TOXICITY AND PHYSIOLOGICAL EFFECTS OF THREE
ESSENTIAL OILS AGAINST
Tribolium castaneum
and
Callosobruchus maculatus
SRI ITA TARIGAN
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY BOGOR
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
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.
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.
© Copyright by IPB, 2016
All Rights Reserved
A Thesis
submitted in partial fulfillment of the requirements for Degree of Masters
in Entomology
TOXICITY AND PHYSIOLOGICAL EFFECTS OF THREE
ESSENTIAL OILS AGAINST
Tribolium castaneum
AND
Callosobruchus maculatus
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY BOGOR
2016
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 SEAMEO-BIOTROP 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
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TABLE OF CONTENTS
LIST OF TABLES i
LIST OF FIGURES ii
1 INTRODUCTION 1
Background 1
Problem Statement 2
Objectives 3
Significant of Research 3
Justification of Study 3
Research Hypotheses 4
2 LITERATURE REVIEW 5
3 MATERIALS AND METHODS 11
Place and Time 12
Materials 12
Insect Culture 12
Preparation Essential Oil Treatment 12
Fumigation Tests for Adults 12
Fumigation Tests for Larvae 12
Fumigation Test for Eggs 12
Biochemical Tests 13
Data Analysis 14
4 RESULTS AND DISCUSSION 15
Mortality Effect of Essential Oils against Adults 15 Mortality Effect of Essential Oils against Larvae 17 Mortality Effect of Essential Oils against Eggs 18 Effect of Essential Oils on Carbohydrate, Protein, and Fat Contents 20 Effect of Essential Oils on EST and GST Enzymes Activity 26
5 CONCLUSIONS AND RECOMMENDATION 31
Conclusions 31
Recommendation 31
REFERENCES 32
LIST OF TABLES
1 Mortality effect of cardamom against T. castaneum and C. maculatus adults 15 2 Mortality effect of cinnamon against T. castaneum and C. maculatus adults 16 3 Mortality effect of nutmeg against T. castaneum and C. maculatus adults 16 4 Mortality effect of cardamom against T. castaneum and C. maculatus larvae 17 5 Mortality effect of cinnamon against T. castaneum and C. maculatus larvae 18 6 Mortality effect of nutmeg against T. castaneum and C. maculatus larvae 19 7 Mortality effect of cardamom against T. castaneum and C. maculatus eggs 20 8 Mortality effect of cinnamon against T. castaneum and C. maculatus eggs 21 9 Mortality effect of nutmeg against T. castaneum and C. maculatus eggs 21 10 Toxicity of essential oils against T. castaneum and C. maculatus 22
LIST OF FIGURES
1 Dorsal view of T. castaneum adult 5
2 The larvae of T. castaneum 6
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 sub-lethal 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, α -pinene, β-pinene, and β-phellandren (Piras et al. 2012). Cinnamomum aromaticum
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).
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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 post-harvest 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
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.
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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 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.
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).
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).
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and to treat rheumatism. Nutmegseed 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
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, 8-cineole, 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 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),
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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 SEAMEO-BIOTROP 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.
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.
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
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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
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
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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). 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
Table 2 The mortality effect of cinnamon oil against T. castaneum and C. 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 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%
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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 Essential Oils against Larvae
To evaluate the effect of essential oil against larvae, the three essential oils were made in different concentrations to analyse the effectiveness of the essential oils against T. castaneum and C. maculatus larvae. It was observed that the
The experimental test showed that comparatively low concentration of cinnamon oil was used to treat T. castaneum and C. maculatus larvae as opposed to concentration of nutmeg and cardamom oils. It was observed that at a concentration of 0.7%, cinnamon oil was capable of causing 100% mortality against T. castaneum and C. maculatus larvae. On the other hand, for cardamom oil 1% concentration caused 100% mortality. The drastic response by both larvae of T. castaneum and C. maculatus were confirmatory test that at the lowest concentration of 0.1% cinnamon oil was capable of causing 50% and 45% of T. castaneum and C. maculatus, respectively (Table 5).
Table 4 The mortality effects of cardamom oil against T. castaneum and C. maculatus larvae
Insects Concentration (%) Mortality (%) at 72 HAT*
Table 5 The mortality effect of cinnamon oil against T. castaneum and C. significantly different by Duncan Multiple Range Test (DMRT) at significant level of 5%
From the experimental analysis, the mortality revealed that cinnamon oil is most effective resulting in highest mortality to both larvae of T. castaneum and C. maculatus compared with cardamom and nutmeg oils. Furthermore, cinnamon oil shown to be more toxic to T. castaneum larvae compared to C. maculatus larvae. This was evidence by lowest concentration of 0.1% which caused 50% and 45% mortality against T. castaneum and C. maculatus larvae, respectively. On the other hand, nutmeg oil caused 50.0% mortality against T. castaneum larvae at a concentration of 0.25%. In Table 6 it was evidence that the highest concentration of 2% nutmeg oil resulted in 100% mortality of T. castaneum larvae whereas for
C. maculatus larvae similar mortality of 100% was achieved at a concentration of 1.5%. From the result it was deduced that nutmeg oil was the comparatively more toxic to C. maculatus larvae than T. castaneum larvae.
Mortality Effect of Essential Oils against Eggs
From the experimental results according to Table 7, the effect of cardamom oil was realized at different concentrations against T. castaneum egg. From the concentrations it was evidenced that to achieve 81.3% mortality against T. castaneum eggs a concentration of 5% was used for treatment within 72 hours. In addition, it was observed that at a lower concentration (3%), a mortality of 52.5% was caused against T. castaneum egg. However, for control no mortality was recorded. Cinnamon oil also recorded a similar pattern with cardamom oil. Note that in this analysis, it was observed that upon treatment of T. castaneum eggs with cardamom and cinnamon oils an increase in concentration resulted to an increase in mortality, in other words the mortality increase with increase in treatment concentration.
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Table 6 The mortality effect of nutmeg oil against T. castaneum and C. maculatus larvae
Insects Concentration (%) Mortality (%) at 72 HAT*
T. castaneum significantly different by Duncan Multiple Range Test (DMRT) at significant level of 5%
For cinnamon oil analysis, it was observed that only at a relatively lower concentration of 0.1% caused 70% mortality against C. maculatus eggs. Note that, in this experiment different concentrations were used to treat T. castaneum and C. maculatus eggs. From this analysis, 90% mortality was caused against C. maculatus eggs at a concentration of 1% cinnamon oil. On the other hand, similar mortality of 90% was recorded to T. castaneum eggs at a concentration 1.8% (Table 8). Contrastly, for cardamom and nutmeg oils similar mortality of 90% was achieved at a concentration of above 5%. From this analysis it was inferred that cinnamon oil had comparatively higher efficacy against T. castaneum than C. maculatus eggs as shown in Table 8.
For nutmeg oil resulted in 45% mortality against T. castaneum eggs at the lowest concentration of 0.25%. Whereas upon treatment with 0.1% concentration mortality of 42.5% was recorded when C. maculatus eggs were treated. Nonetheless, treatment of T. castaneum egg with 1.5% concentration of nutmeg oil resulted in 60% mortality. This was incontrast with treatment of C. maculatus
eggs where similar mortality of 60% was recorded at a concentration of 0.75%. From table 9 again it is clearly evidenced that nutmeg oil was significantly toxic to C. maculatus eggs compared to T. castaneum eggs.
From the Table 10, to better understand the toxicity effect of the essential oils against T. castaneum and C. maculatus the datas were subjected to stastistical analysis using Polo-Plus. Here, the mortality and concentration values were used as input data to generate 50% and 95% lethal concentration. From the analysis, Table 10 was used to represent the probit analysis results.
concentration compared to high concentration. In addition, in C. maculatus larva a concentration of 0.132% was recorded whereas for T. castaneum larva concentration of 0.109% was recorded. Based on these results similar pattern was postulated to be in eggs of both insect. Again, as seen in Table 10 a concentration of 0.019% was recorded for C. maculatus egg in contrast 1.051% was recorded for T. castaneum egg. In summary, it was concluded that at a lower concentration cinnamon oil induced higher mortality effect to both treated insects.
Table 7 The mortality effect of cardamom oil against T. castaneum and C. maculatus eggs
Insects Concentration (%) Mortality (%) at 72 HAT
T. castaneum significantly different by Duncan Multiple Range Test (DMRT) at significant level of 5%.
Effect of Essential Oils on Carbohydrate, Protein, and Fat Contents
The experimental results depict that as the concentration increased the total amount of carbohydrate relatively decreased. For instance, when both insects species were tested for carbohydrate it was observed that as the concentrations increase the total amount of carbohydrate also decrease as shown in Figure 5, we can see that for T. castaneum cinnamon oil had a relatively higher toxicity effect to trigger reduction of total amount of carbohydrate compare to cardamom and nutmeg oils. However, as shown in Figure 6 for C. maculatus cinnamon oil triggered reduction in total amount of carbohydrate.
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Table 8 The mortality effect of cinnamon oil against T. castaneum and C. maculatus eggs
Insects Concentration (%) Mortality (%) at 72 HAT
T. castaneum significantly different by Duncan Multiple Range Test (DMRT) at significant level of 5%
Table 9 The mortality effect of nutmeg oil against T. castaneum and C. maculatus
Table 10 Toxicity of essential oils against T. castaneum and C. maculatus
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carbohydrate and protein decreased while total fat in the third instar larva of T. castaneum increased after the treatment of essential oils. Total carbohydrate, protein, and fat contents against the third instar larva of C. maculatus after receiving the third treatment essential oils is shown in Figure 6.
Figure 5 Amount of carbohydrate content in T. castaneum larva exposed to different concentration of essential oils after 24 h. Different letters indicate significant differences among concentration level of each essential oil according to Tukey test at p=0.05
Treatment of the third instar larva of C. maculatus with cinnamon oil at concentration of 2.5% and 15% resulted in reduction of total amount of carbohydrate. The decrease of carbohydrate to C. maculatus larva treated nutmeg oil occurs at a concentration of 2.5%. Cardamom oil treatment resulted in increasing amount of carbohydrate in C. maculatus larva with increasing concentration. This suggests that cinnamon oil is more toxic to T. castaneum larva to C. maculatus larva.
The third applications of essential oils cause a decline in the number of carbohydrate in the third instar larva of T. castaneum. At the highest concentration (15%) third application of essential oils cause the rate of decline is very significant amount of carbohydrate when compared with control. Cinnamon oil causes a decrease in the amount of carbohydrate of 1.96 times compared to the control. Cinnamon oil is more toxic than nutmeg and cardamom oils.
In the analysis of the effect of essential oils against protein content in both insect species it was observed that total amount of protein decreased with the increase in treatment concentration of essential oils. For instance, in Figure 8 it was observed that at lower concentration (1.5%) tend to trigger reduction the total amount of protein. From the graph in Figure 8 it was observed that cinnamon and nutmeg oils significantly decreased the amount of protein in contrast cardamom oil records no significant effect on total amount of protein in T. castaneum larva.
Figure 7 Amount of protein content in T. castaneum larva exposed to different concentration of essential oils after 24 h. Different letters indicate significant differences among concentration level of each essential oil according to Tukey test at p=0.05
In figure 9, it was observed that the total amount of protein significantly decreased with increased in the concentration of essential oil. From the graph it was observed that all the three essential oils similarly result in a significant decrease in total amount of protein in C. maculatus larva.
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cinnamon oil recorded higher toxicity effect that triggered drastic reduction in total amount of fat content as compared with cardamom oil.
Figure 8 Amount of protein content in C. maculatus larva exposed to different concentration of essential oils after 24 h. Different letters indicate significant differences among concentration level of each essential oil according to Tukey test at p=0.05
Figure 9 Amount of fat content in T. castaneum larva exposed to different concentration of essential oils after 24 h. Different letters indicate significant differences among concentration level of each essential oil according to Tukey test at p=0.05.
cinnamon oils recorded higher toxicity effect that triggered drastic reduction in total amount of fat contents as compared with cardamom oil.
On the other hand for C. maculatus in Figure 10 it was observed that cinnamon oil had significantly higher toxicity to trigger reduction in total amont of fat content compared with cardamom and nutmeg oils, however nutmeg oil significantly had higher toxicity more than cardamom oil.
Figure 10 Amount of fat content in C. maculatus larva exposed to different concentration of essential oils after 24 h. Different letters indicate significant differences among concentration level of each essential oil according to Tukey test at p=0.05
Effect of Essential Oils on Esterase and Glutathione S-transferase Activity
According to Figure 11 it was observed that cinnamon oil significantly result in reduction in esterase activity. This was more effective compared to cinnamon oil. Moreover, this effect was also effective when compared to nutmeg oil. The experimental result for esterase activity in C. maculatus in Figure 11 showed that the cinnamon and cardamom oils had significant effect on esterase activity. On the other hand, nutmeg oil had relatively low effect on esterase activity as shown in Figure 12. It was observed that at a concentration of 5% caused an increase in esterase activity after which at a concentration of 10% esterase activity drastically started reducing. Therefore, based on the concentration used to treat the larvae of both insect species all the essential oils had significant physiology effect on the esterase enzyme activity with cinnamon oil at concentration of 15% lead to high rate of reduction of enzyme activity compared to both nutmeg and cardamom oils. The experimental results of GST activity according to Figure 13 showed that application of the three essential oils against T. castaneum and C. maculatus
larvae induced significant decrease in the enzyme activity. It was observed that GST activity declined at relatively higher rate in T. castaneum and C. maculatus
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observed that in general the activity for (GST) enzyme in C. maculatus larva when treated with cardamom, cinnamon, and nutmeg oils significantly decreased followed with increased the concentration of oils.
Figure 11 Activity of esterase in T. castaneum larva exposed to different concentration of essential oils after 24 h. Different letters indicate significant differences among concentration level of each essential oil according to Tukey test at p=0.05
Figure 12 Activity of esterase content in C. maculatus larva exposed to different concentration of essential oils after 24 h. Different letters indicate significant differences among concentration level of each essential oil according to Tukey test at p=0.05
Nonetheless, cinnamon oil resulted in a significant higher decrease in GST activity compared with cardamom and nutmeg oils. From this analysis it was deduced that the three essential oils resulted in a decrease in the GST activity in
Figure 13 The activity of glutathione s-transferase (GST) in T. castaneum larva exposed to different concentration of essential oils after 24 h. Different letters indicate significant differences among concentration level of each essential oil according to Tukey test at p=0.05
Figure 14 The activity of glutathione s-transferase (GST) in C. maculatus larva exposed to different concentration of essential oils after 24 h. Different letters indicate significant differences among concentration level of each essential oil according to Tukey test at p=0.05
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evidence in figure 11-14. Therefore from the biochemical analysis it was clear to infere the trend of the effect of increase in concentration of essential oils.
Discussion
Essential oils are naturally complex secondary metabolites derived from aromatic plants which can be used as a bioinsecticide for controlling some of the pests in the warehouse (Lopez & Pascual-Villalobos 2010). In this study, it was observed that three essential oils at different concentrations had different toxicity effect against adults, larvae, and eggs for both insect species. Among three tested essential oils, cinnamon and cardamom oils had high toxicity against three phases for both insect species as shown in Table 10. However, nutmeg oil had the lowest toxicity against both insect species. According to Wang et al. (2014) the level of fumigant and contact effect of essential oils largely corresponded to the dose and exposure time. In addition, the toxicity of the essential oil was also influenced by the chemical composition, which highly depended on the place of origin, weather and climatic conditions, methods and period of extraction and plant parts used (Rajendran & Sriranjini 2008).
T. castaneum and C. maculatus were affected by the fumigants depending on the chemical composition. Furthermore, it was observed that all essential oils had different respond based on their chemical composition. The experimental results from this study were similar to the study conducted by Mondal & Khalequzzaman (2009) where the contact effect of cinnamon oil had greater toxicity effect against T. castaneum larvae (LC50=0.074 mg cm-2) and Sitophillus
zeamais adults (LC50=0.196 mg cm -2).
Although, nutmeg oil consists of high aromatic compounds such as: d -pinene, d-champena, dipentena, myristicin, elemicin, and safrole. The primary compound in nutmeg oil is myristicin that acts as a narcotic which may cause damage to the brain by interfering with the activity of acetylcholinesterase (Jaiswal et al. 2009). It was observed by Dhingra et al. (2006) an extract of n-hexane in nutmeg oil at a dose of 100-150 µg ml-1 significantly degrades activity of acetylcholinesterase in white mice. Kasim et al. (2014) observed that cinnamon oil, consists of compounds such as 1,2-naphthalenedione (9.03%), ethanone (1.11%), and borneol (1.03%), where the main toxic compound in cinnamon is cinnamaldehyde (86.67%). This was evidenced by the study conducted with Maina (2013) who observed that cinnemaldehyde compound (LD50=19.0-24.0 mg m-2) had a higher mortality effect against Dermatophagoides
pteronyssinus Trouessart (Acari: Pyroglypidae) adults compared with benzyl benzoate and dibutyl phthalate insecticide.
The study has also shown that cinnamon oil has a fumigant and contact effect against Lasioderma serricorne, Sitophilus oryzae, and C. chinensis at a dose of 0.7 mg cm-2 with a percentage of 100% within 24 HAT (Kim et al. 2003). From the three analyzed essential oils there was different responds by both insect species due to different chemical composition of the fumigants. Cinnamon oil was observed has the highest effect of decreasing the total carbohydrate, protein, and fat contents compared with cardamom and nutmeg oils. This was similar to the study by War et al. (2013). This was consistent with the study by War et al.
body of Helicoverpa armigera. Toxicity of plant extract can be characterized by its ability in decreasing the amount of protein in insects. EST and GST are group of enzymes that play an important role in the detoxification process of toxic compounds that are entering and existing from the insect body. According to Terrie (1984) EST and GST enzymes are made up of protein compounds (85%).
The decrease in total protein in larva was postulated as an indicator of toxic exposure to insecticides. Decrease in the total of protein ultimately decreases total carbohydrate and fat contents. Nath et al. (2011) reported that stress due to exposure to insecticide may interferes with insect physiology that generally translates to a decrease in total amount of protein as a consequent of decline in amino acids formed in TCA cycle due to insufficient fatty acid to generated Adenosine Tryphospate (ATP) energy. According to Smirle et al. (1996) stated that low ATP energy triggered stress in insect resulting in death. They further stated that fat acts as a source of energy stored in the body of insects.
According to study by Chapman (1998) stored fats in fat body tends to increase during feeding process while decrease when insects are inactive (pupa stages). Nonetheless study done by Ebadollahi et al. (2013) reported that the total of carbohydrate, protein, and fat contents in T. castaneum larva subjected to fumigation with A. foeniculum decreased as the concentration of the fumigants increased. From the experimental analysis it was observed that both of larvae tested after fumigated with three essential oils within 24 HAT resulted in a significant decrease in esterase and GST activity compared with control. The experimental results of this study were similar with the study by Ebadollahi et al.
(2013) who reported that essential oil of A. foeniculum decreased the activity of EST and GST enzymes on third instar of T.castaneum larva. A decrease in EST and GST enzymes activity on both insect might have been triggered by low amount of protein in fat body. Moreover, War et al. (2013) reported that the building blocks of EST and GST enzymes consist of 60% protein. Consequently, a decrease in the activity of EST and GST activity in larva subjected the insect unable to resist the toxic compound.
From the literature it is still unknown if the mode of action essential oils can decrease the amount of EST and GST as a defense mechanism to respond to toxic substances. The experiment of this study indicated that a decrease EST and GST activity resulted in decreased in antioxidant activity of P450 gene that responsible for detoxification of toxic compounds in insects. Although there was a decline in enzyme activity in larva stage there is still a gap to evaluate the mode of action of the essential oil that might have induce higher mortality of T. castaneum and C. maculatus. Finally, it was observed that cinnamon and cardamom oils had higher potential to be used as an alternative fumigant for controlling T. castaneum and
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4 CONCLUSIONS AND RECOMMENDATION
Conclusions
Cinnamon oil has higher efficacy on C. maculatus and T. castaneum
compared with cardamom and nutmeg oils. Cinnamon oil has higher levels of toxicity for adult, larva and egg of C. maculatus compared to adult, larva and egg of T. castaneum. Cinnamon oil resulted significantly reduced of total carbohydrate, protein, fat contents, activity of EST and GST enzymes in both insect tested.
Recommendation
REFERENCES
Abbasipour H, Mohammad M, Fahimeh R, Mohammad HH. 2011. Fumigant toxicity and oviposition deterency of the essential oil from cardamon,
Elettaria cardamom, against three stored product insects. J Insects Sci. 11 (165):1-10.
Abdelwahab SI, Manal MET, Faridah QZ, Adil HAA, Shamsul K, Yasodha S, Khalijah A. 2014. Chemical composition and antioxidant properties of the essential oil of Cinnamomum altissimum Kosterm. (Lauraceae). Arabian J Chem. http://dx.doi.org/10.1016/j.arabjc.2014.
Afify AMR, El-Beltagi HS, Fayed SAS, Shalaby EA. 2011. Acaricidal activity of different extracts from Syzygium cumini L. Skeels (Pomposia) against
Tetranychus urticae Koch. Asian Pac J Trop Biomed. 1(5):359-64.
Aggarwal KK, Tripathi AK, Prajapati V, Kumar S. 2001a. Toxicity of 1.8 cineole against three species of stored product coleopterans. Insect Sci Appl. 21: 229-236.
Ahmed SM, Eapen M. 1986. Vapour toxicity and repellency of some essential oils to insect pests. Indian Perfumer. 30:273-278.
Aitken AD. 1975. Travelers, I: Coleoptera, Technical Bulletin 31, HMSO, London.
Alibabaie TM, Mohammad IS. 2015. Occupational IgE-mediated allergy to
Tribolium confusum (confused flour beetle). Allergy. 55:879-882.
Allotey J, Oyewo EO. 2004. Some aspects of the biology and control of
Callosobruchus maculatus stored on some soyabean, Glycine max (L) Merr varieties. J Afric Food Nutri Devel. 4(2):1-13.
Amma KPAP, Maheswari PR, Indu S, Venagopalan NPN. 2010. Chemical composition, flavanoid, phenolic contents and radical scavenging activity of four major varieties of cardamom. Int J Biol Med Res. 1(3):20-24.
Amos TG, Wiliams P, Du-Guesclin PB, Schwarz M. 1974. Compounds related to juvenile hormone: activity of terpenoids selected on Tribolium castaneum
and Tribolium confusum. J Econ Entomol. 67:474-476.
Anderson JA, Coats JR. 2012. Acetylcholinesterase inhibition by nootkatone and carvacrol in arthropods. Pestic Biochem Physiol. 102:124-128.
Anonymous. 1962. Wealth of India: Raw materials. Vol. VI. Council of Scientific and Industrial Research, New Delhi. p. 474-6.
Bakkali F, Averbeck S, Averbeck D, Idaomar M. 2008. Biological effects of essential oils-a review. Food Chem Toxicol. 46:446-75.
Beeman RW, Haas S, Friesen K. 2012. Beetle wrangling tips (an Introduction to the care and handling of Tribolium castaneum. Available on: http: //www.ars.usda.gov/Research/ docs.htm.
Bradford MM. 1976. A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 72:248-54.
Bull DL. 1981. Factors that influence tobacco, Heliothis virescens, resistance to organophosphorous insecticides. Bull EntomolSoc Amer. 27:193-197. Bullington SW. 1998. A new system to protect stored cocoa beans from insects
