Australian Journal of Basic and Applied Sciences

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1 AENSI Journals Australian Journal of Basic and Applied Sciences ISSN: Journal home page: Occurrence of Tephritid Fruit Flies with Intermediate Morphologies of Bactrocera Carambolae and B. Papayae (Diptera: Tephritidae) in Selangor, Peninsular Malaysia. 1 Salim Juma, 1 Rita Muhamad, 1 Nur Azura Adam, 2 Alvin Kah-Wei Hee, 1 Manjeri Gnanasegaram 1 Department of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia, Serdang UPM, Selangor Darul Ehsan, Malaysia 2 Department of Biology, Faculty of Science, Universiti Putra Malaysia, Serdang UPM, Selangor Darul Ehsan, Malaysia A R T I C L E I N F O Article history: Received 19 August 2014 Received in revised form 19 September 2014 Accepted 29 September 2014 Available online 22 October 2014 Keywords: Bactrocera carambolae, Bactrocera papayae, intermediates, methyl eugenol, starfruit A B S T R A C T Background: Tephritid fruit flies, Bactrocera carambolae (Drew and Hancock) and Bactrocera papayae (Drew and Hancock) belonging to the B. dorsalis complex are key pests of valuable fruits in Southeast Asia. Control of these pests is often limited by difficulties on their identification that have been increasing due to recent occurrences of species with morphological characteristics of B. carambolae and B. papayae (hereafter known as intermediates). Whilst field observations of mating between B. carambolae and B. papayae have not been documented, it was believed that this phenomenon had occurred through trapping of adult fruit flies with intermediate morphologies of the two mentioned species. This was also substantiated from results of laboratory crossing between B. carambolae and B. papayae. Objective: A study was conducted to investigate the occurrence of intermediates and its proportion from their parental stocks based on field collection of flies infested starfruits, Averrhoa carambola L. and methyl eugenol-baited traps. Results: A total of 7,144 Bactrocera fruit flies were collected from infested fruits and another 12,522 male flies from traps were collected. A high proportion of intermediates (63%) were trapped whilst 25% of the flies from infested fruits accounted for intermediates. Conclusion: This study suggested that B. carambolae, B. papayae along with their intermediates are pests of economic importance to starfruit in Malaysia. The study also has reported the predominance of B. carambolae from damaged fruits and intermediates from methyl eugenol trapping capture; indicating that B. carambolae is a major fruit fly species infesting starfruit however, intermediates are found in high proportion in fruit field environment AENSI Publisher All rights reserved. To Cite This Article: Salim Juma, Rita Muhamad, Nur Azura Adam, Alvin Kah-Wei Hee, Manjeri Gnanasegaram, Occurrence of Tephritid Fruit Flies with Intermediate Morphologies of Bactrocera Carambolae and B. Papayae (Diptera: Tephritidae) in Selangor, Peninsular Malaysia.. Aust. J. Basic & Appl. Sci., 8(17): , 2014 INTRODUCTION Many species of Bactrocera fruit flies (Diptera: Tephritidae) are major fruit pests in tropics and subtropics (Clarke et al., 2005: Drew et al., 2008). This includes certain members from the B. dorsalis complex that are the most damaging pests of valuable fruits in Southeast Asia especially Malaysia, Indonesia and Thailand (Clarke et al., 2005). Bactrocera dorsalis (Hendel) (Oriental fruit fly) has been studied widely and it has been identified as the major pest of starfruit in Malaysia (Clausen, 1965; Ooi, 1984; Vijaysegaran, 1984). Later on, this species was divided into Malaysia A and Malaysia B (Drew, 1991) or B. dorsalis A and B. dorsalis B (White and Elson Harris, 1992) and now are referred as B. carambolae (Drew and Hancock) and B. papayae (Drew and Hancock) respectively (Drew and Hancock, 1994). These two species are said to be endemic in Peninsular Malaysia (Tan, 1997). They occur in many fruit growing areas (Tan and Serit, 1994; Chua and Khoo, 1995), infest wide range of fruits preferably soft fruits such as starfruit, Averrhoa carambola L. and guava, Psidium guajava L. (Allwood et al., 1999), and greatly affect their production and quality. Besides, they also cause quarantine restriction in exporting of fresh fruits. Bactrocera carambolae and B. papayae that are widely distributed in Southeast Asia (Drew et al., 2008) together with B. philippinensis in Philippines (Schutze et al., 2013) and B. invadens in Africa (Khamis et al., 2012) are economically important and most closely related species among the members of B. dorsalis complex (Drew and Hancock, 1994; Lawson et al., 2003) and are suggested as one biological species (Schutze et al., 2013). A number of techniques have been employed in distinguishing these two species which are of global economic importance. These include adult morphology (Drew and Hancock, 1994), wings, aedeagal and aculeus Corresponding Author: Rita Muhamad, Department of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia, Serdang UPM, Selangor Darul Ehsan, Malaysia Tel: ; rita@upm.edu.my

2 610 Rita Muhamad et al, 2014 morphometrics, (Iwahashi, 2001; Drew et al., 2008; Ebina and Otho, 2006), genetics (Muraji and Nakahara, 2001; Smith et al., 2003), pheromonal and behavioural analyses (Tan and Nishida, 1996; Tan et al., 2013), host preference and geographical distribution (Drew and Hancock, 1994). B. carambolae by its black spot on the foreleg femora and patterns of wings costal bands and abdominal black bands is a morphologically distinct species from B. papayae but probably as a subspecies based on mating compatibility and progeny viability studies (Wee and Tan, 2005; Drew et al., 2008). Similarities in some morphologies (Drew and Hancock, 1994; Drew et al., 2008) and methyl eugenol attraction (Wee et al., 2002) are being observed between B. carambolae and B. papayae. In addition, the production of same sex pheromonal components (Wee et al., 2002; 2007) and presence of similar gene banding patterns (Yong, 1995; Muraji and Nakahara, 2002; Smith et al., 2003; Ebina and Otho, 2006; Schutze et al., 2012) among these sibling species have also been reported and are said to facilitate their mating success in producing fertile offspring (Wee and Tan, 2000; 2005), therefore, leading to occurrences of intermediates. Suspects for the presence of intermediates started from early 1990s then confirmed with laboratory crosses during 2000 s until now (Wee and Tan, 2000; Ebina and Otho, 2006; Schutze et al., 2013). So far however, there is no reliable evidence of field mating for these sibling species. In essence, the intermediate occurrence has increased the complications in morphological discrimination of B. carambolae and B. papayae, because some of their morphological features fall within intermediates. Diagnostic studies have been conducted; yet have not cleared the status of these B. dorsalis complex species due to their many molecular and morphological similarities (Wee et al., 2002; 2007; Schutze et al., 2012; 2013). In addition, information on field surveys on intermediates is scarce (Wee and Tan, 2005). Morphological identification remains fundamental and a preceding tool in the first step of discriminating insect species. Therefore, this survey was undertaken with the objectives to determine the occurrence of adult Tephritid fruit fly species with morphological characteristics between that of B. carambolae and B. papayae and their proportions from parental species population in the field. MATERIALS AND METHODS Sampling sites and time: In this study, two experiments were involved; first to incubate damaged fruits and second to set methyl eugenol traps in fruit orchards to obtain adult fruit flies. Each experiment was conducted at three sites; Universiti Putra Malaysia (UPM), Serdang, (here after known as UPM) (3 o 02 N, 101 o 42 E, 31 m), Department of Agriculture (DOA), Serdang (here after known as DOA) (3 o 02 N, 101 o 42 E, 31 m) and Semenyih (02 o 56 N, 101 o 52 E), all located in the state of Selangor Darul-Ehsan, Peninsular Malaysia. The orchards in UPM and DOA are institution research fields surrounded by many other fruit orchards such as starfruit (Averrhoa carambola L.), papaya (Carica papaya L.), guava (Psidium guajava L.), mango (Mangifera indica L.), durian (Durio zibethinus L.), jackfruit (Artocarpus heterophyllus Lam.) and chempedak (A. integer Thunb.). Semenyih is a plantation area with great diversity of vegetation and many large commercial fruit orchards particularly starfruit. The three sites had nearly similar ecological patterns in terms of natural vegetations. The sampling was carried out between March and June in 2012, a period when starfruits were abundantly available for sampling and development of fruit flies. Host fruit: The starfruit, A. carambola, was used in this study. The fruit is morphologically preferable for breeding of many Bactrocera fruit flies (Sauers-Muller, 2005), and it is available throughout the year for sampling and maintaining the field population of fruit flies (Galán Saúco et al., 1993). However, in Malaysia the major fruiting seasons of the starfruit are between April-June and October-December of each year (Ministry of Agriculture and Agro-Based Industry Malaysia, 2004). Sampling procedures: Experiment 1: Collection and incubation of sample fruits to obtain fruit flies: Forty fruits were randomly collected from 20 trees (2 fruits/ tree) in one hectare of each orchard. Fruits were then packed in small boxes in single layers each with ten fruits in trays with vermiculite as soil mimics for pupation of dropped larvae than transported to the insectarium for incubation. From each orchard, two collections on bimonthly intervals were made to obtain adequate number of fruit flies. Each sampled fruit was kept in plastic containers over 2 cm layer of vermiculite (a soil mimic for pupation of fruit fly larvae) that was regularly moisturized with sterile water and incubated under ambient conditions of o C, 90-99% RH and 12:12 D: L photoperiod ranges for about days (Christenson and Foote, 1960) for development of fruit fly eggs to adults. The containers were covered with nylon cloth at the top for easy feeding, ventilation, and preventing jumping larvae and emerging flying adult fruit flies from escaping as well as preventing the entry of predating lizards. The containers were placed on bench with legs placed on a plastic

3 611 Rita Muhamad et al, 2014 pan smeared with petroleum jelly to prevent immature and adult emerged flies from ant predation. At two days before expected date of adult emergence, pupae were carefully separated from the vermiculite using spatula, placed in Petri dishes containing moistened vermiculite and further incubated in respective containers for adults to emerge. Food (sugar to brewer s yeast (3: 1)), and water (Walker et al., 1997) in soaked sponge were placed on floor of the container to raise emerged adults of fruit flies. Food and water were replenished using new sponges after every 2 days until adults became fully matured and coloured for easy identification. The fruit flies were then further preserved in 70% ethanol for identification. Experiment 2: Capture of male fruit flies using methyl eugenol traps: Three starfruit trees were selected randomly from one hectare of each orchard for sampling. The sampling trees were about 50 m away from each other, to prevent lure interference among the traps. Three traps baited with methyl eugenol insecticidal lure (1 ml in soaked cotton wool) with one trap per tree were set hanging on each of the sampling trees, m above the ground level in shades using metal wire hangers. The hangers were smeared with petroleum jelly to prevent caught flies from ant predation. The traps were made from mineral water plastic bottles (1.5 l), perforated with twelve sided holes of 2 cm 2 around the middle of bottle. In addition, few small holes (not large to prevent fruit fly escape) were randomly made at the bottom of each trap to allow entered rain water to drain. From each location, two collections were made at bimonthly intervals to obtain adequate fruit flies and the traps were set three weeks before collection. The lure in each trap was changed using a new cotton wick after every collection, and captured flies were stored in 70% ethanol for identification. Identification of collected fruit fly species: The adult fruit flies were individually examined under dissecting stereo microscope and image analyzing microscope (Olympus S2 X 10, Japan) connected with camera (Olympus E 450, Japan). In addition, a computer based key tool (CD-ROM) referred from Lawson et al., (2003) with various species of Bactrocera and their associated information on morphological features, hosts and geographical distribution was used. The morphological diagnostic features of each fruit fly were observed and compared with those described in taxonomic keys of Drew and Hancock (1994) and Lawson et al., (2003) for identification to species (B. carambolae and B. papayae). Intermediates were those species with characteristics from both B. carambolae and B. papayae or characteristics in between the two species. Sample images of each species of identified fruit flies with its respective important distinctive features were also taken using Dino-Lite Premier digital microscope (AnMo Electronics Corp. Taiwan). Statistical analysis: Both experiments were conducted based on Completely Randomized Design (CRD). Analysis of Variance (ANOVA) was used to compare the number of total fruit flies of each species as well as the total number of male and female fruit flies collected from each location. Treatment means with significance difference were separated by Tukey s (HSD) Test. All analyses were performed using Statistical Analysis Software (SAS) 9.2. (SAS Institute Inc., 2008). Results: In the present study, two B. dorsalis complex species; B. carambolae, B. papayae along with their intermediates were identified from the incubated damaged fruits collected from different sampling locations. In addition to the above mentioned species, few specimens of Bactrocera umbrosa Fabricius were collected from the methyl eugenol trapping. Figures 1-3 represent species of identified fruit flies with their respective distinctive features. Table 1 shows the mean number of recovered fruit flies of different species per fruit at three locations. Significant differences were observed among the number of fruit flies of different species in UPM (F = 26.7, P < 0.001), and DOA (F = 80.6, P < 0.001), while no significant difference (F = 0.7, P > 0.5) was observed among species in Semenyih. In UPM, the number of B. carambolae (17.6 ± 1.6) was significantly higher than B. papayae and intermediates. Similarly number of intermediates (11.2 ± 1.4) recorded was significantly higher than B. papayae (4.9 ± 0.4). In DOA, significantly higher number of B. carambolae (29.3 ± 2.6) was observed compared to B. papayae and intermediates. Bactrocera papayae (3.7 ± 0.4) and intermediates (6.3 ± 0.6) showed no significant difference (P > 0.05) in number. Regarding the total number of fruit flies of each species, results show that, the B. carambolae collected were significantly higher in number at 59% ± 4 followed by intermediates (25% ± 1.9) and B. papayae (16% ± 0.9) (F = 75.5, P < 0.001). Table 2 shows the mean number of total female and male fruit flies recovered from damaged fruits from different locations. The results show that in all locations the mean number of the females were observed to be insignificantly different (P > 0.05) to those of males.the mean number of total female to male fruit flies in UPM (16.8 ± 1.4: 16.9 ± 1.5), DOA (19.9 ± 1.6: 19.5 ± 1.7) and Semenyih (8.4 ± 0.7: 7.9 ± 0.7) were not significantly different (F = 0.00, P > 0.05, F = 0.03, P > 0.05 and F = 0.3, P > 0.05 respectively). In this study, a

4 612 Rita Muhamad et al, 2014 total of 7,144 fruit flies emerged from recovered pupae from sampled fruits with the average of 29.8 per fruits of which the mean number of females (15.08 ± 0.8) and males (14.7 ± 0.9) were also not significantly different (F = 0.06, P > 0.05). Fig. 1: Bactrocera carambolae. (a) B. carambolae whole body (male), dorsal view. (b) B. carambolae whole body (female), dorsal view. (c) B. carambolae wing. (d) B. carambolae fore leg femora. (e) B. carambolae abdomen, dorsal view. Fig. 2: Bactrocera papayae. (a) B. papayae whole body (male), dorsal view. (b) B. papayae whole body (female), dorsal view. (c) B. papayae wing. (d) B. papayae fore leg femora. (e) B. papayae abdomen, dorsal view.

5 613 Rita Muhamad et al, 2014 Fig. 3: Intermediates. (a) Intermediate species whole body, lateral view. (b) Intermediate species wing. Table 1: Mean number of fruit flies of different species recovered from damaged fruit at different locations. Number of fruit flies ± S.E Fruit fly species UPM DOA Semenyih B. carambolae 17.6 ± 1.6a 29.3 ± 2.6a 5.9 ± 0.6a B. papayae 4.9 ± 0.4c 3.7 ± 0.4b 5.4 ± 0.5a Intermediates 11.2 ± 1.4b 6.3 ± 0.6b 4.9 ± 0.7a Note: Means in same column followed by the same letters are not significantly different (P < 0.05) by Tukey s (HSD) Test Table 2: Mean number of total female and males fruit flies at different starfruit orchard locations. Number of fruit flies ± S.E Gender UPM DOA Semenyih Female 16.8 ± 1.4a 19.9 ± 1.6a 8.4 ± 0.7a Male 16.9 ± 1.5a 19.5 ± 1.7a 7.9 ± 0.7a Note: Means in same column followed by the same letters are not significantly different (P < 0.05) by Tukey s (HSD) Test The data in Table 3 shows the mean number of fruit flies of different species captured in methyl eugenol traps at three locations. Significant difference (P < 0.05) was observed in number of fruit flies of different species collected from different locations. The number of intermediates captured from the methyl eugenol traps was significantly higher than B. carambolae, and B. papayae at all locations (P < 0.05). The mean number of intermediate collected from UPM, DOA and Semenyih were ± 62.7, ± 25.3 and ± 4.3 respectively. Moreover, the number of B. carambolae (88.2 ± 113.5) was significantly higher (P < 0.001) than B. papayae (24.7 ± 3.8) at DOA, however, no significant difference (P > 0.05) was observed between them at other two locations. Overall, from a total of 12,522 trapped fruit flies, the number of intermediates (62%) was significantly greater than B. carambolae (21%), and B. papayae (16%) (F = 45.4, P < 0.001). Table 3: Mean number of fruit flies of different species captured by methyl eugenol traps from different locations. Number of fruit flies ± S.E Species UPM DOA Semenyih B. carambolae ± 26.7b 88.2 ± 13.5b ± 29.7b B. papayae ± 23.9b 24.7 ± 3.8c ± 20.7b Intermediates ± 62.7a ± 25.3a ± 4.3a Note: Means in same column followed by the same letters are not significantly different (P < 0.05) by Tukey s (HSD) Test Discussion: The results have shown recovery of different Bactrocera complex species from incubated fruits indicating that starfruit is a major host of these fruit flies in Malaysia. The two fruit fly species; B. carambolae and B. papayae recovered from starfruit have been reported in earlier studies in Selangor (Vijaysegaran, 1984; Ooi, 1984; Yong and Khoo, 1994), and other parts of Peninsular Malaysia (Chinajariyawong et al., 2000; Wee and Tan, 2000). The observed morphological characteristics of B. carambolae and B. papayae were compatible with descriptions by previous studies (Drew and Hancock, 1994; Drew et al., 2008). Unlike B. papayae, B. carambolae displayed black spot on foreleg femora and costal bands broadened across the apex as also previously described (Drew and Hancock, 1994; Lawson et al., 2003). This highlights that B. carambolae can be

6 614 Rita Muhamad et al, 2014 differentiated from B. papayae as well the other two sibling species (B. dorsalis and B. philippinensis) that look more similar (Drew et al., 2008; Krosch et al., 2013; Schutze et al., 2013) based on its morphology. In addition, the development of fruit flies species with morphological intermediate forms from laboratory crossing of these two siblings of B. dorsalis complex species (B. carambolae and B. papayae) has been documented (Wee and Tan, 2005; Ebina and Otho, 2006). The mating compatibility among other closely related species of B. dorsalis complex such as B. dorsalis and B. papayae and B. philippinensis (Schutze et al., 2013) and other Bactrocera such as Bactrocera tryoni, Froggatt and Bactrocera neohumeralis, Hardy has been also reported. However, the percentage of intermediates of B.tryoni and B. neohumeralis was very low (below 5%) compared to their parental species (Pike, 2004). This highlights the increased occurrence of intermediates among Bactrocera fruit flies. However, no survey that studied mating of B. carambolae and B. papayae in the field has been found. Similarly, the fruit fly species trapped from methyl eugenol traps, they have also been previously reported in Malaysia (Wee and Shelly, 2013). In addition, it highlights the selective nature and specificity of methyl eugenol lure towards fruit fly species capturing (Wee and Shelly, 2013). The host plant species available in and around the sampling farms can determine the composition of fruit fly species caught by methyl eugenol traps, the starfruit has been mentioned being among the most preferable on for B. dorsalis (Sauers-Muller, 2005) and capture of B. umbrosa may be due to the presence of unbagged Artocarpus plant species such as jackfruit farms, Artocarpus heterophyllus Lam. and chempedak (A. integer (Thunb.), its main hosts (Tan and Lee, 1982). The results also showed the recovery of all most similar fruit fly species from all three sites in both experiments. This was probably due to the involvement of the same species of sampling fruit (A. carambola) collected from areas with nearly similar ecological conditions especially of similar natural vegetation as also suggested by Rousse et al., (2006) and Shelly and Edu (2010). However, differences in abundance of fruit flies for each species among the farms were observed. The higher number of B. carambolae compared to B. papayae recorded in all locations from this study is supported by the previous studies (Yong and Khoo, 1994; Chua and Khoo, 1995; Allwood et al., 1999) highlighting the significance predominance of B. carambolae. This further suggests that this species is prevalent on starfruit in Selangor, Malaysia compared to B. papayae and intermediates. Similar to results of this study, the insignificant difference between the number of female and male B. dorsalis fruit flies from different infested fruits has been reported (Vijaysegaran, 1984; Ooi, 1984; Serit and Tan, 1994), however, no study was found in literature that explains the difference between female and male numbers among the species of B. carambolae, B. papayae and their intermediates. Female fruit flies cause fruit damage through oviposition; therefore, sorting the emerged flies by their sexes may provide important information in explaining their infestation level to the fruits. This study reports the variation in abundance of captured fruit fly species in methyl eugenol traps at different localities with intermediates > B. carambolae > B. papayae for all three sites. The large area and availability of various host plants of fruit flies due to powerful attraction of methyl eugenol may have possibly contributed to the higher capture of intermediate fruit fly species in methyl eugenol. Similarly, it may be also has contributed in collection of higher number of fruit flies from the methyl eugenol trapping compared to the damaged fruits in all studied localities. It has been reported that males of B. dorsalis can detect methyl eugenol lure kept at a distance of 1 km (Steiner, 1952), and usually move towards it for mating and food (Tan and Nishida, 1996). Serdang area is full of orchards of various fruit species. These results differ from that obtained from the damaged fruits that recorded majority of B. carambolae, as the experiment involved a limited area of sampling with only starfruit in each orchard. Conclusion: The study results suggested that B. carambolae, B. papayae along with their intermediates are pests of economic importance to starfruit in Malaysia. It also supports other studies for the existence of intermediates and for the consistently mean number of males and females of B. dorsalis fruit flies (1: 1) developed from the infested fruits. The study also has reported the predominance of B. carambolae from damaged fruits and intermediates from methyl eugenol trapping capture indicating that, B. carambolae is a major fruit fly species infesting starfruit however, intermediates are found in high proportion in fruit field environment. The intermediates showed many variations in morphologies from their parental siblings. The results suggest that B. carambolae and B. papayae, morphologically are different but biologically are the same species. Therefore, the similar management strategies from IPM can be applied by the farmers to control these Tephritid fruit flies. Further studies should be focused on field mating occurrence between these sibling species. ACKNOWLEDGEMENTS We express our sincere thanks to the World Bank through the State University of Zanzibar Staff Development Plan under the Higher Science and Technical Education Development Project Tanzania (SUZA/30/2/3) for the financial support to Salim Juma. The assistance of the University Agricultural Park in

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