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Morphological and Biochemical Characterization of Bacteria Associated with the Developmental Stage of the Peach Fruit Fly, Bactrocera zonata (Diptera: Tephritidae)

Bacterial-insects associations including both intracellular and intercellular are very common in nature and known since the last century (Petri, 1909). Associations between them are close and complex ranging from parasitism to mutualism, with a long period of association and co-evolution history (Dillon and Dillon 2004; Dale and Moran, 2006). The bacterial communities in insects mostly reside in the gut, mycetomes, hemocoel and within the cells. The associated bacteria plays an important role in host’s nutrition, digestion, International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 9 Number 10 (2020) Journal homepage: http://www.ijcmas.com

protection, detoxification of insecticides, resistance to pathogens, and semiochemicals production (Dillon and Dillon 2004;Russell and Moran 2006;Oliver et al., 2003;Engel and Moran 2013;Feldhaar, 2011;Douglas, 2015;Hammer and Bowers 2015;Ezenwa et al., 2012;Wingfield et al., 2016;Hosokawa et al., 2017). The bacterial community associated with the insects is likely to be influenced by various factors such as diet and the host on which insects feed (Ferrari et al., 2007;Hosokawa et al., 2017;Wagner et al., 2015;Medina et al., 2011). The variations in the bacterial communities have been reported at different stages of insects' development (Aharon et al., 2013;Malacrinò et al., 2018).
The Tephritid (Dipteran : Tephritidae) fly are commonly known as "fruit flies". The peach fruit fly, Bactrocera zonata (Saunders.) is a polyphgous pests, which is a menace to more than 50 species of fruit crops and wild plants (Duyck et al., 2004) particularly in South Asia (including India), Southeast Asia and some parts of Africa (Kapoor, 1993, CABI/EPPO 2013. B. zonata is considered as one of the most destructive fruit pests species of peach (Prunus persica (L.) Batsch), guava (Psidium guajava L.), mango (Mangifera indica L.) in tropical and subtropical climatic countries (Choudhary et al., 2015). Instead of enormous damage potential of B. zonata, the studies have been limited to its biology, distributions and population genetic structure (Duyck et al., 2004;Choudhary et al., 2015;Choudhary et al., 2018).
To the best of our knowledge, very few studies have been carried out to access the bacterial communities associated with B. zonata adult flies using culture-dependent techniques (Reddy et al., 2014;Naaz et al., 2016) and culture-independent techniques across ontogeny . However, there are no previous reports on the characterization of the cultivable gut bacterial communities associated with the B. zonata across different developmental stages. The present study was undertaken to isolate and morphologically as well as biochemically characterize the dominant bacteria associated with the different developmental stages B. zonata. This information will act as baseline for subsequent studies such as roles of these microorganisms in their development and management.

Sample collection and rearing
Fruit flies infested wood apple (Aegle marmelos L.) fruits were collected from Research farm of ICAR Research Complex for Eastern Region, Research Centre (ICAR-RCER, RC), Ranchi, India (23° 45′N latitude, 85° 30′ E longitude, elevation 620 m AMSL) in May, 2018. Subsequent rearing (up to 6 generations) was carried out in the laboratory at ambient room conditions (25±1°C temperature; 65±5% RH; 12:12 h LD photoperiod). 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 pair (male and female) of B. zonata was released into a smaller rearing cage (30×30×30 cm) provided with their natural host (Bael) 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. After oviposition, the 1 st instar larvae were directly collected by cutting the infested fruits of wood apple with fruit fly. Different stages of B. zonata were reared on same host fruits in the laboratory conditions according to protocol described by  and collected for further experiments. Larvae that emerged from the fruits and moved out to the sand for pupation were collected for 3 rd instar larval stage. A fair number of 3 rd instar larvae were left in the soil to pupate in order to obtain pupal stage samples and other pupae were left for the emergence of adult stage. Five days old adult male and females were collected to study the microbiota associated with the adult stage.

Dissection and isolation of bacteria
The bacteria were isolated from gut of first instar larvae and third instar larvae, mid-aged pupae (whole-body) and 5-day-old male and female adults of B. zonata, following the protocol described by Liu et al., (2016). Before being dissected, adults were anaesthetized at −18°C for 5 min. Samples (larvae, pupae and adults) were washed with ethanol (75%) for 30 s, followed by sodium hypochlorite (1%) for one minute and finally rinsed with sterilized water for three times to remove surface contamination. Five individuals of larvae, five male and five female flies were dissected aseptically in a petri dish under a stereoscope, and then all the dissected tissues and five pupae were placed in a 1.5 mL centrifuge tube containing 400 μL of sterile water, respectively. The tissue suspension was blended under a laminar air flow hood. There were three biological replications for each treatment. To isolate the cultivable gut bacteria, the blended samples were diluted with four concentrations (10 −1 , 10 −2 , 10 −3 , 10 −4 ). One hundred microliters of diluent was separately spread onto PYEA (Peptone yeast extract agar) and NA (Nutrient Agar) medium and incubated at 37±1°C for 24-48 h for bacterial growth. 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).

Isolation of bacterial isolates associated with the developmental stage of the peach fruit fly, Bactrocera zonata
Bacteria associated with the different developmental stage (First Instar larvae, Third Instar larvae, Pupa, male and female Adult) of the peach fruit fly, Bactrocera zonata were isolated on two culture media viz. Peptone Yeast Extract agar (PYEA) and nutrient agar (NA) media (enriched culture media). It was found that bacteria were associated with all the developmental stage of the B. zonata. A total of 35 different bacterial colonies were observed on two different enriched culture media (Table 1).

Morphological characterization
The bacterial isolates isolated from the different developmental stages of B. zonata were mostly rod shaped except two bacterial isolates namely BL1G and Bl3D were coccus and BM5 was Rod-coccus shaped isolated from larval stage and male adult flies. All the bacterial isolated were Gram-negative but few isolates such as BL1E and BL1G in first instar larvae, BL3B and BL3H in third instar larvae, BM4 and BM5 in male adult flies and only BF5 in female adult flies were Grampositive in nature ( Table 2). Most of the isolates were found non-motile, except few isolate in each developmental stage i.e. BL1B and BL1E in first instar larvae, BL3E, BL3F and BL3G in third instar larvae, BP2, BP3 and BP6 in pupa, BM1 and BM4 in male adult flies and BF2 and BF4 in female adult flies were motile. On the basis of growth in broth, all the bacterial isolates of different stages have sediment growth while only bacterial isolate BM5 showed pellical formation in broth medium.

Biochemical characterization
In first instar larvae, Isolate BL1A and BL1C gave a positive reaction for citrate, VP, Indole, catalase and carbohydrate fermentation whereas, a negative reaction for MR, oxidase and TSI test. However, isolate BL1D have similar results for all the biochemical tests except indole negative. Isolate BL1B was citrate, catalase, oxidase and D-glucose positive while MR, VP, Indole, TSI, and gas production in glucose medium negative. A positive reaction for catalase, VP and D-glucose was observed for isolate BL1E and it was negative for MR, Indole and gas production in glucose medium while doubtful for oxidase, citrate and TSI tests. BL1F gave a positive reaction for citrate, MR, Indole, catalase and D-glucose whereas, a negative reaction for VP, oxidase and TSI test. The only cocci isolate BL1G gave positive reaction for VP, D-glucose and few other carbohydrates whereas, a negative reaction for citrate, Indole, catalase, oxidase and TSI test (  Table 2).

Identification of bacterial isolates associated with the developmental stage of the peach fruit fly, Bactrocera zonata
The bacterial community associated with the developmental stage of the peach fruit fly isolated by culture-dependent technique were morphologically and biochemically characterized and belongs to various genera different family. The total 35 different bacterial colonies on the basis of colony morphology were screened and out of which, thirteen different bacterial species belonging to eleven genera (seven families) were identified from the developmental stage (first Instar larvae, third Instar larvae, pupa, male and female adults) of Bactrocera zonata ( were identified to be associated with different life stages. Out of total bacterial isolates, seven bacterial species from first instar larvae, nine from third instar larvae, seven from pupae and six each from male and female adult flies were identified. Genera Klebsiella, Enterobacter and Pseudomonas were found in all the development stages. To the best of our knowledge, this study is the first of its kind to explore the cultivable bacterial community in the different life stages (larvae, pupae, male and female adults) of B. zonata by conventional method. The gut of tephritid fruit flies in general (Behar et al., 2009;Lloyd et al., 1986;Wang et al., 2011) and B. zonata in particular (Reddy et al., 2014;Naaz et al., 2016;Naaz et al., 2020 (Drew and Lloyd 1987), Bactrocera (Capuzzo et al., 2005;Prabhakar et al., 2009;Shi et al., 2012;Wang et al., 2011Wang et al., , 2013, Anastrepha (Kuzina et al., 2001) and Ceratitis (Behar et al., 2008). These findings show that such bacteria are widespread in tephritids, suggesting a stable association with these fruit flies (Jang and Nishijima 1990;Lauzon et al., 2009;Wang et al., 2011).
The bacterial species Enterobacter sp., K. oxytoca, K. pneumoniae and Pseudomonas sp., were common across all the developmental stages of B. zonata suggest the possibility of vertical transmission of these bacterial communities. Vertical transition is common for some of the bacterial communities in tephritid flies (Andongma et al., 2015;Lauzon et al., 2009;Naaz et al., 2020).

Identified as
Bacillus sp. Recently, It has been also 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). Among all the common bacterial species across developmental stages, Pseudomonas sp. was not reported earlier to be associated with B. zonata. However, genus Pseudomonas have been reported from many insect species including fruit flies such as Bactrocera dorsalis, Zeugodacus tau (Brinkmann et al., 2008;Sood and Nath, 2002;Prabhakar et al., 2013, Noman et al., 2020.

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Earlier, K. oxytoca, K. pneumonia, Enterobacter cloacae, Enterobacter sp., Bacillus sp., Microbacterium sp., and Rhodococcus sp. were also reported from the gut of B. zonata (Reddy et al., 2014;Naaz et al., 2016) whereas, Citrobacter freundii, Providencia sp., Pantoea sp., Pseudomonas sp., Enterococcus sp., and Ochrobactrum sp. were newly added in the list of bacterial community associated with the B. zonata. However, genera Citrobacter and Providencia were identified from four Bactrocera species (Lloyd et al., 1986) and B. dorsalis (Gujjar et al., 2017). The bacterial species of genus Pantoea, is a free-living diazotrophic bacterium of family Enterobacteriaceae and was consistently reported from the different organs and stages of Zeugodacus cucurbitae and Z.tau (Sood and Nath 2002;Prabhakar et al., 2009). Ochrobactrum sp. have been reported from many insect species including fruit flies such as B. dorsalis and Z. tau (Brinkmann et al., 2008;Sood and Nath, 2002;Prabhakar et al., 2013, Noman et al., 2020. Thus the present findings get substantial support from the observations of other workers, who consistently observed the association of phylum, Proteobacteria with different organs and developmental stages of other Bactrocera species (Sood and Nath, 2002;Prabhakar et al., 2013, Gujjar et al., 2017, Noman et al., 2020 including B. zonata (Reddy et al., 2014;Naaz et al., 2016;. The present finding added a few more genera to the list of bacterial diversity of the B. zonata, but a vast range of gut bacterial diversity exists in the B. zonata system is still unkown. Therefore, an extensive study to explore the gut bacterial diversity across all the developmental stages of the fly and to understand the host behavior in relation to gut bacterial community are needed using both culture-dependent and -independent techniques (with large number of replicates). However, our study significantly supplements to the available information on the cultivable bacterial diversity associated with the developmental stages of B. zonata. bacteria interactions. Molecular Ecology, 17, 1375-1386. Brinkmann, N., Martens, R., and Tebbe, C.C. (2008. Origin and diversity of metabolically active gut bacteria from laboratory-bred larvae of Manduca sexta (Sphingidae: Lepidoptera: Insecta). The mitochondrial genome of the peach fruitfly, Bactrocera zonata (Saunders) (Diptera: Tephritidae): complete DNA sequence, genome organization, and phylogenetic analysis with other tephritids using next generation DNA sequencing. Gene, 569, 191-202. Dale, C., and Moran, N.A. (2006). Molecular interactions between bacterial symbionts and their hosts. Cell, 126, 453-465.