Detection of Mediterranean Fruit Fly Larvae Ceratitis capitata ( Diptera : Tephidae ) in Different Types of Fruit by HS-SPME GC-MS Method

Timely detection of Mediterranean fruit fly (Medfly) is very important so that eradication action can be taken on time. The larvae stage of this insect is the most dangerous stage as it is within the pulp of the fruit, making it hard to detect by visual inspection. In most countries at ports of entry the inspector check a small sample of fruit by visual inspection or by cutting the produce and searching for fungus and pests. This paper will investigate a quick, reliable and sensitive method to determine the presence of fruit flies. Our research focuses on developing the technology for detecting hidden infestations by using the Head Space-Soild Phase Micro Extraction (HS-SPME) method coupled with Gas Chromatography-Mass Spectrocopy (GC-MS) technique. Five different types of fruit were infested with an early stage of Medfly Ceratitis capitata Wiedemann (Diptera: Tephidae). We investigated to detect the differences in volatile organic compounds (VOC’s) between infested and noninfested fruits by using HS-SPME with (GC-MS). The results indicated that for few chemicals no significant differences between infested and non-infested fruit can be seen, especially in the fruits with first instar. However, in case of third instar larvae infested fruits significant differences in the chemicals can be seen as compare to non infested fruits and other instar infestations. These chemicals include ethyl (Z)-2 butenoate, 2-heptanone, anisole, β-cis-ocimene, 1,3,7-nonatriene,4,8-dimethy-,ethyl octyate, isoamyl caproate and 1β,4βh,10βhguaia-5,11-diene, in apple. Ethyl (Z)-2-butenoate, (+)-2-bornanone, (-)-transisopiperitenol, methyl caprate, caryophyllene and farnesene in orange. Butanoic acid, 3-methyl-,2-methylbutul acetate, sabinene, β-myrcene, octanoic acid, methyl ester, dihydrocarvone, (-)-trans-isopiperitenol and ethyl laurate in mandarin. Butyl 2-methylbutanoate, terpinen-4-ol, P-menth-8-en-2-one, How to cite this paper: Al-Khshemawee, H., Agarwal, M., Du, X. and Ren, Y.L. (2017) Detection of Mediterranean Fruit Fly Larvae Ceratitis capitata (Diptera: Tephidae) in Different Types of Fruit by HS-SPME GC-MS Method. Journal of Biosciences and Medicines, 5, 154-169. https://doi.org/10.4236/jbm.2017.53017 Received: March 13, 2017 Accepted: March 31, 2017 Published: April 3, 2017 H. Al-Khshemawee et al. 155 E-,(3E,7E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene and dodecanoic acid, ethyl ester in lemon. Decane, 3-methyl-, p-menth-1,4(8)-diene, 1-undecene and α-cubebene in avocado. Thus, the VOC’s method could provide a possible tool for detecting tephritid larvae and this method could be adopted by industries importing and exporting fruit.


Introduction
Mediterranean fruit fly Ceratitis capitata Wiedemann is species of invasive pest that affects fruit production and export worldwide. C. capitata attacks approximately 250 different species around the world [1]. United States spends about $57 million per year on Medfly risk management [2]. Furthermore, over the period 2003-2008, Australian industry and government invested around $128 million in the management of Medfly C. capitata [3]. Currently, fruit fly control is almost exclusively carried out with chemicals that are harmful to human health and to the environment [4]. In organic fruit production, the issue is more serious, since the law regarding organic farming prohibits the use of synthetic substances that include pesticides [5]. For this reason, farmers are trying to limit the problems by avoiding infection by Medfly. Results obtained in field and laboratory tests demonstrate different susceptibilities to Medfly damage [6]. Several types of research have demonstrated how different varieties of fruit display a variety of chemical profiles and how the release of their VOCs increases or decreases during the maturation process. Also, much attention has been a focused on the development of trapping systems for detection and monitoring of hidden infection [7]. However, improved methods are needed for detection of the immature stages as well, such as first, second and third instars. Larvae feed and develop while hidden within the pulp, making infestation difficult to detect by the senses, especially for Medfly eggs and first instar larvae which are clear to pale white in colour and hence camouflage well with fruit pulp and only 2-3mm length inside the fruit [8]. In ports of entry, quarantine inspectors check import produce by taking small samples of fruit and checking for any signs of pests such as boring or feeding spots or by opening the fruit to search for Medfly larvae [9].
Research has shown that only 35% of fruit infested with fruit fly were detected by trained agricultural inspectors. If not checked by quarantine inspectors, infested fruits get distributed to consumers [8].
This paper will evaluate the use of Head Space-Solid Phase Micro Extraction (HS-SPME) method coupled with Gas Chromatography-Mass Spectroscopy (GC-MS) technique as a potential technology for improving detection of hidden insect infestation inside fruit. According to [10], medfly can change the volatile compounds of the fruits. It also has been shown that chemical changes can occur within host fruit as a result of insect infestation [11]. In this research, we examined different types of fruits infested with C. capitata to determine if infested fruit give us different chemicals profiles from non-infected fruits. Samples volatiles were collected at various stages of infections (first, second and third instars) of larvae, and chemical analysis was performed by GC-MS equipment.

Insects
Medfly colony were obtained from the Department of Agriculture and Food, Western Australia (DAFWA) and reared in the Murdoch University Laboratory, in Perth Australia. All the flies were reared under conditions: 23˚C ± 2˚C and 75% ± 5% RH, and 12:12-h (L:D) [12]. Adults are placed in screen cages (40cm length ×40 cm height ×40 cm depth) and each cage contained medfly food made from crystaline sugar from (Bidvest, Australia), yeast hydrolysate from (Australian Biosearch) in ratio of 4:1 and water 50 mL. About 10-12 days after adult's emergence from pupae and mating of adults flies, eggs were collected every day, which are deposited on to mesh side and fallen into the water tray kept adjacent to the cage.

Fruits
Royal Gola Apple (Malus domestica) from New South Wales, Valencia orange (Citrus spp.) from Western Australia, Hass avocado (Persea Americana) from South-West of Western Australia, Hicksons Mandarin (Citrus reliculata) from New South Wales and Eureka Lemon (Citrus limon) from New South Wales were obtained from the local fruit and vegetable market. The fruits were stored for 2 days under 2˚C; twenty ripe fruits from each variety were used in our experiment.

Infestation and Sample Preparation
Fruits were cleaned with distilled water to remove any surface contaminants.
Then, 30 eggs with 0.5 ml of water were transferred to each single fruit by using a sterile syringe. Fruits were divided into three groups (5 fruits in each group) for volatiles analysis at different stages of larvae, first, second and third instars.
Each group used two fruits to monitor the progress of larval development by cutting the fruit to determine the larvae stage and to evaluate the level of infestation by using a microscope to see the larvae stages and remaining three fruits were used for volatile analysis. In addition, one group was sampled as non-infested fruits. All the fruits were placed and stored in laboratory at a temperature of 24˚C for 7 -9 days; for the development of the larval stage. After collecting the volatile compounds as described in the next section, the various types of fruits were cut into small pieces and the number of larvae inside each fruit was counted.

Collection/Extraction of Volatile Compounds
The analysis of compounds was focused on whole fruits. Various types of fruit were placed individually into 2 litre jars. One whole fruit was analysed in each jar. Volatiles were collected by solid phase micro extraction (SPME) fibre with 50/30 µm Carboxen/DVB/PDMS (2 cm) (Sigma-Aldrich, Bellefonte, USA) coating. The samples were collected by inserting the fibre into the jar and exposing to the headspace. VOC's were collected on different times depending on the level of larvae inside the fruit. After sealing the jars for 16 hours at a temperature of 24˚C, the fibre was exposed to headspace for 2 hours which the optimized the HP-SPME extraction time. The desorption time of SPME fiber was 10 min in the injection port.

Analysis of Volatile Compounds
VOC's were analyzed with (Gas chromatography Agilent GCMS 7820A equipped with a mass spectrometer detector 5977E (Agilent Technologies, USA) and a DB-35ms column (30 m × 250 µm × 0.25 µm) (Santa Clara, CA 95051, USA). The carrier gas was 99.999% helium supplied by (BOC, gas, Sydney, Australia). The GC-MS operation conditions were as follows: The temperature of the injector port was 270°C. The initial oven temperature was 50°C and increased to 250°C by (5 • C/min). The column Flow rate was 1:1 ml/min and splitless was 20 ml/min at 1.5 min. The total GCMS run time was 45 min. Three experimental replicates were taken for each type of fruit. Compound peaks were deconvoluted by AMDIS version 2.72 and identified by searching the NIST 2014 MS database (the US National Institute of Standards and Technology) with retention index confirmation. Three replications for each type of fruit were analysed, and the experiment were repeated two times to confirm the chemicals.

Method Sensitivity and Limit of Detection (LOD)
The limit of detection was evaluated with alkane standard C7-C30 (Supelco, Bellefonte, USA). One litre Erlenmeyer flasks (Bibby Sterilin, Staffordshire, Cat. No. FE 1 L/3 equipped with cone/screw-thread adapter (Crown Scientific, Code ST 5313) with 1.1 cm blue septa (Grace Davison Discovery Sciences, catalog: 6518 ) were used make stock and diluted standard. The stock standard of concentration was prepared by adding 4 µl of standard into sealed 1L Erlenmeyer flasks. Then, samples were diluted to ppb from ppm, ppt from ppb and ppq from ppt levels by transferring 1 mL of head space by syringe into another flask. After 1 hour of extraction time with 50/30 µm Carboxen/DVB/PDMS (2 cm) (Sigma-Aldrich, Bellefonte, USA) fibre at room temperature, the SPME fibre was injected into GC-MS with 270°C injection port. Each level was repeated two times.

Statistical Analysis
The number of larvae inside the fruit was analysed by one way (ANOVA). For the comparison of volatile compounds between different instars, the peak area was analysed by software using the two way (ANOVA) test [13]. Differences in the result were compared by using the least significant differences test (LSD P ≤ 0.05) for determining the means between different instars with non-infested fruits. The peak area was divided by 10 6 for each single compound. The peaks left after subtracting from the blank run were only analysed.

Level of Infestation
The fruits were dissected immediately after collection of the volatiles compounds for finding out the level of infestation. For the first instar, it was hard to calculate the number of larvae; so the data was calculated by counting second instar larvae. The results indicated there were significant differences in the level of hatching in avocado compared to other types of fruit, like apple, lemon, orange and mandarin in laboratory conditions. Average ± SD number of larvae per fruit was as follow: 8.06 ± 1.58 apple, 13.13 ± 1.21 orange, 11.93 ± 1.77 mandarin, 9.06 ± 1.81 lemon and 18.93 ± 1.10 avocado ( Figure 1). The non-infested fruit was also dissected to confirm there are no natural infestations by Medfly.

Limit of Detection (LOD)
The GC-MS response of the stock and diluted alkanes standard decreased form ppm (Parts per million) level to ppt (parts per trillion) level ( Table 1). Some of the alkanes can be detected less than ppt level with SPME method. Octane, decane, undecane, pentadecane, hexadecane and heptadecane can be detected in ppt level. Octane can be detected in small amount even at ppq level.

The Volatiles Compounds from Fruit
There were many differences between compounds for each type of fruits and also between infested and non infested fruits. Some of the compounds were detected in one type of fruit, was found to be absent in another type. From the GC analysis, about 33 compounds from apple, 45 compounds from orange, 45 compounds from mandarin, 44 compounds from lemon, and 40 compounds from avocado were identified. All these compounds were identified by comparing with the retention index in the literature (NIST) and mass spectra in the NIST. We analysed 23 compounds each from apple (Table 2), orange (Table 3), mandarin (Table 4), and lemon (Table 5), while 17 compounds were analysed from avocado ( Table 6). The comparison of main compounds between different types of fruit infested with three different instars is explained in Figure 2.

The Volatile Compounds from Apples
In case of non infested apples, the main peaks were hexyl acetate, n-butyl 2 methylbutyrate, n-hexyl propionate and isobutyl caproate. Many compounds were detected in fruits infested with third instar larvae but not in fruits infested with first or second instar larvae or in non infested fruit and these included ethyl (Z)-2 butenoate, 2-heptanone, anisole, β-cis-ocimene, 1,3,7-nonatriene,4,8-dimethy-, ethyl octyate, isoamyl caproate, ethyl decylate and 1β,4βh,10βh-guaia-5,11-diene ( Table 2). The most significant compounds comparing with non infested fruits in third instar were propyl isobutyrate, 1-hexanol, hexyl acetate, n-butyl 2 methylbutyrate, n-hexyl propionate and isobutyl caproate. In the second instar, they were propyl isobutyrate, 1-hexanol, hexyl acetate, n-butyl 2 methylbutyrate and n-hexyl propionate, while the significant chemicals which were recorded in first instar were propyl isobutyrate, hexyl acetate and isobutyl caproate ( Table   2). The concentration of 1-hexanol and styrene increased with increase in instars, however, propyl isobutyrate, hexyl acetate, n-butyl 2 methylbutyrate and        n-hexyl propionate were decreased with increase in instars. From these results, it shows that the level of larvae can change the profile of compounds as also reported by [14]. Also, these compounds may impact on medfly larvae behaviour [15]. Infested apple with codling moth (Cydia pomonella L.) larvae gave a high level of esters and farnescene, from first instar infested and other instars, but the amount decreased in healthy fruits [16]. [11] Similar pattern in increase of esters can be found in our findings. In addition, some compounds were presented in high concentration in non-infested apple as compare to infested fruit such as propyl isobutyrate, n-butyl 2 methylbutyrate and others and also vice versa pattern is seen where high concentration of some compounds can be seen in infested fruits as compare to non infested fruits ( Table 2). [11] [17] found that infested mango Mangifera indica (MI) with Anastrepha ludens (Loew) larvae extracts contain many compounds in higher level compared with the other two kinds of mangoes healthy mango (HM) and mechanically damaged mango (MDM). [18] Found nonanol, dodecane, tetradecane, 2-pinene, limonene, farnesene and hexyl carproate in five cultivars of peach infested with Medfly.
Some of these chemicals increase in concentration with increase in infestation
Some of chemicals increased in concentration with increase in the level of infestation like D-limonene. Similar results were observed in case of orange, mandarian and lemon. However, 1-heptanal, E-4,8-dimethyl-1,3,7-noatriene and α-terpineol decreased in concentration with increase in the level of infestation ( Table 6). As mentioned before the number of larvae recorded on avocado fruit was much higher compared to other fruits under laboratory conditions ( Figure   1), and this may be due to the volatiles contents which are favourable for the growth of larvae as compare to other types of fruits. Some of the compound detected by [25] includes, hexyl acetate, 2-pinene, valencen and hexyl carproate.
Our results indicate that these volatiles levels, emitted from fruit with an early stage of larvae infestation can be detected by the HS-SPME GC-MS method. Recently, volatiles compound detection technology has been successfully used in different postharvest cases for early infested detection of insects and fungus.
Fruit infested with Medfly or other insect eggs, release unique volatile compound. These compounds can be exploited to provide tools for improved pest detection. Finally, this research provides the basis for determining the larvae infested by the HS-SPME GC-MS method. Also, it can be used to assess the applicability of this technology for detection of other species of fruit fly, different type of fruits and different number of larvae. We recommend the use this technology in quarantine areas or prior of the importation of fruit for early detection of any infestation in the fruits by fruit flies.