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Original Research Article

Insecticidal effects of Fortunella crassifolia essential oil used against house fly (Musca domestica)

Gehad T. El-Sherbini1* and Nancy Osman hanykamel2

1Department of Parasitology, Faculty of Pharmacy, 2Department of Parasitology, Faculty of Medicine October 6 University Cairo, Egypt

*Corresponding author

A B S T R A C T

Introduction

The house fly, MuscadomesticaL. is considered as one of the most important pests which cause health problems in the environment as it accompanies humans during their daily activity everywhere, both indoors and outdoors. It is recognized as a public health pest causing a serious threat to human and livestock by vectoring several pathogenic organisms such as protozoa cysts, helminthic parasites, enteropathogenic bacteria, and enterovirus

(Emerson, et al., 1999; Douglass and Jesse, 2002; Mian, etal., 2002; Barin, et al., 2010). Many insecticides such as organochlorines and organophosphates, and more recently pyrethroids and spinosad, have been used for housefly control. However, houseflies can develop resistance to these pesticides (Scott, et al., 2000; Shono and Scott, 2003) and health and environmental risks are associated with these compounds; thus, investigators

ISSN: 2319-7706 Volume 3 Number 6 (2014) pp. 1-09 http://www.ijcmas.com

K e y w o r d s

Fortunella crassifolia, essential oil, chemical composition, insecticidal activity, Musca domestica L.

Growing patterns of insecticidal drug resistance laid the foundation for research in exploring novel anti-insect agents from medicinal plants particularly with fumigant action for ease of application.The aim of this study was to determine the some constituents of the essential oil isolated from Fortunellacrassifolia Swingle peel by hydro-distillation, and to test the efficacy of the essential oil on insecticidal activity against house fly, Muscadomestica. Two Fortunellafractions obtained by steam distillation, Soxhlet n-hexane extraction, extraction with aqueous ethanol, and with ethanolic ammonia solution were evaluated in vitro for activity against house fly, Musca domesticaL.These data warrant further study on identifying the components of the extracts with the highest activities.The essential oil showed potent insecticidal activity .It was suggested that the essential oil from Fortunella crassifolia Swingle peel might be used as a natural insecticide against house fly.

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continue to search for alternative methods of fly management. The use of plants in medicine can be dated back to before humans understood the cause of infectious disease (Douglass and Jesse, 2002). In this sense, essential oils (EOs) are potential alternatives and environmental friendly insecticides. Kumquats (Figure.1) have been used in Chinese folk remedies for centuries. Kumquats are preserved using salt to draw out the juice. The juice is then steeped in hot water to relieve sore throats and suppress coughing. Most traditional medicines are still folk remedies and have not been confirmed. Technological advances in the 20th century left traditional medicines to be forgotten. Antioxidant, antimicrobial, and cytotoxic properties have been demonstrated in several Yemeni traditional medicinal plants (Regnault-Roger, 1997). Kumquats (Fortunella spp.) belong to the Citrus genus are fruit, usually eaten raw as a whole fruit together with the peel, excluding the seeds. The peel is sweet and edible with a typical aroma due to the presence of flavonoids and terpenoids (Koyasako and Bernhard, 1983). Kumquats are also an excellent source of nutrients and phytochemicals, including ascorbic acid, carotenoids, flavonoids and essential oils. F. crassifolia, known as jingdan or meiwa kumquat, has a typical citrus character. As with other citrus fruits, F. crassifolia can be candied, prepared as marmalade, added to fruit salad and preserved as a whole in sugar syrup in food industry.It is well known that F. crassifolia is rich in flavonoids. Previously, 3 , 5 -di-C- -gluco- pyranosylphloretin, a characteristic flavonoid, was well-studied in the genus Fortunella (Ogawa, et al., 2001). Recently, the antioxidant activity of F. crassifolia has been reported (Kondo, et al., 2005). However, studies on the chemical composition and biological

activities of F. crassifolia peel oils are limited. From a dietary viewpoint, essential oils represent added value for kumquat fruit because, in addition to their contribution to the flavor, they play an important role in human health, as with other non-nutritive phytochemicals such as polyphenols and flavonoids (Schirra, 2008).

The Citrus essential oils have various functional properties, such as an attractive aroma, a repellant agent against insects and animals, and antioxidant activities. A previous study has reported the antimicrobial action of citrus oils (Subba, et al., 1967). Meanwhile, citrus oils are not only available to the food industry, but are also generally recognized as safe and have been found to be inhibitory, in both oil and vapor form, against a range of both Gram-positive and Gram-negative bacteria (Fisher and Phillips, 2008). Moreover, there are a large number of studies on plant essential oils regarding their antimicrobial properties in order to develop a source of antimicrobial ingredients for the food industry (Mkaddem, et al., 2009). To our knowledge, there are no reports in the literature concerning the insecticidal activity of the essential oil from F. crassifolia until now. The aim of this study was therefore to determine the main constituents of the essential oil of F. crassifolia peel, together with an evaluation of its insecticidal activity against the house fly larvae for the possibility of using these oils as larvicides for controlling the insects by treating insect breeding places which create serious problems mostly in the developing countries. It is odorous components and secondary metabolites that can be extracted from plant tissues through steam distillation. Essential oils and their volatile

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constituents are widely used in the prevention and treatment of human illnesses. They are also documented for exhibition of acute toxicity, anti-feeding and ovi position deterrents against a wide variety of insect-pests. The risk lower level of the volatile essential oils to the environment and their minimal residual activity against predator, parasitoid and pollinator insect populations, making essential-oil-based pesticides compatible with integrated pest management programs (Regnault-Roger, 1997; Isman, 2006; Koul, et al., 2008).

Materials and Methods

Chemicals Isolation of the Essential Oil

Fresh F. crassifolia fruit was purchased from a local market in Cairo in December 2012. The plant was initially identified by morphological features and the database present in the Department of Biology, October 6University. The individual fruits were washed with water and then separated into peel and flesh by hand. The fresh peelings (250 g) were homogenized with a mixer, followed by hydro-distillation using a Clevenger-type apparatus for 4 h. The essential oil was collected over water, dried over anhydrous sodium sulfate and stored at 4°C until analyzed. The oil yield was about 2% (5 g/250 g of fresh peel).

Collection and maintenance of flies

Adults of M. domestica were collected from the garbage site of the Abu Arish area (Cairo), by using a sweeping net and transported into a small cage to the Department of Parasitology, Faculty of Medicine, October 6 University, for identification and reared in laboratory for four generations before experiment. Adult flies were maintained in cages (30x 30 x 30 cm) and provided with granulated

sugar, petri dishes containing cotton pads soaked in milk powder dissolved in water (10% w/v) and jars (500 mL) containing larval media for egg laying. Larval media consisted of yeast, dry milk powder, wheat bran and water according to the method described by Pavela (2006) (Pavela, 2006). The jars were removed from cages after 2-3 days when eggs were visible and were provided with wood dust for pupation and kept in separate cages for fly emergence.

Fumigant toxicity bioassays

Fumigant toxicity of the essential oil was determined by sealing ten adult flies in a 120ml jar with a 1 cm diameter cotton ball treated with 100 µl of 100% essential oil. Essential oil was injected into the center of each ball to allow its volatilization while preventing the insects from coming in contact with the oil. Mortality was recorded at 15 minutes, 30 minutes, 1 hour and 2 hours, respectively for the house flies. Control jar had insects and an untreated cotton ball. The experiment is repeated thrice.

Biological studies

The second instar larvae used in this experiment were 3-days-old after hatching from the same egg batch. Larval treatment carried out in petri dishes according to the method explained by Brady 1966, in which interior of each petri dish treated with 1 ml from each of the aforementioned concentrations of the tested essential oils. Each experiment was conducted in four replicates (20 larvae/ replicate) along with the control group. After treatment, the larvae were transferred to the rearing jar and the mortality assessed by touching each larva with a paint brush (no. 0), and those not responding were considered dead. The LD50 and LD75 toxicity determined based on mortality data at 24 h assessments. Living larvae were further

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examined daily to estimate the effect on the larval duration after treatment, percentage of pupation and the successfully emerged adults according to Sripongpun 2008 and Peydro et al. 1995. In addition, any morphological abnormalities recorded were photographed at all developmental stages (Fig. 3).

Statistical analysis of Data

The results were recorded as means ± standard deviation. Statistical analysis was performed using one-way analysis of variance (1-way ANOVA) using SPSS tatistical package (SPSS 16) and the statistical significance was determined at P< 0.05.

Results and Discussion

Essential oil was obtained from the fresh peel of F. crassifolia by hydro- The amount of the major compound, limonene, in the essential oil obtained from F. crassifolia peel is high (74.6%), followed by myrcene (8.11%), -pinene (0.34%). A previous report showed that the major chemical component of citrus oils is limonene, ranging from 32 to 98%, with sweet orange containing 68 98%, lemon 45 76% and bergamot 32 45% (14-24).In another study on the characteristic odor components in the essential oil from F. japonica peel, limonene was the most dominant component, amounting to 93.73% (Choi, 2005). The limonene content of F. crassifolia peel oil was comparatively less than that reported for F. japonica peel oil. The -pinene content of the essential oil was similar to that reported for F. japonica peel oil (Choi, 2005). However, the contents of myrcene, camphene and -selinene were found to be higher than those reported for F. japonica peel oil (Choi, 2005).

Larval duration

Results in Table 4 revealed that the

duration of the control larvae of M. domesticawas 4.46 ± 0.51 days; a significant prolongation was observed in the duration of larvae treated with LC50 and LC75 of the examined oils, which were 5.61 ± 0.69 and 5.56 ± 0.73 days.

Pupation percent and pupal duration:

Data in Table 4 showed a high significant reduction in pupation percent (62.50 and 42.50%) at LC50 and LC75 of F. crassifolia, comparable to 95.00% for control group. Data in the same table revealed that the pupal duration of the control group was 4.43 ± 0.50 days which significantly prolonged in groups treated with LC50 and LC75 of tested oils and reached 5.44 ± 0.53 and 5.00 ± 0.67 days at LC75 .The current results are in agreement with findings of Kumar et al. 2012 during their evaluation of insecticidal activity of E. globulus (Myrtales: Myrtaceae) against the house fly.

The adult emergence percentage

Adult emergence reduced from 95% for control group to 57.5 and 30% for groups treated with LC50 and LC75 of F. crassifolia (Table 5). High reduction in M. domestica emergence was also reported by Abdel Halim and Morsy 2005 after using volatile oils of C.macrocarpa and A. officinarum against Synthesio myianudiseta. Also, Kumar et al. 2011 found that crude oils of M. piperita and E. globulus suppressed the emergence of adult flies by 100%. Fortunella essential oil has shown good insecticidal as shown in the (Table2,3,4,5). Itpossesses moderate toxicity to variousinsects. The mortality ranged from 25 to100%during the first 12hours in the fumigant toxicity bioassay of the essential oil, mortality ranged from 25 to 55% during the first 3 hours.

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Fig.1 Fortunella margarita, kumquat,is in the Rutaceae family

Table.1 The main chemical components of the essential oil in the fresh Fortunella crassifolia Swingle peel.

NO RIa Compound Identificationb

(%)c

1 936 -Pinene MS,RI 0.34 2 993 Myrcene MS,RI 8.11 3 1035 Limonene MS,RI 74.6

aRI, retention indices as determined on TR-5 capillary column using homologous series of n-alkanes. bMethods of identification: MS, by comparison of the mass spectrum with those of the computer mass libraries; RI, by comparison of RI with those from the literature.(%)crelative percentage obtained from peak area.

Table.2 Contact toxicity of the essential oil of fortunella to adult M. domestica

Mean mortality (%) Essential

oils Conc.%

15min 30min 60min 2 hr 3 hr 6 hr 12 hr 24 hr Limonene 5

10 100

0 0

33.3

0 25

53.3

25 50

63.3

35 65 70

55 75

100

75 80

100

100 100 100

100 100 100

Myrcene 5 10 100

0 0

15,1

0 20 34

20 25

45.3

40 54 60

50 65 75

65 85

100

100 100 100

100 100 100

-Pinene 5 10 100

0 0

8.5

0 18 25

20 20

33.3

34 44 50

44 55 62

55 67 80

100 100 100

100 100 100

Control 0 0 0 0 0 0 0 0

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Table.3 Fumigant toxicity of the essential oil of fortunella to M. domestica insect stages

Mortality % Time Larva Pupa Adult

Control

15 min 0 5 4 0 30 min 0 10 8 0 60 min 10 15 12 0

2 hrs 12 20 25 0

3 hrs 25 25 33.5 0

6 hrs 30 50 50 0

12hrs 100 100 100 0

24hrs 100 100 100 0

. Table.4 Effect of the tested plant oils on the larval duration of M.domestica

treated as 3rd larval in star at 29°C

Oil Dose Larval duration

fortunella LC50 LC75

5.61 ± 0.69b 5.56 ± 0.73 b

Control 4.46 ± 0.51 a

Data expressed as mean± SD, significance at p<0.05 is between different superscripts

Table.5 Effect of the tested plant oils on the pupulation percentage, and pupal duration (days) of M. domestica treated as 3rd larval in star at 29°C

Es. oil Dose Pupation (%±SD

Pupal duration

fortunella LC50 LC75

62.50±9.57b

42.50±9.57b

4.82± 0.53b 5.00 ± 0.67b

Control 95.00±5.77a

4.43 ± 0.50a Data expressed as mean± SD, significance at p<0.05 is between different superscripts

Table.6 Effect of the tested plant oils on the adult emergence percent M. domestica treated as 3rd larval instar at 29°C

Es. oil Dose Adult emergence (%±SD) fortunella

LC50 LC75

57.50±9.57b 30.00±8.16b

Control 95.00±5.77a Data expressed as mean± SD, significance at p<0.05 is between different superscripts

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Plate.1 Adult Pupa, Larva and adult house fly

Plate.2 The control figure represented the normal pupa and figures (1-11) represented the malformations

Plate.3 The control figure represented the normal larva and Figures 1-7 represented the malformations at larval stage

.

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Although, the insecticidal activity of essential oil is much lessthan that of synthetic insecticides such as dichlorvos, propuxorand permethrin, they are quite harmless. They are not toxic to mammals. Their volatility as well as their non-toxicity makes them an ideal candidate to be used in restaurants, hospitals and schools against various insects. There is a need to assess various essential oils for insecticidal activity against insects that cause diseases and damage food products.

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