Introduction

The class Insecta, composed of over a million species, is regarded as the most diverse group of animals found almost in every habitat (Vilmos and Kurucz 1998; Park et al. 2004). Insects are estimated to harbor endosymbionts including viruses, bacteria, fungi, protozoa, nematodes and multicellular parasites (Buchner 1965; Douglas 1998; Moran et al. 2005). Associations of insect with micro-organisms are complex and intimate ranging from parasitism to mutualism, with a long period of association and co-evolution history (Dillon and Dillon 2004; Dale and Moran 2006). Interactions between hosts and their microbes can be parasitic, such as the interaction of the bacterium, Paenibacillus larvae (American foulbrood) in honeybees (Schmid-Hempel 1998; Müller et al. 2015) and mutualistic, such as the interaction between termites and their gut microbes (Breznak and Brune 1994; Schmitt-Wagner et al. 2003). Compared to mammals, there is a lack of literature regarding the functions of microorganisms in insect guts but in several insect species, such as desert locusts and termites gut microorganisms have been extensively studied (Dillon and Charnley 2002; Ohkuma 2003; Hongoh et al. 2005).

Tephritid flies are commonly known as “fruit flies” because a number of species infest a wide variety of fruits and vegetables in tropical and subtropical regions of the world. Symbiotic associations between tephritids and their gut bacteria have been studied well particularly for viz., Anastrepha, Bactrocera, Ceratitis and Rhagoletis (Kuzina et al. 2001; Raghu et al. 2002; Petri 1909; Capuzzo et al. 2005; Kounatidis et al. 2009; Daser and Brandl 1992; Lauzon et al. 1998; Lauzon 2003; Prabhakar et al. 2009, 2013). Among Bactrocera, the olive fly Bactrocera oleae (Rossi) (Subfamily Dacinae), was the first species for which a bacterial symbiosis was described (Petri 1909). A range of bacteria belonging to different genera viz. Acetobacter, Agrobacterium, Arthrobacter, Bacillus, Citrobacter, Defluvibacter, Delftia, Enterobacter, Escherichia, Erwinia, Flavobacterium, Hafnia, Kluyvera, Klebsiella, Listeria, Lactobacillus, Micrococcus, Ochrobactrum, Pantoea, Pectobacterium, Pseudomonas, Proteus, Providencia, Staphylococcus, Stentrophomonas, Streptococcus, Raoultella, Serratia and Xanthomonas has been isolated and characterized from the gut of tephritid flies (Lloyd et al. 1986; Drew and Lloyd 1987; Jang and Nishijima 1990; Lauzon et al. 1998, 2000; Zinder and Dworkin 2000; Bergey et al. 2001; Kuzina et al. 2001; Marchini et al. 2002; Belcari et al. 2003; Behar et al. 2005, 2008, 2009; Capuzzo et al. 2005; Sacchetti et al. 2008; Kounatidis et al. 2009; Prabhakar et al. 2009; 2013; Crotti et al. 2010; Thaochan et al. 2010; Wang et al 2011; Reddy et al. 2014). These associated bacterial symbionts play a very important role in the host’s nutrition, development, reproduction, resistance to pathogens, and semiochemicals production (Brand et al. 1975; Brune 2003; Moran et al. 2005; Dillon and Dillon 2004). They also provides digestive enzymes or vitamins (Nakabachi and Ishikawa 1999) lacking in fruit tissues of their host and improves digestion efficiency which are ultimately manifested in the development, fecundity, and survivability of their hosts. Furthermore, cultivable gut bacteria have also been implemented in pest management strategies; as bacteria were found to be involved in the degradation of the toxic compound ingested by the host insect leading to insecticide resistance (Eutick et al. 1978; Fukatsu and Hosokawa 2002; Bousch and Matsumara 1967; Prabhakar et al. 2008). Certain components of bacterial odour play an important role in fruit flies behaviour in feeding or ovipositional stimulants (Drew and Lloyd 1987; Lauzon et al. 2000) and have also been exploited in the pest management strategies in the form of baits or traps (Sacchetti et al. 2007; Robacker 2007; Thaochan and Chinajariyawong 2011; Sood et al. 2010).

The peach fruit fly, Bactrocera zonata (Saunders) is a serious and polyphagous pest of fruit crops and wild plant species in many parts of the world (Duyck et al. 2004). At present, it is widely distributed in Bangladesh, Bhutan, India, Iran, Laos, Myanmar, Nepal, Oman, Pakistan, Saudi Arabia, Sri Lanka, Thailand, United Arab Emirates, Vietnam and Yemen from Asia; and part of Africa (White and Elson-Harris 1992; Kapoor 1993; Choudhary et al. 2012; 2015). Reddy et al. (2014) reported bacterial population of genus Bacillus, Enterobacter, Klebsiella and Stenotrophomonas from the gut of laboratory reared and field collected B. zonata (Reddy et al. 2014). Laboratory studies on these cultivable bacterial odours suggest their possible implications in fruit fly management programmes (Shi et al. 2012; Reddy et al. 2014). However, there is still a dearth of knowledge on the bacterial communities attracting B. zonata under field conditions. The cultivable gut bacteria in particular will be more useful than non-cultivable bacteria while searching for a good fruit fly attractant. In this study, the gut symbionts of B. zonata were isolated and characterized using culture-dependent methods and molecular technique (16S rRNA) for their identification and also to establish the phylogenetic position. In this study, we also evaluated the chemo-attraction potential of dominant cultivable gut bacteria of B. zonata male and female adult flies under field conditions.

Materials & Methods

Origin and initiation of stock culture of B. zonata

Bactrocera zonata adult flies used in this study were reared with infested mango fruits collected from the research farm of ICAR Research Complex for Eastern Region, Research Centre, Ranchi, India (23° 45′ N latitude, 85° 30′ E longitude, elevation 620 m AMSL). Subsequent rearing was carried out at ambient room conditions (25±1°C temperature; 65±5% RH; 12:12 h LD photoperiod) in the laboratory. The infested fruits were kept individually in 20 × 15 cm cage with 5 cm of thick sterile fine sand until emergence of the adults. The adults were identified on the basis of morphological descriptions given by Drew and Raghu (2002), Madhura and Verghese (2004) and Prabhakar et al. (2012) and a pair (male and female) of B. zonata was released into a smaller rearing cage (30×30×30 cm) provided with their natural host (Mango/Guava) for oviposition. The feeding was supplemented with adult diet [(glucose and protein hydrolyzate (Protinex®, Pfizer Ltd., India) in the ratio of 1:1 in Petri plates)] and water ad-libitum through soaked cotton swabs in a 50 ml beaker. The food supplements were replaced weekly. Six inbreeding generations were reared for isolation of predominant and closely associate bacterial symbionts.

Dissection of B. zonata and isolation of gut bacteria

The bacteria were isolated from the gut of 11 days old adult flies reared in laboratory as described by Lloyd et al. (1986). Before dissection, adults were anesthetized at -20°C for 5 min. Flies were surface sterilized with ethanol (70%) for 30 seconds followed by sodium hypochloride (0.25%) for one minutes and then washed three times with sterilized distilled water (SDW) to remove external contaminations. Five adults of each male and female were dissected aseptically with two pairs of sterilized tweezers in a plate containing physiological saline to remove fly gut under laminar air flow. Gut content was streaked separately on Peptone Yeast Extract Agar (PYEA) and Nutrient Agar (NA) for bacterial growth at 30±1°C for 48-72 h. A single colony of each of the bacterial isolates was separated with the inoculation loop and streaked onto respective PYEA and NA plates for their growth. Predominant bacterial isolates were obtained through repeated sub-culturing to ensure their purity. The purified bacterial isolates with respective medium were maintained on PYEA slants and/or plates at 4-8°C for further use.

Morphological and biochemical characterization of gut bacteria

Morphological (Shape, Gram's staining), cultural (Pigment production, growth in broth medium) and biochemical (citrate, methyl red, Voges–Proskauer (V.P.), triple sugar iron (TSI), catalase, oxidase and carbohydrate fermentation tests) characterization of the pure culture was done by standard techniques and isolates characteristics were compared with Bergey's Manual of Determinative Bacteriology (Holt et al. 2000).

Molecular characterization

Genomic DNA extraction

Total genomic DNA of each bacterial isolate was extracted following the method of Prabhakar et al. (2009). The 48 h old bacterial cultures were multiplied on Peptone Yeast Extract Broth (PYEB) and transferred to 1.5 ml microtube and spun at 10,000 rpm for 12 min. After discarding the supernatant, the microtubes containing bacterial pellets (approx. 50 mg) were immersed in liquid nitrogen container for one min and the pellets were ground to fine powder immediately using micro pestle. To this, 700 μl of cetyltrimethylammonium bromide (CTAB) extraction buffer was added and incubated at 65o C for 1 h in a water bath. An equal volume (700 μl) of chloroform: isoamyl alcohol (24:1 v/v) was added and contents were mixed thoroughly. Tubes were spun at 10,000 rpm for 12 min in high speed refrigerated centrifuge (REMI India) at 4°C. The aqueous phase was transferred to new tubes and 450 μl pre-chilled isopropanol was added and kept at -20°C for 20-30 min to precipitate the DNA. Tubes were then spun at 10,000 rpm for 12 min and supernatant was decanted. The DNA pellet was washed with 70 per cent ethanol (three times), dried and dissolved in 100 μl of Tris EDTA buffer (10mM Tris HCl and 147 1mM EDTA, pH 8.0). RNAse @ 10 μl/ ml (Hi-media, Mumbai, India) was added and emulsion was incubated for 30 min at 37°C. Genomic DNA obtained was visualized after electrophoresis in a 1.0% agarose gel in 1× TAE and stored at -20°C for further use in PCR amplification.

PCR amplification of 16S rRNA genes

The 16S rRNA universal bacterial primers fD1 (5’-AGAGTTTGATCCTGGCTCAG-3’) and rP2 (5’-ACGGCTACCTTGTTACGACTT-3’) (Weisburg et al. 1991) were used for Polymerase chain reaction (PCR), yielding an amplicon of approximately 1450bp. The PCR amplification was carried out in 0.2 ml PCR tubes with 25 μl reaction volume containing 10 ng of DNA template, 20 pmol of each primer in 25 mM MgCl2, 10 mM of each deoxyribonucleoside triphosphate (Fermentas), 5 units of taq polymerase (Fermentas) and 10X reaction buffer. Amplifications were performed using Flexigene 9700 thermal cycler (QIAGEN India Pvt Ltd.) with an initial denaturation step of 5 min at 94°C followed by 35 cycles at 94°C for 1 min, 55°C for 1 min, 72°C for 1 min 30 sec and a final extension step at 72°C for 5 min. The product was separated in a 1% (w/v) agarose gel in TAE buffer (40 mM Tris-acetate, 1mM EDTA). PCR products of 16S rRNA gene of three gut bacteria obtained through amplification with specific primers were freeze dried (CHRIST ALPHA I-2LD) and were custom sequenced (ABI PRISM 310 TM 165 Genetic Analyzer, Applied Biosystems, USA) using the same upstream and downstream primers (Xcelris Labs Limited, India).

Nucleotide sequence analysis

The sequences of different bacterial isolates were blasted using online NCBI Blastn program (http://www.ncbi.nih.gov/blast). Sequences with >98% identity, considered to be from same species and Sequences with >97% identity, considered to be form the same genera (Schloss and Handelsman 2005). Twenty-seven 16S rRNA sequences of different bacteria (free living and insect symbionts including fruit fly symbionts) of high sequence similarity were selected for sequence comparison from GenBank Nucleotide Database, NCBI. The selected sequences along with three submitted bacterial sequences were aligned into the MEGA 6.0 alignment utility (Tamura et al. 2013), and a multiple sequence alignment was constructed using ClustalW using default parameters. The phylogenetic tree was inferred using the neighbour-joining (NJ) algorithms (Saitou and Nei 1987) in MEGA 6.0 software with Burkholderia pseudomallei kept as outgroup, and the confidence level of the tree topology was tested using 1000 bootstrap replicates (Felsenstein 1985).

Population kinetics of bacterial symbionts within B. zonata

The bacterial flora of adult fruit flies as affected by their age and sex was studied by bacterial count of whole fruit flies in different age groups of both sexes by the serial dilution method procedure as followed by Prabhakar et al. (2009). Three pairs (male and female) of fruit flies were randomly picked for the study. The adult flies (male and female) were anesthetized using cold treatment at 4°C for 10 min and flies were surface sterilized in sequential washing with 70% (v/v) alcohol for 30 s, 0.25% (v/v) sodium hypochloride for 1 min and sterile distilled water (SDW) for 30 s. The sterilized flies were crushed individually in a test tube with a sterile glass rod in 1 ml SDW. The final volume was made to 10 ml by adding SDW (stock solution). The bacterial population in the whole fruit fly was enumerated on PYEA plates using the serial dilution method. A known quantity (0.1 ml) of bacterial suspension from different dilutions was spread on the PYEA plates with the help of glass spreader. The plates were incubated at 30°C for 48 hrs. The process was repeated three times for 1-,2-,3-,4-,5-,7-,9-,11-,13-, 15-,17-, 19- and 21 day old flies. Data obtained were expressed in colony forming units (cfu’s) after emergence of adult fruit flies.

Bioassay for attractancy of adult B. zonata flies to bacterial culture

The bacterial culture preparation and bioassay was performed as described by Martinez et al. (1994). In brief, bacterial isolates from the gut of B. zonata were inoculated to 100 ml of sterile PYE broth. The conical flasks were incubated in a shaker at 150 rpm for 6 days at 30±1°C. Bacterial cultures were centrifuged at 10,000 rpm for 15 min to separate pellet and supernatant. The pellet was discarded because most of the bacterial cells contained in the pellet were supposed to be less attractive, as shown in experiments described by Robacker and Garcia (1993).

The field bioassays were conducted in the mango orchard of ICAR Research Complex for Eastern Region, Research Centre, Ranchi, India during the month of June, 2014. Eight treatments were made comprising of three bacterial filtrates, three different combinations of bacterial filtrates, Methyl Eugenol (ME) trap and a control (uninnoculated PYE Broth). To test attraction of B. zonata adults under field conditions, 5 ml filtrate of each bacterium + 1 drop of Dichlorvos (Nuvan®) was soaked onto the cotton balls. These cotton balls were tied with the help of thread and passed through the hole of the trap (plastic container) and lid was closed. Each trap was a cylindrical plastic container with wide holes on the surface to allow the entry of fruit flies inside it, one side having a lid and a small hole on the opposite side to pass the thread holding bacterial filtrates. After that, these traps were placed in the field fastened well on lower branches, positioned 1.0 m above the ground keeping 20 m distance between each other in three replications. The observations were taken after 24 hrs, 48 hrs and 72hrs. The same procedure was repeated three times in the same orchard.

Data analysis

The statistical analyses of the results were performed using the SPSS 16 statistical software (SPSS Inc., Chicago, IL, USA, 2007). The number of fruit flies attracted towards the treatment sources in the field attraction bioassay was converted into percentages and compared by one-way analysis of variance (ANOVA) with square-root transformation to meet the homogeneity of variances. Significant ANOVAs were followed by t-tests for multiple comparisons of mean values (P≤0.05).

Results

Characterization of gut bacteria

The three dominant cultivable bacterial isolates (BZM1, BZM2 and BZM4) from the mid gut of B. zonata were isolated, characterized and analyzed for attractancy towards fruit fly, B. zonata. Characterization based on morphological and biochemical tests showed that the isolated colonies were belonging to various genera, including rods of both Gram positive and Gram negative. Bacterial isolates BZM2 and BZM4 were gram positive while BZM1 was gram negative in reaction. With reference to biochemical characteristics, isolates BZM2 and BZM4 showed negative reaction for V.P., indole, cellobiose, raffinose and gas production in glucose medium while, found positive for catalase, growth in 10% NaCl and D-sorbitol tests. Morphological and biochemical characteristics were compared with the Bergey’s Manual of Determinative Bacteriology (Holt et al. 2000) and the three bacterial isolates (BZM1, BZM2 and BZM4) were tentatively identified as Klebsiella sp., Microbacterium sp. and Rhodococcus sp., respectively (Table 1).

Table 1 Morphological, biochemical and molecular characteristics of predominant gut bacterial symbionts of peach fruit fly, Bactrocera zonata
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16S rRNA gene sequencingof three bacterial isolates viz., BZM1, BZM2 and BZM4, using eubacterial universal primer resulted in the sequences of length 1337, 1336 and 1340bp, respectively. Homology analysis of bacterial symbionts with the blastn program, GenBank, NCBI revealed that BZM1 showed maximum homology with Klebsiella oxytoca strain 127 (KF254665, E-value 0.0, Total score 2,379, Maximum score 2,379 and Maximum identity 99.47%) identified as Klebsiella oxytoca, whereas, BZM2 with Microbacterium sp. (JN867365, E-value 0.0, Total score 2,412, Maximum score 2,412 and Maximum identity 97.00%) and BZM4 with Rhodococcus sp. (EU741198, E-value 0.0, Total score 2,304, Maximum score 2,304 and Maximum identity 98.35%) were identified as Microbacterium sp. and Rhodococcus sp., respectively. 16S rRNA gene nucleotide sequences of these bacteria were submitted to GenBank database under accession number KR024407 (Klebsiella oxytoca), KR024408 (Microbacterium sp.) and KR024409 (Rhodococcus sp.).

The phylogenetic analysis also confirmed a similar relationship pattern of the three symbionts. The phylogenetic analysis with α-proteobacteria, Burkholderi pseudomallei as an outgroup placed Klebsiella oxytoca within gram negative γ-proteobacteria. Microbacterium sp. and Rhodococcus sp. were clustered under gram positive Actinobacteria group within the separate families, Microbacteriacea and Nocardiaceae, respectively (Fig 1).

Fig 1
figure 1

Neighbor-joining tree of bacterial symbionts of Bactrocera zonata based on 16S rRNA gene sequences. GenBank accession numbers are followed by names of bacteria. Burkholderia pseudomallei was taken as an outgroup. The arrows indicate gut symbionts of B. zonata in present study

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Population kinetics of bacterial symbiont of the fruit fly

Symbiotic bacteria were found associated with the newly emerged adult B. zonata flies and the total bacterial count in one-day-old flies were 7.00 x 106 cfu in male and 9.00 x 106 cfu in female. The cfu values indicated that there was a significant rise in bacterial population in the adult flies with the increase in age up to the 11th day (383.33 x 106 cfu in male and 436.67 x 106 cfu in female). After that, the bacterial population did not vary significantly and fluctuated to around 383.33 x 106 cfu and 436.67 x 106 cfu in male and female, respectively. The bacterial symbionts of adult fruit flies follow a characteristic S- shaped growth curve with respect to age of fruit flies in both male and female.

The population of gut bacterial isolates of B. zonata i.e. K. oxytoca, Microbacterium sp. and Rhodococcus sp. were increased significantly upto the 11th day and thereafter fluctuated between 168.00 x 106 to 182.33 x 106, 133.00 x 106 to 137.00 x 106, 53.67 x 106 to 58.00 x 106 cfu upto the 21st day, respectively. The characteristic S- shaped growth curves were obtained for the population of different bacteria with respect to the age of B. zonata (Fig 2).

Fig 2
figure 2

Growth curve of three bacterial symbionts and total bacterial population within the adult male and female B. zonata against the age of adult B. zonata

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Attractancy bioassay

The results of field evaluation of bacterial supernatant showed a considerable decrease in the number of B. zonata flies captured to bacterial attractants from 24 h to 72 h of observations. A significant difference was observed in the attractancy of male and female fruit flies at different time intervals under field conditions (Table 2). As described in the methods, the effect of this change in population size was eliminated by transforming the actual counts of flies captured in traps into percentages of flies captured after every 24 hrs. Bacterial isolate BZM1 (K. oxytoca) attracted the highest number of females (8.33±0.33%, 5.67±0.33% and 3.33±0.33%) and males (6.67±0.33%, 3.67±0.33% and 1.33±0.33%) at 24, 48 and 72 h intervals, respectively. Whereas, the maximum number of adult male flies were trapped on ME traps (36, 42 and 34 males at 24, 48 and 72 hrs interval, respectively). Combination of BZM1+BZM2 also showed similar trend in the attractancy of B. zonata flies. Over the course of time during 72 hrs of experiment, all the treatments containing bacterial supernatant, alone or in combinations, attracted more females of B. zonata than males (Table 2). This suggests that volatile(s) produced by these bacteria may contain chemical(s) that are more specific for attracting females.

Table 2 Number of adult flies of Bactrocera zonata captured in traps baited with bacterial supernatant from cultivable bacterial broth culture
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Discussion

In this study, morphological, biochemical reactions and 16S rDNA sequence analysis were used to identify dominant cultivable gut bacterial populations of the peach fruit fly, B. zonata. Earlier, many workers suggested that the nucleic acid sequences, mainly 16S rRNA genes, have proved to be an important tool to know the taxonomic position of the microbial community of insects (Brauman et al. 2001; Toth et al. 2001; Thaochan et al. 2010; Wang et al. 2011). The gut of tephritid fruit flies in general (Lloyd et al. 1986; Drew and Lloyd 1987; Belcari et al. 2003; Behar et al. 2005, 2008, 2009; Capuzzo et al. 2005; Sacchetti et al. 2008; Kounatidis et al. 2009; Crotti et al. 2010; Thaochan et al. 2010; Wang et al 2011) and B. zonata in particular (Reddy et al. 2014) is a store house of bacterial community. In this study, three dominant bacterial symbionts viz., K. oxytoca, Microbacterium spp. and Rhodococcus spp. were identified. The Gram-negative bacteria i.e. Klebsiella belongs to the family Enterobacteriaceae, while Gram-positive bacteria (Microbacterium sp. and Rhodococcus sp.) were from the families Microbacteriaceae and Nocardiaceae. Earlier, K. oxytoca, was also reported from the gut of B. zonata and Bactrocera tau (Walker) (Sood and Nath 2002; Reddy et al. 2014; Prabhakar et al. 2009). Moreover, Enterobacteriaceae have frequently been identified as the dominant species in the gut of several other tephritids, such as Dacus (Drew and Lloyd 1987), Bactrocera (Capuzzo et al. 2005; Prabhakar et al. 2009; Shi et al. 2012; Wang et al. 2011, 2013), Anastrepha (Kuzina et al. 2001) and Ceratitis (Behar et al. 2008). Recently, It has been postulated that gut enterobacteria are dispersed into the female reproductive system, where they are subsequently transferred to the eggs, then to fruit during oviposition and finally passed to the fly offspring (Behar et al. 2008; Shi et al. 2012).

Microbacterium spp. of phylum Actinobacteria was also identified in the 16S rDNA cloned libraries from the intestinal tract of laboratory-reared and field-collected populations of oriental fruit fly, B. dorsalis (Wang et al. 2011). In the present study, Rhodococcus spp., which belongs to family Nocardiaceae, was first time reported from the gut of tephritids. Ngugi et al. (2005) reported, Rhodococcus opacus a gram-positive resorcinol degrading bacteria in the gut of termite, Macrotermes michaelseni whereas two strain of Rhodococcus triatomae were isolated from a blood-sucking bug of the genus Triatoma (Yassin 2005). Vertebrate pathogenicity of Microbacterium sp. and Rhodococcus sp. cannot be denied as some of these bacterial species have earlier been reported as vertebrate pathogens (Laffineur et al. 2003; Muscatello et al. 2007). However, the present findings get substantial support from the observation of other workers, who reported the gut association of K. oxytoca and Microbacterium sp. with different species of tephritids.

The significant rise in total bacterial population within the adult flies shows up to the 11th day (383.33 x 106 cfu in male and 436.67 x 106 cfu in female) and did not differ significantly thereafter. The bacterial population reached its maximum on the 11th day, coinciding with the reproductive maturity of fruit flies (gonad development), indicating a possible role in meeting the protein requirements of the flies. Bacterial symbionts have a vital role in the adult survival (Capuzzo et al. 2005), nitrogen fixation and in pectin degradation (Behar et al. 2005). The bacterial cells may also be consumed as food, providing amino acids, supplementary nitrogen compounds and other nutrients which are scarce in fruits. The exact role of gut bacteria in the life-cycle of different fruit fly species is not well understood and further study is required.

According to the results of field studies, significant higher numbers of female flies were captured than male flies in all the bacterial based baits. The attractiveness of these isolates to fruit flies however suggests their possible role in the fruit fly nutrition and physiology. However, concrete and concentrated efforts across the fruit fly species are needed to elucidate the complex phenomenon, to draw conclusions. The present findings are similar with earlier reports where attraction of female flies was higher than male flies towards metabolites from bacterial isolates (Wang et al. 2012). Male B. zonata flies were strongly attracted by methyl eugenol (ME). ME is widely used as an attractant to control fruit flies (Hee and Tan 2004). ME attracted only males while in present study, metabolites produced by the cultivable bacteria of B. zonata trapped both females and males. The bacterial species are also known to produce volatiles as a metabolic by-product when cultured on medium (Kai et al. 2009). Thus, it can be drawn that bacteria emitting volatile chemical constituents play an important role in luring female flies in the field. To identify the bioactive chemicals in the metabolites of bacterial symbionts further, gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC) is required. With improvement of the emitted volatile compounds, novel compounds supplement to methyl eugenol even for female targeted trapping can be developed. The behavioral responses of B. zonata were documented by the field bioassays in this study. Consequently, the current study significantly supplements to the available information on bacterial isolates and also illustrates the potential to develop a female-targeted strategy to control this polyphagous pest as well as the expansion of our understanding of insect-bacteria symbiosis.