
essential oils were bought from Al-Gomhuria Company of drugs, chemicals and medical
supplies in Egypt. Garlic oil (Allium sativumL); Basil oil (Ocimumbasilicum);Eucalyptus
(Eucalyptus globulus); and Pine (Pinus pinaster L.) The fumigant toxicity of these oils
was tested to the adults and 4th instar larvae of T. castaneum .
Table (1): The essential oils used in the investigation.
Common name
Scientific name Family Used part
English Arabic
1 Allium sativumL. Amaryllidaceae Garlic oil ‫الثوم‬ Seeds
2 Ocimum basilicum Lamiaceae Basil oil ‫الريحان‬ Leaves
3 Eucalyptus globulus Myrtaceae Eucalyptus ‫الكافور‬ Leaves
4 Pinus pinaster L. Pinaceae Pine ‫الصنوبر‬ Fruits

Fumigant Toxicity of Essential Oils:

Ten grams of each pure oil was diluted with 50 ml. acetone to obtain 20% (w/v)
stock concentration which diluted to obtain 10,50, 2.5,1.25,and .625% (w/v)
 Toxicity of some Essential oils and its Biochemical effect against Red Flour Beetle, 29

concentrations. In this experiment, 200 ml glass jars with tied covers were used as
fumigation chambers for the plant oil. The tested dosages of oil inside the jars were 62.5,
125, 250, 500, and 1000 mg/l.air. Six jars were taken in each treatment. Inside every jar
one filter paper was inserted at the bottom. Then one ml. from each oil concentration of
the different prepared concentrations (10; 5; 2.5,1.25 and 6.25 % w/v) was taken and
added to every glass jar on a filter paper for achieving the mentioned oil conc. inside the
well-closed jars. Thirty adults and larvae were put inside each jar in cotton bags (2×1 cm)
with a few amounts of crushed wheat. The jars well closed and incubated at 28 ±1 ºC and
60±5% R.H. The same steps were followed in the control treatment using only acetone
without oil. Mortality was calculated after 1, 2, 3, 5 and 7 days post-treatment. Percentage
of insect mortality was calculated using Abbott formula Abbott (1925). The fumigation of
sub-lethal-time of insect was done as described above, using the method with
concentrations viz. LT50 , LT90 and LT95 after 3, 5 and 7 days from treatment.
Biochemical Studies:
A known weight of 4thinstar larvae of T. castaneum(0.5g) which was treated with
LC50 of garlic oil and the same weight of untreated ones were kept in the deep freezer
until used for the certain physiological purpose as follows: frozen larvae were
homogenized in 1ml. distilled water by using chilled glass Teflon homogenizer (ST- 2
Mechanic-Preczyina, Poland). Homogenates were centrifuged at 8000 r.p.m. for 15 min
at 5 ºC and the supernatant was used for biochemical parameters assay.
Determine the Effect of Different Treatments on the Activity of some Insect Enzymes:
1-Acetylcholinesterase Determination (AChE):
Acetylcholinesterase (AChE) activity was estimated according to the method
described by Simpson et al.(1964) using acetylcholine bromide (AChBr) as substrate .
The reaction mixture contained 200 ul enzyme solution / 0.5 ml 0.067 M phosphate
buffer (PH7) and 0.5 ml AchBr (3 mM). The test tubes were incubated at 37°C for exactly
30 min. 1 ml of alkaline hydroxylamine (equal volume of 2 M hydroxylamine chloride
and 3.5 M NaOH ) was added to the test tubes , Then 0.5 ml of Hcl (1 part of cone. Hcl
and 2 parts of ∆ H2O) was added. The mixture was shaken vigorously and allowed to
stand for 2 min. 0.5 ml of ferric chloride solution (0.9 M Feels in 0.1M Hcl) was added
and mixed well . The decrease in AChBr resulting from hydrolysis by AChE was read at
515 nm .
2-Determination of Phosphatases Enzymes (AcP&AlkP):
Acid phosphatases (AcP) and alkaline phosphatases (AlkP) were determined
according to the method described by Powell and Smith (1954). In this method, the
phenol released by enzymatic hydrolysis of disodium phenyl phosphate reacts with 4-
amino antipyrine, and by the addition of potassium ferricyanide, the characteristic brown
color is produced .The reaction mixture consisted of 1 ml carbonate buffer (pH10.4) for
alkaline phosphatase or 1 ml citric buffer (pH 4.9) for acid phosphatase, 1 ml of 0.01 M
disodium phenyl phosphate (substrate), mix with 0.1 ml sample and incubate for exactly
30 min at 37° C. At the end of incubation period, 0.8 ml of 0.5 N NaOH was added to
stop the reaction .Then add 1.2 ml of 0.5 N NaHC03, followed by the addition of 1 ml of
4-aminoantipyrine solution (1%) and 1 ml potassium ferricyanide (0.5%). The produced
color was measured immediately at 510 nm. The enzyme activity is expressed by unit (U)
where 1 unit will hydrolyze 1.0 u mole of p-nitophenyl phosphate per minute at 37 C°/
and pH 10.4 and 4,8 for alkaline and acid phosphatases , respectively .
3-Transaminase Enzymes (GOT & GPT):
Glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase
(GPT) enzyme activities were determined colorimetrically according to the method of
Reitman and Frankle (1957). GOT transfer the amino group from L-aspartate to α-ketyo
30 El-Gizawy, K. KH et al.

acid (α-ketoglutaric acid) producing a new amino acid (L-glutamate) and a new keto acid
(oxaloacetic acid). GPT transfer the amino group from D,L alanine to α-keto acid (α-
ketoglutaric acid), resulting in a new amino acid (L-glutamate) and a new keto acid
(pyruvic acid). Oxaloacetate or pyruvate reacts with 2, 4-dinitrophyenylhedrazine
forming oxaloacetate or pyruvate hydrazone which in alkaline medium form a brown
colour which can measure by spectrophotometerically. The reaction mixture consisted of
1ml of a mixture of phosphate buffer (PH 7.4) 0.2 mM α-ketoglutaric and 200 mM D-L
alanine or L-aspartate, 0.2 ml of larval homogenate was then added to the reaction
mixture. The mixture was incubated for 30 min. then after, 10 ml of 0.4 N NaOH was
added. The optical density of the produced brown color is measured after 5 min using a
spectrophotometer at 520 nm. The enzyme activity is expressed as M Pyruvate/gm body
weight/min.
4-Determination of Non-Specific Esterases Activities:
Alpha esterases (α-esterases) and beta esterases(|3-esterases)were determined
according to Van Asperen (1962) using a-naphthyl acetate or β-naphthyl acetate as
substrates ,respectively.Napthol produced as a result of substrate hydrolysis can be
measured by the addition of diazoblue sodium lauryl sulphate solution which produces a
strong blue colour in case of α-naphthol or strong red color in the case of β-naphthol. The
color was measured by spectrophotometrically. The reaction mixture consists of 5 ml
substrate solution (3x10-4 M α-or β-naphthylacetate, l% acetone and 0.1M phosphate
buffer,pH7) and 20 ul of larval homogenate . The mixture was incubated for exactly 15
min at 27°C, then 1 ml of diazoblue color reagent (prepared by mixing 2 parts of l%
diazoblue B and 5 parts of 5% sodium lauryl sulphate) was added. The developed color
was read at 600 or 555 nm for a- and P-naphthol produced from hydrolysis of the
substrate respectively.
5-Carbohydrate Hydrolyzing Enzymes:
The methods used to determine the digestion of trehalose, starch and sucrose by
trehalase, amylase and invertase enzymes respectively, were similar to those described by
Ishaaya and Swiriski (1976). The free aldehydic group of glucose formed after trehalose,
starch and sucrose digestion was determined using 3, 5 dinitrosalicylic acid reagent.The
trehalase reaction mixture consisted of 0.2 ml of 3% trehalose (substrate), 0.2 ml
phosphate buffer (pH 5.4) and 0.2 ml larval homogenate. The invertase reaction mixture
consisted of 0.2 ml of 4% sucrose (substrate), 0.1 ml phosphate buffer (pH 5.4) and 0.2
ml of larval homogenate. The amylase reaction mixture consisted of 0.2 ml of 2% starch
(Substrate), 0.160 ml phosphate buffer (pH 5.4) and 0.2 ml of larval homogenate. The
dinitrosalicylic acid reagent was prepared by dissolving one gram of 3, 5-dinitrosalicylic
acid in 20 ml of NaOH and 50 ml of distilled water with the aid of a magnetic stirrer.
Potassium sodium tartar ate (30 gm.) was added, and magnetic stirring was continued
until a clear solution was obtained. Distilled water then added to bring the final volume to
100 ml. All test tubes were incubated at 37°C for exactly 60 min, 0.8 ml of 3, 5
dinitrosaleylic acid reagent were then added. The reaction mixture was heated 5 min at
100°C in a boiling water bath followed by immediate cooling in an ice bath. The optical
density (OD) of the produced color is measured at 550 nm using a spectrophotometer.
The enzymatic activity was expressed as mg glucose released/gm. body weight/min.
Determination of the Main Metabolites:
The main metabolites (total proteins, total lipids and total carbohydrates) were
determined in the body homogenates.
1-Total Proteins:
Total proteins were determined by the method of Bradford (1976). Protein reagent
was prepared by dissolving 100 mg of Coomassie Brilliant blue G-250 in 50 ml of 95%
 Toxicity of some Essential oils and its Biochemical effect against Red Flour Beetle, 31

ethanol.100 ml of 85% (W/V) phosphoric acid were added to this solution . The resulting
solution was diluted to a final volume of one liter. Sample solution (50 ul) or for
preparation of standard curve 50 ul of serial concentrations containing 10 to 100 ug
bovine serum albumin were pipetted into test tubes. The volume in the test tube was
adjusted to one ml with phosphate buffer (0.1M, PH 6.6), Five millimeters of protein
reagent were added to test tube and the contents were mixed either by inversion or
vortexing. The absorbance at 595 nm was measured after two min and before one hr
against blank prepared from one ml of phosphate buffer and five ml protein reagent.
2-Total Lipids:
Total lipids were estimated by the method of Knight et al. (1972) using
phosphovanillin reagent prepared by dissolving of 0.6 gm pure vanillin in 10 ml ethanol
and completed to 100 ml with distilled water. Then 400 ml of cone. Phosphoric acid was
added.
3-Total Carbohydrates:
The method was based on the digestion of trehalose. starch and sucrose by
trehalase, amylase, and invertase, respectively, according to the method described by
Ishaaya and Swiriski (1976). The free aldhydic group of glucose formed after
trehalose, starch and /or sucrose digestion was determined using 3,5 dinitrosalicylic
acid reagent. The trehalase reaction mixture consisted of 0.2 ml 3% trehalose
(substrate) , 0.18 ml (0.2 M ) acetate buffer (pH 5.4) and 20µl of adults homogenate. The
amylase reaction mixture consisted of 0.2 ml of 2% starch (substrate), 0.16 ml (0.2 M)
phosphate buffer (pH 6), and 20 µl of adults homogenate. The invertase reaction
mixture consisted of 0.2 ml of 4% sucrose (substrate), 0.18 ml (0.2 M) acetate buffer
(PH 5.4) and 20 µl of adults homogenate.

RESULTS AND DISCUSSION

1-Fumigant Toxicity of some Essential Oils against the Adults and 4th instar Larvae
of T. castaneum :
The results of the effect of fumigation toxicity of plant oils (Garlic oil (Allium
sativum), Basil oil (Ocimum basilicum), Eucalyptus oil(Eucalyptus globulus), and Pine
oil (Pinus pinaster) on 4th instar larvae and adult of T. Castaneum. The results showed
that mortality was increased by increasing the plant oil concentration and period of
exposure. The lethal concentration of Garlic and Basil essential oils to adult and larval
stage of T. castaneum are shown in Table (2).The results showed that after 3 days post-
treatment for the adult stage the LC50 values were 126 and 1667% mg/l. air. The
corresponding values at 7 days were significantly lower and amounted 47and 209% mg/l.
air. For Garlic and Basil essential oils, respectively. While 4thinstar larvae the LC50
value was 7, and 881 % mg/l. air. The corresponding values at 7 days were significantly
lower and amounted 41and 205% mg/l. air. For Garlic and Basil essential oils,
respectively. The LC90 value of adult stage was 4436and 75088 % mg/l. air at 3 days and
declined to 198and 1590% mg/l. air at 7 days post-treatment Garlic and Basil essential
oils, respectively. while The LC90 value of larvae stage were 839and 41849% mg/l. air
at 3 days and declined to 162and 7010% mg/l. air at 7 days post-treatment Garlic and
Basil essential oils, respectively. The LC95 value were 12163and 221062 % mg/l. air at 3
day and reduced to 297and 2827% mg/l. air at 7 days from treatment for Garlic and Basil
essential oils, respectively. while The LC95 value of larvae was 1639and 128027 % mg/l.
air at 3 days and reduced to 238, 276 and 19081% mg/l. air at 7 days from treatment for
Garlic and Basil essential oils, respectively. The lethal concentrations of Eucalyptus and
Pine essential oils to the adult and 4th larvae stage of T. castaneum are shown in Table
32 El-Gizawy, K. KH et al.

(3). The results showed that the lethal concentrations are exposure period dependent. The
higher the exposure period was the lower the LC values. At 3 days post-treatment for
adult stage the LC50 value was 861, and 857 % mg/l. air. The corresponding values at 7
days were significantly lower and amounted 55 and 86 % mg/l. air. For Eucalyptus and
Pine essential oils, respectively. while4th larvae the LC50 value was 238 and % mg/l. air.
The corresponding values at 7 days were significantly lower and amounted 70 and 51 %
mg/l. air. For Eucalyptus and Pine essential oils. respectively. The LC90 value of the adult
stage was 37081 and 37815 % mg/l. air at 3 days and declined to 1030 and 9507% mg/l.
air at 7 days post treatment Eucalyptus and Pine essential oils, respectively. while The
LC90 values of 4th larvae stage were 2617 and 105547 % mg/l. air at 3 days and declined
to 415 and 1377% mg/l. air at 7 days post treatment Eucalyptus and Pine essential oils,
respectively. The LC95 values were 107787, 110681 and 548427 % mg/l. air at 3 days and
reduced to 2365 and 36104 % mg/l. air at 7 days from treatment for Eucalyptus and
Pine essential oils. respectively. While, the LC95 values of 4th larvae were 5163 and
486788 % mg/l. air at 3 days and reduced to 687 and 3504 % mg/l. air at 7 days from
treatment for Eucalyptus and Pine essential oils. Respectively

Table (2): Lethal concentrations of some essential oils in the fumigant toxicity against
adult and 4th larval stage of T. castaneum and various exposure periods.
Exposure Lethal concentrations and their confidence limits
period Stage Slope ±SD R
LC50 LC90 LC95
(days)
Garlic oil(Allium sativum)
126 4436 12163
adult 0.82±0.009 0.992
(69-232) (855-23006) (1400-105663)
3 days
79 839 1639
larvae 1.25±0.06 0.978
(47.34-132) (427-1649) (661-4062)
53 627 1265
adult 1.19±0.008 0.996
(26-104) (326-1206) (508-3147)
5 days
62 227 327
larvae 2.28±0.30 0.969
(43.72-88) (167-307) (222-483)
47 198 297
adult 2.06±0.34 0.956
(28-78) (145-271) (199-444)
7 days
41 162 238
larvae 2.17±0.03 0.996
(24-70) (116-225) (154-367)
Basil oil(Ocimumbasilicum)
1667 75088 221062
adult 0.77±0.03 0.971
(501-5538) (2542-2217906) (3947-132380)
3 days 128027
881 41849
larvae (3165- 0.74±0.01 0.985
(340-1934) (1989-880209)
5177340)
416 4143 7947
adult 1.28±0.05 0.981
(286-605) (1474-11642) (2262-27913)
5 days 80224
450 25532
larvae (2524- 0.73±0.02 0.976
(231-876) (1567-415867)
2549327)
209 1590 2827
adult 1.45±0.04 0.988
(153-284) (807-3132) (1211-6597)
7 days
205 7010 19081
larvae 0.83±0.01 0.989
(123-342) (1132-43417) (1856-196166)
R= Correlation Coefficient of regression line SD= Standard deviation of the mortality
regression line.
 Toxicity of some Essential oils and its Biochemical effect against Red Flour Beetle, 33

2-Toxicity Index of Various Essential Oils against Adults of T. castaneum .

The toxicity effect of various essential oils against adults of T. castaneumat the LC50
after 7 days post-treatment at 28±1° C could be arranged in descending order as follows:
Garlic oil, Eucalyptus oil, Chili pepper oil, Pine oil, Basil oil and Nigella oil which was
the least effective. Table (4) The toxicity effect of various essential oils against larvae of
T. castaneumat the LC50 after 7 days post-treatment at 30±1° C could be arranged in
descending order as follows: Garlic oil, Pine oil, Eucalyptus oil and Basil oil which was
the least effective. Table (5).The results indicated that Garlic oils were much more
effective as fumigants than other used oils against T. castaneum adults. Meanwhile, this
results demonstrated the probability of using these essential plant oils as grain
protestants’ as well as or as Fumigants to control T. castaneum stages. According
to the results obtained from the present study insects mortality increased with
increasing concentration levels and exposure time.
Table (3):Lethal concentrations of some essential oils in the fumigant toxicity against
adult and 4th larval stage of Tribolium castaneum and various exposure
periods.
Exposure Lethal concentrations and their confidence limits
Slope ±SD R
period (days) Stage LC50 LC90 LC95
Eucalyptus oil (Eucalyptus globulus)
861 37081 107787
adult 0.78±0.08 0.934
(364-2035) (2151-639158) (3446-639158)
3 days
238 2617 5163
larvae 1.23±0.008 0.996
(168-338) (1016-6738) (1580-16868)
177 2886 6370
adult 1.05±0.0009 0.999
(115-270) (929-8970) (1490-27229)
5 days
113 1367 2770
larvae 1.18±0.03 0.987
(72-178) (598-3123) (935-8199)
55 1030 2365
adult 1.00±0.03 0.982
(25-120) (414-2563) (658-8492)
7 days
70 415 687
larvae 1.66±0.04 0.991
(46-107) (266-648) (381-1238)
Pine oil(Pinuspinaster)
857 37815 110681
adult 0.77±0.01 0.982
(364-2015) (2137-669152) (3414-3587856)
3 days
481 105547 486788
larvae 0.54± 0.015 0.972
(194-1192) (905-106223) (1283-1845)
332 26869 93372
adult 0.67±0.02 0.971
(174-633) (1285-561728) (2042-4268589)
5 days
149 7516 22831
larvae 0.75±0.002 0.997
(80-278) (953-59237) (1552-335752)
86 9507 36104
adult 0.62±0.001 0.998
(32-227) (673-134217) (1037-1256877)
7 days
51 1377 3504
larvae 0.89±0.011 0.992
(20-126) (452-4193) (727-16873)
R= Correlation Coefficient of regression line SD= Standard deviation of the mortality
regression line.

This kind of is in agreement with Sahaf and Moharramipour (2008). However,

generally, the effectiveness of gas decreased over time that can be related to their high
volatility and low stability (Mikhaiel, 2011; Ogendo et al., 2008; Rozman et al., 2007;
Sahaf et al., 2007). Isman (2006) Observation of T. granarius exposed to C. copticum
essential oil indicated their knockdown, hyperactivity, convulsion, paralysis; and finally
insect death and mentioned that fast effect of essential oils is because of their neurotoxic
mode of action. Insect's susceptibility to the same essential oil differs from species to
species. In line with the Sahaf and Moharramipour (2008), LC50 values of C. copticum
34 El-Gizawy, K. KH et al.

essential oil against adults of C. maculates after 24 h exposure time was 0. 90? L-1 air. In
the other study Sahaf et 's., (2007) explained that zero. 91? L-1 air of C. copticum oil was
required to obtain 50% mortality of S. oryzae, and 33. 14? L-1 air in the case of T.
castaneum. Shojaaddini et al., (2008) also estimated 257. 83 and 91. 36? L-1 air of C.
copticum oil to control 50% of G. interpunctella adults and larvae, respectively. Based on
LC50 values, S. granariusseems to be more tolerant of C. copticum oil than other
Coleopteran species. Most of the plants and herbal products are locally available and is
applied for pest control programs in small scales. However , there is a need to conduct
further studies to examine their insecticide efficacy against stored-product insect pests.

Table (4):Toxicity index of various essential oils for the adults of T. castaneum after 7
days post-treatment.
LC50 after 7 days Toxicity index at
Essential oils Slope ± SD R.
(mg/l.air) LC50
Garlic oil 47.49 100 2.06±0.34 0.956
Eucalyptus oil 55.04 86.28 1.00±0.03 0.982
Pine oil 86.03 55.20 0.62±0.001 0.998
Basil oil 209.23 22.69 1.45±0.04 0.988

R= Correlation Coefficient of regression line SD= Standard deviation of the mortality

regression line

Table (5):Toxicity index of various essential oils for 4thinstar larvae of T. castaneum
after 7 days post treatment

LC50 after 7 days Toxicity index at

Essential oils Slope ± SD R.
(mg/l.air) LC50
Garlic oil 41.67 100 2.17±0.03 0.996
Pine oil 51.12 81.51 0.89±0.011 0.992
Eucalyptus oil 70.35 59.23 1.66±0.04 0.991
Basil oil 205.29 20.29 0.83±0.01 0.989

R= Correlation Coefficient of regression line SD= Standard deviation of the mortality

regression line

3-Effect of Garlic Essential Oil on the Total Proteins, Lipids and

Carbohydrates of 4th larval of T. castaneum:
Data on the total proteins, carbohydrates and lipids are shown in Table (6) revealed
that, no significant increase was found under treatment garlic oil which recorded 26.66
mg/g.b.w., in compared to the control, the rate of increase was 21.49%. On the other
hand, the obtained results in the same table indicated that the rate of total protein was of
decrease was -63.14% meanwhile insignificant reduced under garlic oil which recorded
211.33 mg/g.b.w. in compared to the control. Total lipids content were insignificantly
reduced to garlic oil treatments which recorded 16.76 mg/g.b.w., compared to control
treatment which recorded 16.96 mg/g.b.w. these of decrease was -1.19% for garlic oil.
4-Effect of Different Treatments on the Activity of the Enzymes:
The treatments of controlling T. castaneum have significantly affected the activity
of different enzymes.Table (6) indicated that, the decrease of Alkaline phosphatase was -
8.79% while on the contrary no significant increase of enzyme activity under garlic oils
 Toxicity of some Essential oils and its Biochemical effect against Red Flour Beetle, 35

treatment was recorded (491.66) in compared to the control treatment, this increase was
1.02%. On the other hand, the obtained results of Acid phosphatase indicated that the
decrease of enzyme was -70.94%, while no significant increase of the enzyme activity
under garlic oils treatment was recorded (116.76 Ux103/g.b.wt) in compared to the
control.

Table (6): Activity of certain enzymes in T. castaneum larvae exposed to different

treatments.
percent of
percent of increase or
Activity increase or Activity
decrease
(Mean ± S.E.) decrease (Mean ± S.E.)
Treatment (%)
(%)
Alkaline phosphatase Acid phosphatase
Ux103/g.b.wt Ux103/g.b.wt

Garlic oil 491.66±33.42 a 1.02 116.0±10.76 a 0.29

Control 486.66±33.42 a - 115.66±10.76 a -

α-esterase β-esterase
mg/g.b.w. mg/g.b.w.

Garlic oil 3404.33±351.66 b 5.33 1230.0±39.79 a 14.22

Control 3222.66±351.66 b - 1055.0±39.79 b -

(GOT) (GPT)
uM/g.b.wt. uM/g.b.wt.

Garlic oil 4722.7±414.13 b -53.72 808.67 ±40.19 ab 15.16

Control 7260.0 ±414.13 a - 686.00 ±40.19 a

Acetylcholinesterase Trehalase
µg/ Br/g/min. µgGlu./g/min

Garlic oil 290.33 ±8.01 a 26.52 1157.00±92.22 a 4.84

Control 213.33 ±8.01 b - 1101.00±92.22 a -

Invertase Amylase
µgGlu./g/min µgGlu./g/min

Garlic oil 1786.66±130.91 b 19.51 140.66±19.50 b 9.00

Control 1438.00±130.91 b - 128.00±19.50 b -

Total carbohydrates Total proteins Total lipids

-
Garlic oil 26.66±1.98b 21.49 211.33±8.71a -11.67 16.76±0.84a
1.19

Control 20.93±1.98b - 236.00±8.71a - 16.96±0.84a -

This increase was 0.29%.A Significant increase in activity of Alpha esterases and
Beta esterases with garlic oils treatments was recorded 3404.33 mg/g.b.w., compared to
control treatment which recorded 3222.66 mg/g.b.w., the increase was 5.33 for the
activity of Alpha esterases for garlic oil,. while an increase to Beta esterases for garlic
oils treatments was 14.22%.A high significant decrease in the activity of Glutamic
Oxaloacetic Transaminase (GOT) and Glutamic Pyruvic Transaminase (GPT)for garlic
36 El-Gizawy, K. KH et al.

oil treatments was recoded 4722.7uM/g.b.wt. in compared to control treatment which was
7260.0 uM/g.b.wt. this decrease was -53.72% for garlic oil. The activity of
Acetylcholinesterase enzyme showed significant greatest under garlic oils treatments
was290.33 µg/ Br/g/min.,the increase of this enzyme was 26.52%.Trehalase activity
showed no significant increase in garlic oil treatments which were 1157.00 µg
Glu./g/min. in compared to control treatment 1101.00 µgGlu./g/min. the increase was
4.84 % for garlic oil. No significant increase in the activity of Invertase enzyme with
garlic oil treatments which was 1786.66 µg Glu./g/min compared to control treatment.
The rate of increase was 19.51%.Amylase activity showed no significant increase with
garlic oil treatments was recorded (140.66 µgGlu./g/min.) compared to control treatment,
the increase was 9.00%. These results were in harmony with (Nathan, et al., 2005)
indicated that the Cytochrome P450 monooxygenases (CYPs), glutathione-S-transferases
(GSTs) and esterases (ESTs) are three major detoxifying enzymes in most organisms.
Abd El-Raheman (2013) Found that the activity of trehalase, acid phosphates, acetyl
cholinesterase, phenoloxidase and lactate dehydrogenase enzymes increased in treated
Sitotroga cerealella larvae with modified atmospheres while the activity of amylase,
alkaline phosphatase enzymes decreased. Abotaleb (2013) showed that total proteins,
lipids and carbohydrates contents were significantly decreased in Sitophilus oryzae adults
feeding in wheat grain treated with different oils compared to control. Furthermore,
different levels of significant changes in the carbohydrase's, protease, phosphatases and
acetylcholine esterase activity, The decrease in the protein content in treated larvae might
be due to inhibition of DNA and RNA synthesis as suggested by Mitlin and Haynes
(1977).
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ARABIC SUMMARY

‫سمية بعض الزيوت العطرية وتاثيرها البيوكيميائى ضد حشرة خنفساء الدقيق الصدئية‬
**‫ سامية عبد الواحد محمد‬،**‫ اشرف لحظى مهني‬،*‫ صفاء محمود حالوة‬،*‫كرم خميس الجيزاوى‬
‫*قسم وقاية النبات – كلية الزراعة بمشتهر – جامعة بنها – مصر‬
‫ هيئة الطاقة الذرية – ابو زعبل – مصر‬- ‫**قسم بحوث النبات – مركز البحوث النووية‬

.‫ حبة البركة‬،‫ الكافور‬،‫ الصنوبر‬،‫ الريحان‬،‫ الفلفل االحمر‬،‫تم اختبار فاعلية ست زيوت نباتية هما زيت الثوم‬
‫ درجة مئوية ضد العمر اليرقى الرابع‬28±1° ‫ ايام عند درجة حرارة‬7 ،5 ،3 ،2 ،1 ‫وذلك بتركيزات مختلفة بعد‬
‫ واظهرت النتائج ان نسبة الموت تزداد بزيادة تركيز‬.‫وطور الحشرة الكاملة لخنفساء الدقيق الصدئية وذلك بالمعمل‬
‫الزيت وطول مدة التعريض وكان اكثر الزيوت النباتية سمية هو زيت الثوم للحشرة تحت الدراسة وذلك بعد اسبوع‬
‫ من خالل تلك‬.‫من المعاملة ايضا تم اختبار تاثير زيت الثوم على فاعلية نشاط بعض االنزيمات للعمر اليرقى الرابع‬
‫الدراسة نوصى باستخدام الزيوت النباتية داخل برامج المكافحة المتكاملة الفات الحبوب المخزونة نظرا لسمية‬
.‫ابخرتها العالية‬