183

International Journal of Human and Health Sciences Vol. 07 No. 02 April’23 

Original article:

The Pesticidal Influence of Clove Extract Against the Rusty Flour Beetle, Tribolium castaneum
Gadah Al-Zarie1, Noorah Saleh Al-Sowayan1

Abstract
Background: The use of conventional insecticides to repel or kill insects leads to 
environmental pollution and harms human and animal health. Researchers are thus actively 
attempting to find natural ways of getting rid of insect pests in more environmentally 
sustainable ways. Objective: Behavioral response of the rusty flour beetle, Tribolium 
castaneum, to aqueous extract from clove was investigated here. Methods: In our study, 
the effect of the aqueous extract of cloves was tested for its potential to repel rusty flour 
beetle as the chemicals present in clove extract can trigger an olfactory response in this 
common pest. Results: Our findings indicate that the aqueous extract of cloves is a an 
effective repellant against the rusty flour beetle. Conclusion: This is a natural way to get 
rid of insect pests in more environmentally sustainable ways, as the use of traditional 
insecticides to repel or kill insects pollutes the environment and harms human and animal 
health.
Keywords: Rusty flour beetle, Tribolium castaneum, Clove plant, Syzygium aromaticum, 
Insect repellant

Correspondence to:  Noorah Saleh Al-Sowayan, Department of Biology, Faculty of 
Science, Qassim University, P.O. Box 30230, Buraydah (51477), Saudi Arabia. 
Email: nsaoiean@qu.edu.sa

1. Department of Biology, College of Science, Qassim University, Buraydah-51477, Saudi Arabia.

Introduction

The world depends upon safe and healthy 
agricultural crops for food; to protect fields and 
post-harvest crops, synthetic chemicals have 
become pesticides of choice. However, they pose 
immense risks to human and animal health, as 
well as cause significant environmental pollution. 
Therefore, there is an urgent need to search for 
alternative methods of controlling agricultural 
pests.1-3 Natural insect and/or pest repellants have 
low environmental impact and are safe for biota. 
These are generally natural extracts and secondary 
compounds derived from plants to fight against 
pest attacks.  

A number of studies have reported testing plant 
extracts as pesticides. Taluker et al.4 evaluated the 
potential of extracts from seeds of Aphanamixis 
polystachya to repel the rusty flour beetle, 
Tribolium castaneum. The authors demonstrated 
that the extract was highly repellent, although 
mildly nutritive, and was toxic to rusty flour 

beetles. Similarly, Islam et al.5 studied the 
biological activity of the essential oil extracted 
from Coriandrum sativum L. against the eggs, 
larvae, and adult stages of the rusty flour beetle. 
Biological tests have shown that essential oils 
perform fumigation activity against eggs, and 
their toxicity increases gradually as concentrations 
are strengthened and repeated doses are applied. 
Coriander oil is reported to have a strong 
repellant activity against adult stages of the rusty 
flour beetle. Iqbal et al.6 confirmed that ethanol 
extracted from sugar cane, Acorus calamus, and 
turmeric, Curcuma longa, can effectively repel the 
rusty flour beetle. 

Elham et al.7 experimented with the toxicity of 
acetylcholine seed extract against several types of 
insect pests to demonstrate that the extract had a 
repellant effect on the rusty flour beetle. Jema et 
al.8 used essential vegetable oils from bay leaf 
plant, Laurus nobilis, to evaluate their repellant 
and toxicity effect against two main pests of stored 

International Journal of Human and Health Sciences Vol. 07 No. 02 April’23
DOI: http://dx.doi.org/10.31344/ijhhs.v7i2.572

mailto:nsaoiean@qu.edu.sa


International Journal of Human and Health Sciences Vol. 07 No. 02 April’23 

184

products: the smaller grain bore, Rhyzopertha 
dominica, and  the rusty flour beetle. They 
observed that volatile oils in the bay leaf plant not 
only repelled but were toxic to adult smaller grain 
bores and rusty flour beetles as well. Garcia et al.9 
reported essential oil from Baccharis salicifolia 
to be toxic to adult rusty flour beetle. Other than 
essential oils and extracts, direct use of powdered 
plant parts has been demonstrated to be effective 
against insecticides by Tripathi et al.,10 who used a 
100% clove powder at a dose of 1.5 g/50 g against 
rusty flour beetles. At a dose of 5g/100 g, powdered 
large cardamom, cinnamon, and clove completely 
inhibited the reproduction of Callosobruchus 
maculatus and the rusty flour beetle.

Methods

Insect collection and rearing: The rusty flour beetle 
was obtained from several sources (flour shop and 
a baker’s warehouse) in Al-Qassim region of the 
Kingdom of Saudi Arabia. The rusty flour beetle 
was reared under laboratory conditions of 30°C 
temperature, 60% relative humidity, and 24 hours 
of complete darkness, following Al-Shuraym.11 
The experimental setups were prepared in 50  cm3 
glass jars that were filled with 1 kg of flour and 
500 gm of yeast. Intact insects of the rusty flour 
beetle were isolated from an infested flour; 50 
pairs were introduced into glass jars. The jars were 
covered with a mucilin cloth to prevent the escape 
of insects and to ensure adequate ventilation; the 
jar was fixed with a rubber strap.

Plant species and chemical composition: 
Flower buds of the carnation plant, clove, that 
is scientifically known as Syzygium aromaticum, 
were used in this study. Table 1 shows the major 
chemical constituents released in the clove bud 
that is generally used for a variety of purposes.

Table 1: Major chemical constituents and their 
average quantities in clove flower buds

Compound Percentage
Furan, tetrahydro-3-methyl 2.5
2-propanone, methylhydrazone 5.6
Cyclopentane, methyl 4.0
Pyrrolidine, 2-butyl-1-methyl 0.1
2H-Pyran-2-one, tetrahydro-6,6-dimethyl 0.4
Eugenol 49.0
Copaene 0.5
Caryophyllene 7.5
Alpha-caryophyllene 1.4

Preparation of Clove Extract – Cold method: 

Fifty grams of cloves were added to 500 mL of 
distilled water and stirred on a magnetic stirrer at 
room temperature for half an hour. The solution 
was then filtered with a cloth to remove plant 
parts; the plants were then put in the centrifuge 
device at 3000 rpm for a period of 15 minutes to 
obtain a clear solution. The solution was poured 
into a glass Petri dish and placed in a drying oven 
at 35°C. Once all water had evaporated, the residue 
was scraped off the bottom of the Petri dish.12 We 
obtained 0.2 grams of the residue that we scraped 
off after drying and placed it inside a graduated 
cylinder; the volume was then made up to a 100 
mL total. 

Experimental Design to Observe the Olfactory 
Response of Rusty Flour Beetle: The design of 
the experimental treatments was adapted from 
the study of Stamopoulos et al.13 A custom-built 
small-scale device was prepared by using three 
plastic petri dishes for three treatments as shown 
in Figure 1. In our study, Dish 2 was prepared with 
two holes on the edge and was placed in the center 
of two other dishes, Dish 3 and 1. Each hole had a 
diameter of 6 mm; Dishes 1 and 3 were connected 
via plastic tubes of 75 mm length. In Dish 3, 
we had a treatment containing a semi-industrial 
environment made up of 5 grams of food (flour 
and yeast); this Petri dish had a fixed cover. On its 
inside, a piece of sponge measuring 1×1 cm was 
placed with the test substance (residue prepared 
before). This dish had one hole to connect it to 
one of the holes in Dish 2. Dish 1 was the control 
experiment that contained the same components 
as Dish 3, except that the test substance was not 
placed on the sponge. 

The treatments: The aqueous extract of cloves 
(0.5μL) was placed in a graduated measuring flask, 
and a drop of acetone was added to it; the volume 
was made up to 1 mL with distilled water. This 
gave a final concentration of 0.05%. Following 
rigorous shaking, 1μL of the solution was placed 
on the sponge piece installed in the Petri Dish 3. 
In the control dish (Dish 1), no test material was 
placed. The number of intact rusty flour beetles 
was calculated in each dish after two hours had 
passed from the time that the set up was completed 
and insects were introduced in Dish 2. The rate 
of attraction and expulsion of the insects was 
calculated according to the following equation as 
described previously by Gunn & Cosway.14

Intensity of reaction =100    



185

International Journal of Human and Health Sciences Vol. 07 No. 02 April’23 

Figure 1. A custom-built device for the study comprising three petri dishes

Where, S is the number of insects attracted to a 
substance, A is the number of insects attracted to 
the control. The classification of attractiveness or 
repulsion of a material is done using the following 
criteria: <1% = non attractive repellent; 1-10%  
= very weakly attractive; 11-20%  = weakly 
attractive; 21-40% = attractive;  and ≤41% =  very 
attractive. 

Results

The results presented in Table 2 show that the 
aqueous extract of cloves led to an expulsion of 
the rusty flour beetle from the treatment in Dish 
3; while some attraction of beetles was recorded 
in the control, i.e., Dish 1. Table 2 and Figure 2 
depict the average ejection ratio according to the 
method of Gunn & Cosway.14 Table 3 presents a 
summary of the response of the rusty flour beetle 
to the aqueous extract of cloves.

Table 2: Expulsion of the rusty flour beetle from 
the treatment in Dish 3 (aqueous extract of cloves)

Intensity of 
reaction (%)

S+CS-CC.S.
Replicate 
number

-100%2-220Replicate 1

0%2011Replicate 2

0%6033Replicate 3

0%6033Replicate 4

25%82
35

Replicate 5

-100%2-220
Replicate 6

S: The number of insects attracted to the scent 
of the test substance; C: The number of insects 
attracted to the control.

Table 3: Response of the rusty flour beetle to the 
aqueous extract of cloves

  - 
Replicate 
number

5016.89-70.83-100Replicate 1

850.8929.170Replicate 2

850.8929.170Replicate 3

850.8929.170Replicate 4

2934.3954.1725Replicate 5

5016.89-70.83-100Replicate 6

15520.84

x Result of duplicate; x ̅  The result of the 
arithmetic mean; N: Replicate number

Figure 2. Intensity of reaction in percent with 
respect to the expulsion of insects by the aqueous 
extract of cloves

Discussion

The efficacy of allomones from the aqueous extract 
of cloves in repelling rusty flour beetle is clear 
from the results presented in Table 3. We tested 
the release of allomones with an olfactometer, 



International Journal of Human and Health Sciences Vol. 07 No. 02 April’23 

186

which revealed repellent compounds to make up 
29.17% of the total constituents in the aqueous 
extract of cloves.  Our results are in agreement 
with previous research by Natalia et al.15 that 
reported volatile oils to have a repellent effect on 
larvae and adult insects of the rusty flour beetle. 
On the contrary, Saim & Meelan16 stated that 
the α-pinene complex present in laurel leaves 
has attractive properties. Numerous scientific 
studies have proven that insects have receptors 
that control their olfactory response, e.g., Jonsson 
& Anderson17 demonstrated that the antennae of 
the cotton leaf worm are equipped with sensory 
filaments through which they can detect plant 
odors with high sensitivity. These odors are 
generally caused by volatile chemical compounds. 
Hansson et al.18 studied olfactory receptor nerves 
in male scarab beetles, Phyllopertha diversa, 
that were capable of detecting and responding to 
volatile compounds emitted by green leaves, as 
well as the pheromones. The researchers indicated 
that the olfactory receptor nerves had the ability to 
distinguish between these volatile substances and 
molecules with a high degree of specificity and 
sensitivity. All these chemical compounds play 
an important role in the insect’s behavior toward 
its plant hosts, which ensures its survival and 
reproduction in a given environment. This also 
explains the behavioral response of rusty flour 

beetles to many of the compounds that have been 
tested till now in our study, as well as others, such 
as essential oils, extracts, or terpenes. The different 
sensory properties are incurred by different organs 
distributed all over the body of the rusty flour 
beetle.

Conclusion

Pesticides extracted from plants have been 
demonstrated to repel insect pests; this is a safe and 
environmentally friendly method of preventing 
pest attack on agricultural crops at small and large 
scales. Based on our findings, we propose that 
aqueous extract of clove buds can effectively be 
used to protect storage warehouses and to keep 
insects away from stored food and prevent them 
from laying eggs.

Conflict of interest: The authors declare no 
conflict of interest.

Funding statement: No funding.

Ethical approval: The experiment was approved 
by the Departmental Ethical Committee for Care 
and Use of Animals.

Author’s contribution: Both the authors 
contributed to the concept, design, literature 
search and drafting of the manuscript. They also 
revised and approved the final manuscript.



187

International Journal of Human and Health Sciences Vol. 07 No. 02 April’23 

References
1. Tabashnik BE. Evolution of resistance to Bacillus 

thuringiensis. Ann Rev Entomol. 1994;39:47-79.
2. Isman MB. Plant essential oils for pest and disease 

management. Crop Protection. 2000;19:603-8.
3. Yang P, Yajun MA, Shuiqing Z. Adulticidal 

activity of five essential oils against Culex pipiens 
quinquefasciatus. J Pesticide Sci. 2005;30:84-9.

4. Taluker FA, Howse PE. Evaluation of Aphanamixis 
polystachya as asource of repellents, antifeedants 
toxicants and protectants in storage against Tribolium 
castaneum (Herbst). J Stored Products Res. 
1995;31(1):55-6.

5. Islam MS. Fumigant and repellent activities 
of essential oil from Coriandrum sativum (L.) 
(Apiaceae) against red flour beetle Tribolium 
castaneum (Herbst) (Coleoptera: Tenebrionidae). J 
Pest Sci. 2009;82(2):171-7.

6. Iqbal J, Qayyum A, Mastafa SZ. Repellent effect 
of ethanol extracts of plant materials on Tribolium 
castaneum (Herbst) (Tenebrionidae:  Coleoptera). 
Pak J Zoology. 2010;42(1):81-6.

7. Elham S, Kamal A, Zamani DR, Amin P, Mohammad 
TH. Toxic and repellent effect of Harmal (Peganum 
harmal L.) acetonic extract on several aphids and 
Tribolium castaneum (Herbst). Chilean J Agric Res. 
2012;72(1):147-51.

8. Jema JMB, Tersim N, Toudert KT, Khouja ML. 
Insecticidal activities of essential from leaves of 
Laurus nobilis L. from Tunisia, Algeria and Morocco, 
and comparative chemical composition. J Stored 
Products Res. 2012;48:97-104.

9. Garcia M, Donadel OJ, Ardanaz CE, Tonn CE, 
Sosa ME. Toxic and repellent effects of Baccharis 
salicifolia essential oil on Tribolium castaneum. Pest 
Manag Sci. 2005;61(6):612-8.

10. Tripathi AK, Singh AK, Upadhay S. Contact and 
fumigant toxicity some common spices against the 
storage insects callosobruchus maculates (Coleoptera: 

Bruchidae) and Tribolium castoreum (Coleoptera: 
Tenebrionidae). Int J Trop Insect Sci. 2009;23(3):151-
7.

11. Al-Shuraym LAM. Evaluate the toxic effect of 
Cyfluthrin on the biochemical mechanism and 
histological strtures of some body systems in the red 
flour beetle, Tribolium castaneum (H.) (Coleoptera: 
Tenebrionidae). [M.S. Thesis]. Girl’s College of 
Education, Riyadh, Saudi Arabia. 2004.

12. Harborne JB. Phytochemical Methods: A Guide to 
Modern Techniques of Plant Analysis. 2nd ed. New 
York, USA: Chapman & Hall; 1984.

13. Stamopoulos DC. Effects of four essential oil vapours 
on the oviposition and fecundity of Acanthoscelides 
obtectus (Say) (Coleopteran: Bruchidae): Laboratory 
evaluation. J Stored Products Res. 1991;27(4):199-
203.

14. Gunn DL, Cosway CA. The temperature and 
humidity reactions of the Cockroach. J of Exp Biol. 
1938;16:555-63.

15. Natalia S, Teodoro S, Adriana F. Composition and 
toxic repellent and feeding deterrent activity of 
essential oils against the stored-grain pests Trifolium 
castaneum (Coleoptera: Tenebrionidae) and 
Sitophilus oryzae (Coleoptera: Curculionidae). Pest 
Manag Sci. 2011;67(6):639-46.

16. Saim N, Melaan CE. Compounds from leaves of bay 
(Laurus nobilis) as repellents for Tribolium castaneum 
(Herbst) when added to wheat flour. J Stored Products 
Res. 1986;22(3):141-4.

17. Jonsson M, Anderson P. Electrophysiological 
response to herbivore‐induced host plant volatiles 
in the moth Spodoptera littoralis. Physiol Entomol. 
1999;24(4):377-85.

18. Honsson BS, Lorsson MC, Leal WS. Olfactory 
receptor neurons detecting plant odours and male 
volatiles in Anomala cuprea beetles (Coleoptera: 
Scarabaeidae). J Insect Physiol. 2001;47(9):1065-76.