Toksisitas dan Efek Fisiologi Tiga Minyak Atsiri terhadap Tribolium castaneum dan Callosobruchus maculatus

TOXICITY AND PHYSIOLOGICAL EFFECTS OF THREE
ESSENTIAL OILS AGAINST Tribolium castaneum and
Callosobruchus maculatus

SRI ITA TARIGAN

GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2016

STATEMENT OF THESIS AND SOURCES OF
INFORMATION AND COPYRIGHT
With this statement, I declare that the thesis entitled “Toxicity and
Physiological Effects of Three Essential Oils against Tribolium castaneum and
Callosobruchus maculatus” is the result of my work with guidance and advice
from the supervisory committee and have not been submitted to any other
universities, in any form. Sources of information that were quoted in this thesis
have been written in the reference section. I here by sign the copyright of my
papers to Bogor Agricultural University.

Bogor, December 2016

Sri Ita Tarigan

SUMMARY
SRI ITA TARIGAN. Toxicity and Physiological Effects of Three Essential Oils
against Tribolium castaneum and Callosobruchus maculatus. Under supervision
of DADANG and IDHAM SAKTI HARAHAP.
During storage of post-harvest products in warehouse, usually there is
emence damage of stored products due to insect infestations. However, much
effort has been made to control and to manage such insect pests using synthetic
pesticides. Recently studies have shown that majority of fumigants and synthetic
insecticides have resulted in development of resistance in most stored product
insect pests. Postharvest and manufacture products such as wheat, beans, maize,
and flour are usually infested by Tribolium castaneum (Coleoptera:
Tenebrionidae) and Callosobruchus maculatus (Coleoptera: Bruchidae) resulting
in mass damage and wast. Therefore to address those problems there is need to
explore alternative fumigant to control and to manage the stored product insect
pests.
The aims of this research were to determine the effectiveness of cardamom

(Ellateria cardamomum: Zingiberaceae), cinnamon (Cinnamomum aromaticum:
Lauraceae) and nutmeg (Myristica fragrans: Myrtaceae) essential oils against T.
castaneum and C. maculatus and to study the physiological effects of essential
oils against T. castaneum and C. maculatus. The experimental results showed that
cinnamon oil had higher efficacy against the egg, larva and adult of C. maculatus
with LC50 values were 0.019%, 0.132%, 0.186%, respectively whereas LC50 of
egg, larva, and adult of T. castaneum were 1.051%, 0.109%, 1.239%,
respectively. Cinnamon oil was more effective to both insect spesies compared
with cardamom and nutmeg oils. Three essential oils had affected the
physiological process by triggering reduction in the total amount of carbohydrate,
protein, fat contents, esterase and glutathione s-transferase activity during the
third instar larvae of both T. castaneum and C. maculatus. Cinnamon was the
most effective essential oil to control and to manage both treated insects. Its
application was environmental friendly and economically affordable for local
user.
Keywords: cardamon, cinnamon, mortality, nutmeg, toxicity

RINGKASAN
SRI ITA TARIGAN. Toksisitas dan Efek Fisiologi Tiga Minyak Atsiri terhadap
Tribolium castaneum dan Callosobruchus maculatus. Dibimbing oleh DADANG

dan IDHAM SAKTI HARAHAP.
Selama penyimpanan produk pasca panen di pergudangan, seringkali
ditemukan kerusakan pada produk simpanan yang disebabkan oleh infestasi
serangga. Meskipun demikian, berbagai usaha telah dilakukan untuk
mengendalikan dan mengelola serangga hama seperti penggunaan pestisida
sintetik. Studi sebelumnya telah menunjukkan bahwa beberapa insektisida dan
fumigan mengakibatkan terjadinya resistensi pada beberapa serangga gudang.
Produk pasca panen dan olahan seperti gandum, kacang, jagung, dan tepung
seringkali diinfestasi oleh Tribolium castaneum (Tribolium: Tenebrionidae) dan
Callosobruchus maculatus (Coleoptera: Bruchidae) sehingga mengakibatkan
kerusakan berat dan kehilangan hasil. Oleh karena itu dalam upaya menghadapi
masalah tersebut di atas, perlu mencari alternatif fumigan untuk mengendalikan
dan mengelola serangga hama gudang.
Penelitian ini bertujuan untuk mengetahui keefektifan minyak atsiri
kapulaga (Ellateria cardamomum: Zingiberaceae), kayu manis (Cinnamomum
aromaticum: Lauraceae), dan pala (Myristica fragrans: Myrtaceae) terhadap T.
castaneum dan C. maculatus dan untuk mempelajari efek fisiologi ketiga minyak
atsiri terhadap T. castaneum dan C. maculatus. Hasil penelitian menunjukkan
bahwa minyak atsiri kayu manis memiliki efikasi yang lebih tinggi terhadap telur,
larva, dan imago C. maculatus dengan nilai LC50 berturut-turut adalah 0.019%,

0.132%, 0.186% sedangkan nilai LC50 telur, larva, dan imago T. castaneum
berturut-turut adalah 1.051%, 0.109%, 1.239%. Minyak atsiri kayu manis lebih
efektif terhadap kedua spesies serangga dibandingkan dengan minyak atsiri
kapulaga dan pala. Ketiga minyak atsiri mempengaruhi proses fisiologi dengan
cara memicu terjadinya penurunan kandungan karbohidrat, protein, lemak,
aktivitas enzim esterase dan glutation s-transferase pada larva instar ketiga T.
castaneum dan C. maculatus. Kayu manis merupakan minyak atsiri yang paling
efektif dalam mengendalikan kedua serangga uji dan dalam aplikasinya bersifat
ramah lingkungan dan efisien bagi masyarakat lokal.
Kata kunci: kapulaga, kayu manis, mortalitas, pala, toksisitas

© Copyright by IPB, 2016
All Rights Reserved
No part of this study may be used or reproduced in any manner whatsoever (print,
photograph, microfilm, etc) without written permission from Bogor Agricultural
University

TOXICITY AND PHYSIOLOGICAL EFFECTS OF THREE
ESSENTIAL OILS AGAINST Tribolium castaneum AND
Callosobruchus maculatus

SRI ITA TARIGAN

A Thesis
submitted in partial fulfillment of the requirements for
Degree of Masters
in
Entomology

GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2016

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Thesis examiner : Dr. Ir. Teguh Santosa, DEA

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4

ACKNOWLEDGEMENTS
First I express thank to Almighty God for making me success in completion
for this thesis. I also extend my sincere gratitude to my supervisors Prof. Dr. Ir.
Dadang, MSc and Dr. Ir. Idham Sakti Harahap, MSi for giving me the guidance,
insightful comments and expertise to see this thesis a success.
I would wish to acknowledge:
1. Dr. Ir. Pudjianto, MSi as a Head Study Program of Entomology at Bogor
Agricultural University
2. Dr. Ir. Teguh Santoso, DEA as a thesis examiner for insightful
comments
3. Director of LPDP Scholarship who has giving me chance to get granted
to finish my study at Bogor Agricultural University.
4. Ir. Sri Widayanti, MSi as Head Laboratory of Entomology at SEAMEOBIOTROP for granting me chance to use the Laboratory to conduct my
research.
5. Bogor Agricultural University, Entomology Study Program, and its
entire academic staff for support, motivation and expertise guidance.
6. My fellow friends from Department of Plant Protection for great
moments during constructive and thoughtful discussion during my study

period at ENT-IPB.
Finally, I am also deeply indebted and fortunate to have support of family,
friends, and colleagues who have been contributing their thoughts, advices, and
prayers. For those whose names I did not mention here, I offer my apologies but
know that those who have made my master journey special and will never been
forgotten.

Bogor, December 2016

Sri Ita Tarigan

5

TABLE OF CONTENTS
LIST OF TABLES
LIST OF FIGURES
1 INTRODUCTION
Background
Problem Statement
Objectives

Significant of Research
Justification of Study
Research Hypotheses
2 LITERATURE REVIEW
3 MATERIALS AND METHODS
Place and Time
Materials
Insect Culture
Preparation Essential Oil Treatment
Fumigation Tests for Adults
Fumigation Tests for Larvae
Fumigation Test for Eggs
Biochemical Tests
Data Analysis

i
ii
1
1
2

3
3
3
4
5
11
12
12
12
12
12
12
12
13
14

4 RESULTS AND DISCUSSION
Mortality Effect of Essential Oils against Adults
Mortality Effect of Essential Oils against Larvae
Mortality Effect of Essential Oils against Eggs

Effect of Essential Oils on Carbohydrate, Protein, and Fat Contents
Effect of Essential Oils on EST and GST Enzymes Activity

15
15
17
18
20
26

5 CONCLUSIONS AND RECOMMENDATION
Conclusions
Recommendation

31
31
31

REFERENCES
CURRICULUM VITAE

32
39

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LIST OF TABLES
1
2
3
4
5
6
7
8
9
10

Mortality effect of cardamom against T. castaneum and C. maculatus adults
Mortality effect of cinnamon against T. castaneum and C. maculatus adults
Mortality effect of nutmeg against T. castaneum and C. maculatus adults
Mortality effect of cardamom against T. castaneum and C. maculatus larvae
Mortality effect of cinnamon against T. castaneum and C. maculatus larvae
Mortality effect of nutmeg against T. castaneum and C. maculatus larvae
Mortality effect of cardamom against T. castaneum and C. maculatus eggs
Mortality effect of cinnamon against T. castaneum and C. maculatus eggs
Mortality effect of nutmeg against T. castaneum and C. maculatus eggs
Toxicity of essential oils against T. castaneum and C. maculatus

15
16
16
17
18
19
20
21
21
22

LIST OF FIGURES
1
2
3
4
5
6
7
8
9
10
11
12
13
14

Dorsal view of T. castaneum adult
The larvae of T. castaneum
C. maculatus infestation and their eggs laid on the surface of green beans
Insect culture of C. maculatus and T. castaneum
Effect of essential oils on total carbohydrate in T. castaneum larvae
Effect of essential oils on total carbohydrate in C. maculatus larvae
Effect of essential oils on total protein in T. castaneum larvae
Effect of essential oils on total protein in C. maculatus larvae
Effect of essential oils on total fat in T. castaneum larvae
Effect of essential oils on total fat in C. maculatus larvae
Effect of essential oils on esterase activity in T. castaneum larvae
Effect of essential oils on esterase activity in C. maculatus larvae
Effect of essential oils on GST activity in T. castaneum larvae
Effect of essential oils on GST activity in C. maculatus larvae

5
6
7
11
23
23
24
25
25
26
27
27
28
28

1 INTRODUCTION
Background
Storage of cereal products in warehouse is the effort to slow down or
maintain the physical and chemical characteristics of food or feed, in order to
avoid decay and damage to the stored products. Recently studies have estimated
that approximately 30% of feeds and foods are lost annually due to poor storage
or insect attack. In addition, magnitude of loss and damage levels depend on the
storage technology applied. A commonly applied control technology to manage
pest infestation in warehouses is phosphine fumigation. However, longterm
exposure and improperly practiced of phosphine fumigation will cause resistance
problems in target insects. This case has been postulated and experienced in
variety of countries such as Malaysia, Singapore (Yusof & Ho 1992), Brazil,
China, Australia, the United States and Indonesia (Rahim et al. 2011). Based on
those cases there is urge for development of alternative approach for pest
management and control in warehouses.
Essential oils are strong volatile aromatic compounds with a unique odor,
flavor or scent extracted from the plant. Moreover, they are metabolic by-products
and so-called volatile plant secondary metabolites. Their aromatic characteristics
often play an important role by making them attract or repel insects, protection
from cold or heat and their chemical used to develop defendant material of
insecticides (Mohan et al. 2011). Due to their distinctive chemical and physical
properties, essential oils have been widely applied as an alternative insecticide. In
addition, bioactivities of botanical essential oils have shown variety of used in
controlling agricultural pests and medically important insect species extending
from toxicity of ovicidal, larvicidal, pupicidal and adulticidal activities to sublethal effects on; oviposition deterrence, antifeedant activity and repellent actions
as well as their effect on biological process like growth rate, lifespan and
reproduction (Bakkali et al. 2008; Isman 2008; Tripathi et al. 2009; Ebadolahi
2011; Regnault et al. 2012).
Eletaria cardamomum Maton (Zingiberaceae) is an herbaceous plant; the
fruits are often used as a spice for cooking and medicinal purposes. In addition,
the chemical compounds in cardamom include; limonene, cineol, terpineol,
borneol acetate terpinyl, and some other types of terpenes (Keezheveettil et al.
2010). Myristica fragrans Houtt (Myristicaceae), also known as nutmeg
commonly is found in Banda Islands in Maluku, Indonesia. Despite nutmeg
known for its commercial value, it has also been used as a cooking spice and has
been utilized as a bactericide (Radwan et al. 2014) and insecticide (Tripathi et al.
2015). The chemical composition of nutmeg includes; sabinen, terpinen 4-ol, αpinene, β-pinene, and β-phellandren (Piras et al. 2012). Cinnamomum aromaticum
(Lauraceae) is one of the indigenous plant used as cooking spice and has also
been used for medicinal purposes (Ranasinghe et al. 2013). Some of the important
compounds in cinnamon oil are limonene, cineol, terpineol, borneol, acetate
terpinyl and other numerous types of terpenes (Abdelwahab et al. 2014). Studies
indicated that the monoterpenoid compound caused the death of insects by
inhibiting the activity of the enzyme acetyldholinesterase (AChE) (Houghton et
al. 2006). However, other monoterpenoid compounds have shown no effect of

2

inhibiting enzyme activity (Grundy & Still 1985; Dohi et al. 2009). Later studies
reported that the fumigant of essential oils of terpene compounds (ZP 51 and SEM
76) in plants. Labiatae and (+) - limonene exuberate inhibition of AChE in the
Ryzopertha dominica adults by 65% (Kostyukovsky et al. 2002; Anderson &
Coats 2012). Furthermore, studies have shown that most xenobiotics tend to cause
enzymatic transformation after penetration to binding sites of protein and
transportation of biological interaction. Glutathione S-transferase (GST) is one of
the most significant enzymes for detoxification mechanism owing to its
engagement intolerance to pesticides (Gui et al. 2009; Afify et al. 2011). Studies
have also indicated that esterases (EST) play a crucial role in the detoxification of
xenobiotics to nontoxic materials (Afify 2011).
The aims of this study were to investigate the toxicity effects of essential oils
against egg, larva and adult of T. castaneum and C. maculatus and to evaluate the
effects of essential oils on total carbohydrate, protein, and fat contents as well as
EST and GST activity.
Problem Statement
Approximately 10-30% of post-harvest products are produced world wide
each year experiencing yield losses due to infestations of insect pests in storage
warehouses (White 1995). T. castaneum and C. maculatus are the most serious
pests causing damage to stored products in the world. The insect species attack
different stored products and expand the range of food products, especially on
stored products (Aitken 1975). Most insects usually tend to develop resistance
faster on infested grain product specially when fumigated with phosphine (Zettler
1991). Essential oils contain lead compounds monoterpenoid potential as an
insecticide, as repellent and antifeedant (Amos et al. 1974; Grundy & Still 1985;
Shaaya et al. 1997; Lee et al. 2003; Ketoh et al. 2005; Rozman & Karunic 2007;
Cosimi et al. 2009). These compounds cause disruption of metabolic processes,
biochemical, physiological and behavioural functions of insects (Nishimura
2001). In addition, the compounds in the essential oil can also affect the activity
of detoxifying enzymes in insects (Nathan et al. 2011).
There are 3 types of enzymes which significant role in detoxification process
of insects (Bull 1981). Enzyme-monooxigenase, cytochrome P450 enzymes
involved are most important for insects in an attempt to detoxify the insecticide
(Zhou & Huang 2002). These enzymes have functions that vary depending on the
synthesis and degradation in the body of the insect (Feyereisen 2005). The GST
enzyme acts as detoxification to organophosphate insecticides, pyrethroid through
metabolic processes (Wei et al. 2001) and the use of secondary toxins through
increased lipid peroxidation (Dou et al. 2009). Group of general esterase enzyme
plays a role in detoxification of insecticides (Gacar & Tasksn 2009). In addition,
these compounds can also affect the content of fat, protein, and carbohydrate
contents in insects. Research results by Ebadollahi et al. (2013) showed that the
essential oil extracted from Agatasche foeniculum (Pursh) Kuntze resulted in
decreasing of carbohydrate, protein, and fat contents in the third instar larvae of T.
castaneum along with the increasing concentration of essential oils that were used.
Therefore, this research was conducted to test effect of three essential oils as
alternative fumigant to control T. castaneum and C. maculatus.

3

Objectives
General objective
The main objective of this research was to conduct the toxicity and
physiological effects of essential oils against T. castaneum and C. maculatus.
Specific objectives
1. To evaluate the toxicity effect of the three essential oils (cardamon,
cinnamon and nutmeg)
2. To evaluate the effect of essential oils on total of carbohydrate, protein,
and fat contents
3. To evaluate the effect of essential oils against enzymes activity
Significant of Research
This research was aimed at providing significant information on potential
efficacy of essential oils (cardamom, cinnamon and nutmeg) as insecticide and
environmental friendly alternative fumigant to control T. castaneum and C.
maculatus infestation in warehouses products.
Justification of Study
Shelf life is considered as a factor for stored products and for most postharvest products, shelf life often depends on the type of fumigant used. Type
fumigant used during post-harvested plays a critical role especially when
agricultural products are transferred from the farm to storage facilities. Previously
researches have showed that essential oils can be a potential fumigant when
prepared at right dose. Based on induction and preliminary studies most
researchers have advocated for the use of essential oils as a potential alternative
fumigant to control and to manage insect pests.
Moreover, development of resistance by insect pest due to higher exposure
time to phosphine has prompted the exploration alternative fumigant from variety
of botanical extracts with an intention to develop fumigant with higher efficacy,
less development of resistance and environmental friendly fumigant. For example,
in Singapore and Malaysia applications of phosphine fumigant during storage of
post-harvest products has presented a great challenge to control insect pests due to
development of insect resistance. Nonetheless, there is also a great challenge of
residual impact due to accumulation of fumigants on deposit products. Studies
have shown that secondary metabolites of botanical extracts can be used as
fumigant and antifeedant (Kim et al. 2013). Pepper extract using hexane and
acetone solvent have shown toxic effect to some insect pest in warehouse (Seo et
al. 2009).
Yang et al. (2010) reported that garlic extract has larvicidal effect against
several species of mosquitoes. In addition, an extract from garlic mixed with ethyl
acetate solvent has been reported to have repellent activity against T. castaneum
and S. zeamais (Donpedro et al. 1989). Thereby, there is still a gap to develop
suitable essential oils with higher efficacy to control stored products insect in
warehouse. Therefore, based on the preliminary studies and investigations, this

4

research explored the toxicity and physiological effects of essential oils extracted
from cardamom, cinnamon, and nutmeg as alternative fumigant to control T.
castaneum and C. maculatus.
Research Hypotheses
This research was based on two hypotheses; there are three types of essential
oils and at a given effective concentration, they inhibit the physiological processes
of T. castaneum and C. maculatus hence controlling damage to grain products.
H0:μ1=μ2: The null hypotheses states that the variance of various treatment
mean was the same, no significant effect of the administered treatment on the
adult, larva, and egg, total carbohydrate, protein and fat contents as well as EST
and GST enzymes activity in T. castaneum and C. maculatus.
H1:μ1≠μ2: The alternate (research) hypotheses this support the argument
that, among the three essential oils there was effective oil that was inhibit egg,
larva, adult and reduce EST and GST activity in T. castaneum and C. maculatus.

5

2

LITERATURE REVIEW

Taxonomy and Biology of Tribolium castaneum Herbst
T. castaneum belongs to class Insecta, order Coleoptera, family
Tenebrionidae and genus Tribolium (Prakash et al. 1987) with a complete
metamorphosis (egg, larva, pupa, and adult). T. castaneum eggs are whitish and
cylindrical microscopic with small bits of flour stuck on their surface, making
them difficult to see and incubation period ranges from 4 to 7 days.
Beeman et al. (2012) reported that the window period for egg development is
between 2-3 days at temperature of 340C with egg diameter ranging from 0.54 to
68 mm with a mean of 0.59±0.02 mm. Leelaja et al. (2007) further reported that
the egg with the 0.61 mm × 0.3 mm fluoresce at 365 nm UV spectrum.
At intial phase, the first instar larva is creamy white body with translucent
light brown head with dark brown eyes. The abdominal segment is partly or
completely concealed ventrally with a pair of pseudo pods. The duration of first
instar is 16 to 18 days. The length of grub is approximately 0.94 to 0.99 mm with
a mean of 0.96±0.02 mm, whereas the width is approximately 0.18 to 0.25 mm
with a mean of 0.19±0.02 mm, respectively.
The second instar larva has yellow-whitish body with slender and cylindrical
body covered with fine hairs. The head is pale brown and last segment of
abdomen have two upturn dark, pointed structures. The duration of this phase is
10 to 14 days. The length of second instar is approximately 1.57 to 2.16 mm with
a mean of 1.83±0.08 mm, whereas the width is 0.27 to 0.41 mm with a mean of
0.26±0.03 mm, respectively.

Figure 1 Dorsal view of T. castaneum adult

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Figure 2 The larvae of T. castaneum
The third instar larvae are structurally similar to second instars except in size.
The duration of third instar is about 8-10 days. The dark brown patches are
developed in last two-three abdominal segments. The length of third instar larva is
1.89 to 2.79 mm with a mean of 2.44±0.13 mm, while the width ranges from 0.40
to 0.65 mm with a mean of 0.49±0.03 mm, respectively.
After third moulting, the fourth instar larva will emerge from exuviae of the
third instar larva. The fourth instar larvae are resemble to third instar in colour.
However, they differ in size and shape. The duration of fourth instar is 8 to 10
days. The body length of fourth instar is 3.10 to 3.42 mm with a mean of
3.27±0.09 mm, whereas the width is 0.50 to 1.16 mm with a mean of 0.55±0.02
mm, respectively.
The duration of fifth instar is 8 to 10 days. The body length of fifth instar
ranges from 4.34 to 5.16 mm with a mean of 4.68± 0.13 mm, while the width
ranges from 0.73 to 0.96 mm with a mean of 0.83±0.04 mm, respectively.
The duration of sixth instar is 8 to 10 days. The body length of fully grown
grubs range from 5.06 to 5.63 mm with a mean of 5.27±0.09 mm, whereas the
width is 0.68 to 0.96 mm with a mean of 0.87±0.12 mm, respectively.
The duration of seventh instar is 9 to 11 days. Before pupation the last instar
larvae will stop feeding. The body length of seventh instar is 5.12 to 6.37 mm
with a mean of 6.22±0.06 mm, while the width is 0.82 to 0.84 mm with a mean of
1.07±0.03 mm, respectively.
Intially before pupation the pupa has dark wings, sclerotized legs with fully
developed eyes however, with absence of cocoon with white colour. Furthermore,
the body gradually turns yellowish and finally brown in colour. During this stage
the pupa is dormant. The male and female pupal periods are 6-7 days and 7-9
days, respectively. William (2000) reported that the pupal period to be
approximately 8 days. The length of male pupa is 3.81±0.03 mm whereas width is
1.07±0.03 mm. The length and width of female pupa are 4.12±0.01 mm and
1.15±0.01 mm, respectively.
Adult beetles are reddish-brown in colour, flattish curved-sided body. The
head is visible from above, with absence of beak while the thorax has slightly
curved sides. The antennae are enlarged at the tip (capitates) with the last three
segments wider than preceding segments. Male has a setiferous patch on the
posterior side of the fore femur, while female luck setiferous patch. The longevity
of breeding between male and female adults is approximately 45-50 days and 75-

7

80 days. The length of male is 3.06±0.03 mm and width is 1.28±0.30 mm whereas
the female length and width is 3.70±0.01 mm and 1.28±0.03 mm, respectively.
Taxonomy and Biology of Callosobruchus maculatus Fabricus
According to Rada & Susheela (2014) C. maculatus belongs to class Insecta,
order Coleoptera, family Bruchidae and genus Callosobruchus with a complete
metamorphosis (egg, larva, pupa, and adult).
C. maculatus has white egg-shaped oval. Eggs are laid individually on the
surface of green beans (Giga & Smith 1983). The eggs hatch in 9-10 days. The
infestation by C. maculatus begins from the field however, most of the damage
occurred during storage in a warehouse (Soutage 1979). In India it is reported that
damages by C. maculatus range from quality of the grain to the quanity of crops
and products resulting in 60% yield loss. Studies have reported that approximately
20-50% of yield loss is caused by C. maculatus (Alloey & Oyewo 2004).
C. maculatus larvae undergo complete metamorphosis. First instar larvae of
C. maculatus have shaped camboid with well-developed teeth, followed by the
second instar phase, cruciform-shaped larvae, the larvae undergo molting for 5
times, transition to the pupal phase complete metamorphosis which mark the exist
from the grain leaving a hole on the grain surface (Rada & Susheela 2014).

Figure 3 C. maculatus infestation and their eggs laid on the surface of green beans
The adults reddish brown, measuring 2-3 mm with slightly round body
shapes that can distinguish it from bruchidae members and there are two
brownish black spots on the pronotum. The shape of head is hypognatus, equipped
with a pair of antennae serrated. The male insects have longer antenna than female
with both sexes having biting mouth apparatus. The adults dewell and develop in
the grain. Adults lay eggs within a period of 5-20 days. Furthermore, C.
maculatus adult has life cycles between 21-25 days under a temperature of 230C
and 23-26 days at a temperature of 280C. A female adult can produce 100-120
eggs depending on the type of bean as feed and the development from egg to adult
in 2-3 weeks (Maina 2013).

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EST and GST Enzymes Activity
Esterase is an enzyme that presents in a large number of insects. The enzyme
contains carboxylic esters, amides, and thioesters which acts as defense against
insecticide compounds. In addition, this enzyme also plays an important role as a
defense against adverse environmental conditions (Hemingway & Karunatne
1998). Mahboukar et al. (2015) reported that the essential oil of Artemisa annua
(L.) resulted in decreasing the EST and GST activity in the fourth instar larva of
Helicoverpa armigera after being treated for 24 h. A. foeniculum essential oil at
concentrations of 1.5% and 2.5% can lead to increase enzyme activity in support
of the insect ability to detoxify the insecticide compound. A. foeniculum oil on
the other concentrations (5%, 10%, 15%, 20% and 25%) also showed a decrease
esterase activity on T. castaneum larvae (Ebadolahi et al. 2013). Some essential
oils are reported capable of inhibiting the enzyme activity of esterase in some
insects warehouse (Nathan et al. 2008, Caballero et al. 2008, Mukanganyama et
al. 2003). GST is an enzyme that has many functions and plays an important role
in detoxifying some compounds and organochlorine insecticides such as
organophosphates example xenobiotic mechanisms in insects that resulted in the
insects become resistant due to the induction of the enzyme activity of GST. In
insect, GST also is highly related to insecticide resistance, which could directly
detoxify the insecticides. In addition, insecticides entered into the body could
destroy the redox balance, and cause the oxidative stress reaction and produce the
lipid hydroperoxides, such as phospholipid hydroperoxides, fatty acid
hydroperoxides, 4-hydroperoxynonenal (Giordano et al. 2007).
Essential oils, Mode of Action and Components
Elettaria cardamomum Maton (cardamom), the Queen of all spices has a
history as old as human race. It is one of the high priced and exotic spices in the
world. It is the dried fruit of an herbaceous perennial plant belonging to the ginger
family, Zingiberaceae. The plant is indigenous to southern India and Sri Lanka.
The major use of Cardamom on world wide is for domestic culinary purpose and
in medicine. The seeds have a pleasant aroma and a characteristic warm, slightly
pungent taste (Amma et al. 2010). The composition of cardamom oil has been
studied by various workers (Nirmala 2000; Marongiu et al. 2004) and the major
compounds found were 1, 8 cineole (20-60 %) and á-terpinyl acetate (20-55 %). It
has been established that the oils and extracts from spices usually used to flavour
dishes are excellent source of natural antioxidant and they also find use as
nutraceuticals, due to the presence of hydroxyl group in their phenolic compounds
(Jayaprakasha 2006; Politeo et al. 2006).
Myristica fragrans, which is commonly known as nutmeg, belongs to the
family Myristicaceae and is a medium sized, evergreen aromatic tree (Anonymous
1992). It is distributed in India, South East Asia, North Australia and Pacific
islands. The nutmeg tree is indigenous to Banda islands in the Moluccas in east
Indonesia (Peter 2001). The nutmeg seed and its fleshy aril (mace) are used as
spices. It contains 4% myristicin. The nutmeg spice has been recognized in
Europe since 12 century when it was used as condiment and fumigant
(Rosengarten 1969). Nutmeg butter, a fat derived from the seed is used in
perfumery, tobacco and toothpaste. Medicinally, it is used to support digestion

9

and to treat rheumatism. Nutmeg seed is also used for diarrhoea, mouth sore and
insomnia. It has been proved that nutmeg has inhibitory activity against several
kinds of anaerobic and aerobic microorganisms (Shinohara 1999). The major
chemical constituents of nutmeg are alkyl benzene derivatives (myristicin,
elemicin, safrole), myristic acid, alpha-pinene, terpenes, beta-pinene and
trimyristin (Qiu et al. 2004; Yang et al. 2008). Nutmeg contains about 10%
essential oil, chiefly composed of terpene hydrocarbons (sabinene and pinene),
myrcene, phellandrene, camphene, limonene, terpinene, myrcene, pcymene and
other terpene derivatives (Jaiswal et al. 2009). Nutmeg also yields nutmeg butter
which contains 25 to 40% fixed oil and is a semi-solid reddish brown fat having
the aroma of nutmeg. Nutmeg butter contains trimyristin, oleic acid, linoleic acid
and resinous material. The fixed oil of nutmeg butter is used in perfumes and for
external application in sprains and rheumatism (Peter 2001). Trimyristin is the
major glycoside bearing anxiogenic activity (Sonavane 2002).
Cinnamomum aromaticum (cinnamon) is a common spice used by different
cultures around the world for several centuries. The volatile oils obtained from the
bark, leaf, and root barks vary significantly in chemical composition, which
suggests that they might vary in their pharmacological effects as well (Shen et al.
2002). The different parts of the plant possess the same array of hydrocarbons in
varying proportions, with primary constituents such as; cinnamaldehyde (bark),
eugenol (leaf) and camphor (root) (Gruenwald et al. 2010). The oil were found to
contain cinnamaldehyde, linalool, camphor, terpinen-4-ol and 1,8-cineole,
eugenol, safrole, c-muurolene, acadinol, germacrene D, a- terpineol, a-cadiene,
1,6-octadien- 3-ol,3,7-dimethyl and 1-phenyl-propanr-2,2-diol diethanoate as
major compounds (Jantan et al. 2005; Abdelwahab et al. 2010).
Toxicity of Essential Oils on Stored Product Insects
Previous research showed that there is a positive effect of essential oils
against warehouse pests for instance a decrease in the number of eggs, egg
viability, and reduction in larval development, number of offspring, α-amylase,
cytochrome P450, GST and acetylcholinesterase (AChE) (Huang et al. 2000;
Huang et al. 1999; Grundy & Still 1985; Lum & Zheng 1991). In addition,
Alibabaie & Mohamad (2015) reported that nutmeg oil at a concentration of 4.232
ml L-1 at exposure time of 3 days resulted in 100% mortality of C. chinensis
adults. This showed there was a positive correlation between exposure time and
dose concentration. Again, essential oil extracted from lime leaves at a
concentration of 16 ml g-1 resulted in high repellency activity against S. oryzae
adults. Furthermore, the F1 generation decreased successively in number when
treated with 2 ml g-1, 4 ml g-1and 6 ml g-1 lime leaves oil (Fajarwati et al. 2015).
In addition, Ellateria cardamomum (cardamom) oil resulted into 25% lose in
number of S. zeamais and Ephestia kuehniella eggs. Mona et al. (2009) reported
that application of Sesanum indicume oil against seventh instar larvae of T.
castaneum resulted in decrease in the number of α-esterase, β esterase, mixed
function oxydase (MFO) and GST enzyme. Nonetheless, E. cardamomum oil
against E. kuehniella showed higher sensivity comparatively to C. chinensis and
T. castaneum. However, T. castaneum showed tolerant to cardamom oil at a
concentration of 55.27 mg ml-1 leading to 80% mortality of T. castaneum adults
within 24 hours after treatment (HAT). Cardamom oil at a concentration of 64.26

10

mg ml-1 resulted in reduction of eggs laid by C. maculatus comparatively with
control (Abbasipour et al. 2011).
Compounds di-n-propyl disulphide derived from neem seeds had fumigant
effect against S. oryzae adult, T. castaneum (adult and larva). S. oryzae adults are
more tolerant than T. castaneum larvae. In addition, toxic vapor of essential oil
from neem extract has been applied to control Ephestia kuehniella and
Lasioderma serricorne (Bullington 1998). Generally Ryzopertha dominica and
Callosobruchus spp adults were more vulnerable to volatile oil compared to E.
kuehniella and L. serricorne (Ahmed & Eapen 1986, Tripathi et al. 2003, Lee et
al. 2004). Lemon oil at concentration of 2% resulted in differences in
susceptibility between males and females (Papachristos & Stamopoulos 2002a,
Papachristos et al. 2004). Furthermore, the third instar larvae of C. maculatus are
more tolerant than pupae (Don Pedro 1996b), and E. kuehniella larvae are more
tolerant than eggs when fumigated with oil component containing arvacrol, 1, 8cineole, menthol, g-terpinene, and terpinen-4-ol in 24-96 (Erler 2005).
Most monoterpenoid are cytotoxic and animal tissue, causing a drastic
reduction in the number of intact mitochondria and golgi bodies, impairing
respiration and photosynthesis and decreasing cell membrane permebiality. At the
same time they are volatile and many serve as chemical messengers for insects
and other animals. Furthermore, most monoterpenes serve as signal of relatively
short duration, making them especially useful for synomones and alarm
pheromones. The doses of essential oils needed to kill insect pests and their
mechanism of action, are potentially important for the safety of humans and other
vertebrates. The target sites and mode of action have not been well elucidated for
the monoterpenoids and only a few studies have examined these questions
(Watanabe et al. 1990; Rice & Coats 1994; Lee et al. 1997).
Little is known about the physiological actions of essential oils on insects, but
treatments with various essential oils or their constituents cause symptoms that
suggest a neurotoxic mode of action (Kostyukovsky et al. 2002). A
monoterpenoid, linalool has been demonstrated to act on the nervous system,
affecting ion transport and the release of acetylcholine esterase in insects (Re et
al. 2000).
Well known essential oils with bioactivity, either as an insecticide or
repellent, are clove, thyme, mint, lemon grass, cinnamon, rosemary and oregano
oils. Bioactivity can vary greatly because of variability in chemical composition
but despite of these varibilities, certain plant species, namely thyme, oregano,
basil, rosemary and mint are consistently bioactive (Isman & Machial 2006).
Elucidation of mode of action of essential oils is important for insect control
because it may give useful information on the most appropriate formulation,
delivery means and resistance management (Sim et al. 2006). Many plant
essential oils and their isolates have fumigant action (Kim et al. 2003). Essential
oils A. annua L. (Trippathi et al. 2000), Anethum sowa (Tripathi et al. 2001),
Curcuma longa (Tripathi et al. 2002), Lippia alba (Verma et al. 2001) and
isolates like d-limonene (Tripathi et al. 2003a), carvones (Tripathi et al. 2003b)
and 1,8-cineole (Aggarwal et al. 2001a) have been well documented as fumigants.
These findings indicate that the route of action for the oils was largely in the
vapour phase via respiratory system, although the exact mode of action of these
oils remains unknown.

11

3 MATERIALS AND METHODS
Place and Time
This research was conducted from December 2015 to June 2016. The
mortality tests were conducted at the Entomology Laboratory, while the
biochemical analysis at the Biotechnology Laboratory both at SEAMEOBIOTROP Bogor, West Java, Indonesia.
Materials
The equipments used in this study were petri dishes, spectrophotometer,
pipette Mohr, Whatman filter papers (9 cm diameter), plasticine, stationery,
rubber bands, gauze, labels, soft brush, elisa plate, strainer plastic, and glass jars.
The materials used in this study were Tribolium castaneum and Callosobruchus
maculatus, green beans, flour, essential oils of cardamom, cinnamon, and nutmeg,
acetone, 1-chloro-2,4-dinitrobenzene (CDNB), α-naphthyl acetate, salt RR,
distilled water, H3PO4, chloroform, vanillin, and sodium sulphate.
Insect Culture
A population of 500 adults of T. castaneum or C. maculatus was inserted into
glass jar (5 cm x 20 cm) containing wheat flour or green beans for T. castaneum
and C. maculatus, respectively. After two weeks, all adults were removed from
the glass jar and further incubated for 4 weeks. This was aimed at producing a
uniform of the F1 generation. Adults between the ages 7-14 days were used for the
mortality test while the third instar larvae were used for biochemical test.

a

b

Figure 4 Insect culture of C. maculatus (a) and T. castaneum (b) in the laboratory
Preparation Essential Oil Treatment
Three essential oils were obtained from Aromatic Medicinal Plant Research
Center, Bogor. These included essential oils of Elateria cardamomum
(cardamom), Cinnamomum aromaticum (cinnamon) and Myristica fragrans
(nutmeg). The preparation of the essential oil concentrations used dilution
methods where the stock concentration further subjected to additional acetone
solution to lower the concentration.

12

Fumigation Tests for Adults
To test toxicity effect of essential oils, preliminary test was conducted to
asses the LC50 and LC95 of each essential oil. In this case, the concentration was
prepared to match the mortality range of 5-99%. Here, for cardamom oil the
concentrations used to treat T. castaneum were 3%, 3.5%, 4%, 4.5%, and 5%
whereas for C. maculatus were as follows; 0.1%, 0.25%, 0.5%, 0.75%, and 1%.
For cinnamon oil, the concentrations used to treat T. castaneum were 1.2%, 1.4%,
1.6%, 1.8%, and 2% whereas for C. maculatus were as follows; 0.1%, 0.25%,
0.5%, 0.75% and 1%. Again for nutmeg oil, the concentrations used to treat T.
castaneum were 2%, 4%, 6%, 8%, 10% whereas for C. maculatus were as
follows; 0.1%, 0.25%, 0.5%, 0.75% and 1%. In this test, five replications were
conducted with each essential oil consisted of different treatment concentrations,
excluding the placebo. A population of 30 adults was placed on whatman filter
paper on the surface of petri dish lid. Then, 0.5 ml of each essential oil was
dispensed on to the surface of whatman filter paper.
On the other hand, 0.5 ml acetone was used as the control. The petri dish was
sealed tightly using plasticine to prevent a effeversence of the fumigant. This was
followed by evaluation of the mortality 72 hour after treatment (HAT). The data
obtained were then analysed using probit analysis.
Fumigation Tests for Larvae
Five concentrations of 1.5%, 2.5%, 5%, 10% and 15% of essential oils were
prepared to treat the larvae with acetone as solvent. This was followed by
uniformly admixing 1000 µl of each concentration with 0.5 g of wheat flour for T.
castaneum and 0.5 g of green beans C. maculatus in a 7-cm diameter petri dish.
The Whatman filter paper was then left to dry at room temperature for 1 minutes.
Control samples were treated only with pure acetone and dried in the same way.
A total of twenty third instars larvae were randomly selected placed with treated
diets and kept at 27 ± 2ºC and 60 ± 5% RH. The experiment was replicated four
times and larvae mortalities were recorded after 72 hours of treatment. Toxicity of
larvicidal activity was then calculated based on the 50% mortality of subjected
insects (LC50) 72 HAT. The mortality was then analysed using probit analysis.
Fumigation Tests for Eggs
In this test, a population of 30 eggs of T. castaneum or C. maculatus was
placed into different petri dish containing wheat flour or green beans for T.
castaneum and C. maculatus, respectively. Here, for cardamom oil, concentrations
used to treat T. castaneum were 3%, 3.5%, 4%, 4.5%, 5% whereas for C.
maculatus were as follows; 0.1%, 0.25%, 0.5%, 0.75% and 1%. For cinnamon oil,
the concentrations used to treat T. castaneum were 1.2%, 1.4%, 1.6%, 1.8%, and
2% whereas for C. maculatus were as follows; 0.1%, 0.25%, 0.5%, 0.75% and1%.
Again for nutmeg oil, the concentrations used to treat T. castaneum were 0.25%,
0.5%, 0.75%, 1%, 1.5% whereas for C. maculatus were as follows; 0.1%, 0.25%,
0.5%, 0.75% and 1%. This was followed by addition of 0.5 ml essential oil then
the petridish was sealed. Lastly, the placebo were only treated with 0.5 ml
acetone, After 14 days, the mortality of eggs were counted under stereo

13

microscope. Sterile eggs (eggs that fail to hatch) counted as mortile or dead. The
egg mortality was evaluated using probit analysis.
Biochemical Tests
A population of 10 third instar larvae was kept in a Petri dish followed by
ascending concentration of the essential oils according to Ebadolahi et al. (2013)
method. In this test a concentration of 1.5, 2.5%, 5%, 10% and 15% were used to
treat T. castaneum and C. maculatus larvae. In this analysis the larvae were
exposed to treatment to a period of 24 hours after which the surviving larvae were
used to analyse the total carbohydrate, protein, and fat contents.
Fat content
The surviving larvae from the fumigation test for larvae were subjected to
analysis of fat content. The surviving larvae were kept in a vowel then mixed with
100 µl sodium sulphate (2%) and 750 µl chloroform: methanol (2:1), then stirred
until homogeneous. The resulting mixture was then centrifuged (10 minutes, 8000
rpm 40C). After which 250 of the supernatant was obtained and added to 500 µl of
H2SO4 then the mixture was placed into water bath at 900C. Subsequently, 30 µl
of vanillin solutions (600 mg vanillin in100 ml distilled water and H3PO4 (400 ml,
85%) was added to the mixture, then the adsorbance at 545 nm was recorded
using spectrophotometer to determine the concentration of fat content (Van
Handel & Day 1998).
Carbohydrate content
To analyse total carbohydrate the surviving larvae in larvacidal bioassay were
placed in a vowel and mixed with stock solution prepared during analysis of fat
content then 150 µl anthrone (500 mg anthrone in 500 µl H2SO4) was added.
Subsequently, the resulting mixture was placed in water bath at 900C. The
concentration of carbohydrate was then recorded using spectrophotometer at an
absorbance of 630 nm (Yuval et al. 1994).
Protein content
For protein analysis six surviving larvae were put in 350 µl distilled water
and then centrifuged for 5 minutes at 10.000 rpm at temperature of 4ºC. Then, 10
µl of supernatant was mixed with 90 µl distilled water and 2500 µl dye. The
concentration was then recorded using spectrophotometer at absorbance of 630
nm (Bradford 1976).
Enzyme Analysis
Esterase analysis
To analyze the esterase activity, six third instar larvae were kept in vowel,
then 1ml 0.1M phosphate buffer solution was then added and stirred until
homogeneous to stabilize the pH. This was then followed by centrifugation for 10
min at 10.000 rpm at a temperature of 40C. After which, 75 µl α-naphthyl acetate
and 75 µl of saline RR (CH3CH2-Na) were again added. The concentration was
then recorded using a spectrophotometer at 450 nm (Han et al. 1995).

14

Glutathione s-transferase analysis
For determining activity of glutathione s-transferase (GST), the method of
Habing et al. (1974) was adopted. In this study 1-chloro-2,4-dinitrobenzene
(CDNB) (20 mM) was used as substrate. First six larvae were homogenized in 20
μl distilled water, then the homogenized solution was centrifuged at 12500 g for
10 minutes at 4ºC. Fifteen μl of supernatant was mixed with 135 μl of phosphate
buffer (pH = 7), 50 μl of CDNB and 100 μl of GST. Finally the concentration of
the solution was read using spectrophotometer at an adsorbance of 340 nm.
Data Analysis
The mortality data were analysed using probit analysis (POLO-Plus) while
the biochemical data, such as total carbohydrate, protein, and fat content, as well
as esterase and glutathione s-transferase activities were analysed by analysis of
variance (ANOVA) using SPSS program and Tukey’s test with confidence level
of 95% was incorporated to further elucidate the difference in the treatments.

15

4 RESULTS AND DISCUSSION
Results
Mortality Effect of Essential Oils against Adults
The mortality test showed that all essential oils at different concentrations
resulted in different mortality against T. castaneum and C. maculatus adults. The
highest concentration (5%) of cardamom oil after 72 HAT resulted in 95%
mortality of T. castaneum adults. At the lowest concentration (3%) of cardamom
oil resulted in 38% the mortality of T. castaneum adults. Furthermore, the highest
concentration (1%) after 72 HAT, which caused 100% mortality of C. maculatus
adults whereas at the lowest concentration (0.1%) resulted in 40% the mortality
(Table 1). Similar mortality effect was recorded when T. castaneum and C.
maculatus adults were treated with cinnamon oil, since there was an increase in
mortality of T. castaneum and C. maculatus adults with an increase in essential oil
concentrations (Table 2).
Table 1 The mortality effect of cardamom oil against T. castaneum and C.
maculatus adults
Insects

T. castaneum

C. maculatus

Concentration (%)

Mortality (%) at 72 HAT*

control
3.00
3.50
4.00
4.50
5.00
control
0.10
0.25
0.50
0.75
1.00

0.0d**
38.0c
57.0bc
68.0b
81.0ab
95.0a
0.0d
40.0c
55.0bc
63.0b
92.0ab
100.0a

*

HAT: hour after treatment
The percentage of mortality followed by the same letter on the same species of insect is not
significantly different by Duncan Multiple Range Test (DMRT) at significant level of 5%
**

Cinnamon oil was capable of causing mortality at comparatively lower
concentrations than cardamom and nutmeg oils against T. castaneum adults. It
was evidenced that at a concentration of 2.0%, cinnamon oil was capable of
causing 100% mortality against T. castaneum adults. To achieve 100% mortality,
a concentration more than 4.5% of cardamom oil used to treat T. castaneum and
C. maculatus adults although for cinnamon oil to achieve 52% mortality at a
concentration of 1.2% used for treatment (Table 2).

16

Table 2 The mortality effect of cinnamon oil against T. castaneum and C.
maculatus adults
Insects

T. castaneum

C. maculatus

Concentration (%)
control
1.20
1.40
1.60
1.80
2.00
control
0.10
0.25
0.50
0.75
1.00

Mortality (%) at 72 HAT*
0.0c**
52.0b
65.0b
71.0ab
93.0ab
100.0a
0.0d
35.0c
51.0bc
76.0abc
96.0ab
100.0a

*

HAT: hour after treatment
The percentage of mortality followed by the same letter on the same species of insect is not
significantly different by Duncan Multiple Range Test (DMRT) at significant level of 5%
**

Table 3 The mortality effect of nutmeg oil against T. castaneum and C. maculatus
adults
Insects

T. castaneum

C. maculatus

Concentration (%)
control
2.00
4.00
6.00
8.00
10.00
control
0.10
0.25
0.50
0.75
1.00

Mortality (%) at 72 HAT*
0.0a**
30.9b
50.9c
63.4cd
84.0d
90.9e
0.0a
45.0b
61.0c
67.8cd
93.8d
100.0e

*

HAT: hour after treatment
The percentage of insect mortality at 72 hours after treatment. The percentage of mortality
followed by the same letter on the same species of insect is not significantly different by Duncan
Multiple Range Test (DMRT) at significant level of 5%
**

Mortality effect of nutmeg oil against T. castaneum and C. maculatus adults
indicated that at a concentration of 10%, nutmeg oil was able caused 90.9%
mortality on T. castaneum adults comparatively nutmeg oil at concentration of 1%
caused 100% mortality to C. maculatus adults. However nutmeg oil at a lower
concentration of 2.0% only caused 30.9% mortality to T. castaenum adults, this
was apparently different with C. maculatus adults where it resulted to 45.0%

17

mortality at concentration of 0.10%. From this analysis it was evidence that a very
low concentration of nutmeg oil caused higher mortality to T. castaneum than C.
maculatus adults.
Mortality Effect of Es