Abstract: The bacteria in fruit flies alimentary tract have been known in advantages in their biology of the insect hosts. Different biochemical, gram reaction and motility tests was performed to identify the mid-gut bacterial community of laboratory host reared pumpkin fly, Bactrocera tau (Walker) (Diptera:Tephritidae). Colony characterization of the isolated bacteria was conducted on nutrient agar and MacConey agar plates. Isolated gut-bacterial species viz., Proteus rettgeri and Klebsiella oxytoca was examined by incorporated with protein (casein:yeast extract:sugar, 1:1:2) and sugar diet to study the effect of bacteria supplemented diets on the ovariole number and egg production of B. tau. Eight genera and nine bacterial species were identified under the family Enterobacteriaceae. The identified bacterial genera were Proteus, Klebsiella, Streptobacillus, Alcaligenes, Haemophilus, Erwinia, Chromobacterium and Flavobacterium. The mean ovariole number per ovary was recorded as 20.66±2.51, 20.56±3.53 and 22.41±3.75 for B. tau fed on P. rettgeri, K. oxytoca incorporated protein diets and only protein diet, respectively. Experimental result revealed no significant influence of gut bacteria added adult diets on egg/female/day of B. tau fed on above mentioned diet treatments.
INTRODUCTION
Insects form an extremely large group of animals and bear a consequently large variety of associated microbes (Jurkevitch, 2011). Many insects species harvour microbial communities in their digestive system (Dillon and Dillon, 2004). Insect symbionts have been broadly categorized into two main types: primary and secondary symbionts. In many cases, the relationship is mutualistic and the bacteria contribute to the fitness of their insect host (Baumann, 2005; Bourtzis and Miller, 2003; Dillon and Dillon, 2004). Bacterial contribution to fitness, particularly in insects that rely on inadequate food source, is frequently nutritional. These microorganisms may provide certain amino acids (Nogge, 1981), essential vitamins (Douglas, 1998), nitrogen and carbon compounds (Benemann, 1973; Dillon and Dillon, 2004). A significant proportion of this bacterial community was found to actively fix nitrogen within the gut of live adults (Behar et al., 2005) and these could be supplementary their host diet with available nitrogen. De Vries et al. (2004) reported that the gut microbiota may have alternated between mutualism/commensalism and parasitism in response to changes in their host’s diet. Removal of bacteria affects measurable physiological and behavioural parameters related to fly fitness (Ben-Yosef et al., 2008) and conversely, inculcation of original bacteria in the diet contributes to a longer life span (Behar et al., 2008b) and enhanced mating competitiveness (Ben Ami et al., 2010). Some bacteria also provide resistance against natural enemies and parasites (Oliver et al., 2003), promote host immunity (Muniz et al., 2006) and enhance social interactions (Dillon et al., 2002).
Tephritidae is a large family that includes many fruit pests and these are usually adopted for housing large quantities of bacteria in their digestive tract. The Petri (1909, 1910) described one of the first bacterial symbiotic associations in an insect species, the olive fly, Bactrocera (Dacus) oleae (Rossi). In the genus Bactrocera adult fruit flies use a combination of their fluid-centered mode of feeding and their labellar filtering mechanism to feed on fruit juices, leachates and bacteria (Enterobacteriaceae) which constitute their primary source of food in nature (Vijaysegaran et al., 1997). The Queensland fruit fly, Bactrocera (Dacus) tryoni (Froggatt) and Dacus cacuminatus (Hering), regurgitated and reingested alimentary canal contents which included bacteria (Drew and Lloyd, 1987). Explorations on different fruit fly’s associated bacterial community revealed that most of fly’s gut microbiota is dominated largely by free-living bacteria of the Enterobacteriaceae, notably by species of Enterobacter, Klebsiella and Pectobacterium (Behar et al., 2008b; Daser and Brandl, 1992; Drew and Lloyd, 1987). These components of the bacterial community remain stable throughout the fly’s life cycle and between geographical regions and some are inherited vertically (Aharon et al., 2013; Behar et al., 2008a; Sood and Nath, 2002).
Knowing the intestinal bacteria is important in the context of developing our understanding of symbiotic relationships, multitrophic interactions between insects and plant or animal host and in the developing new strategies for controlling insect pests (Dillon and Dillon, 2004). Behar et al. (2008b) reported that the Enterobacteriaceae community within the med fly’s (Ceratitis capitata, Wiedemann) gut have an indirect contribution to host fitness by preventing the establishment or proliferation of pathogenic bacteria. Hamden et al. (2013) proved that the addition of beneficial bacteria (Klebsiella pneumonia, Enterobacter spp. and Citobacter freundii) to the larvae’s diet of C. capitata lead to a significant increase in the number of Enterobacteriacae communities inhabiting the sterile male’s gut and a subsequent significant increase in the size of males and other morphometric traits and enhanced sexual performance of males at emergence. Community and functional analyses showed that the microbiota of med fly and olive fruit flies contribute to their diet and affect host fitness parameters. The analysis of the microbiota’s community structure of mass-reared, sterilized medfly males used in the sterile insect technique revealed a strong reduction in Klebsiella spp. compared with non-sterile and wild flies (Yuval et al., 2013). Inoculation of sterile males with the gut population affected female mating behviour as they preferentially mated with inoculated versus non-inoculated males. The studies suggested that control can be significantly improved by manipulating symbionts in pest animals.
The Pumpkin fly, Bactrocera tau (Walker) (Diptera:Tephritidae) is a polyphagous insect that seriously damages fruit production worldwide including Bangladesh. Females oviposit in unripe fruits, within which the larvae develop, leading to fruit degradation and crop losses. Infestation can lead to total crop failure and to quarantine restrictions. The control strategies remain almost exclusively based on insecticides, despite the awareness of a need for the use of more environment friendly control methods. Although, much research has already been done on the biology (Singh et al., 2010), host susceptibility and behaviour (Khan et al., 2011) and genetics/molecular phylogeny (Jamnongluk et al., 2003) of B. tau, yet relatively little is known of its microbial community (Prabhakar et al., 2009, 2013; Sood and Nath, 2002, 2005; Sood and Prabhakar, 2009) and their possible impact on fly fitness parameters.
The present study was therefore undertaken to identify the gut bacterial community of laboratory host reared female B. tau using different morphological, gram reaction and biochemical tests. The effect of gut bacteria incorporated adult diets (protein and sugar) on the ovariole number and fecundity of B. tau was also examined under controlled laboratory condition.
MATERIALS AND METHODS
Insect rearing: Adult B. tau culture originated in 2010 from infested sponge gourd, Luffa cylindsica (L.) collected from Atomic Energy Research Establishment (AERE) campus. Rearing of B. tau was maintained in the Laboratory of Insect Biotechnology Division, Institute of Food and Radiation Biology (IFRB), AERE, Savar, Dhaka, Bangladesh for more than 40 generations using natural hosts. About 3,000 adult flies were maintained in steel framed cages (76.2x66x76.2 cm) covered with wired net. The flies were supplied with protein based diets both in the liquid and dry form viz., (1) Baking yeast: sugar: water at 1:3:4 ratio and (2) Casein: yeast extract: sugar at 1:1:2 ratio. Water was supplied in a conical flask socked with cotton ball. The temperature and the relative humidity of the rearing room maintained at 27±1°C and 75±5% and 14:10 h dark and light cycle.
Isolation and identification of gut bacteria: The gut bacteria of B. tau were isolated and identified according to standard method (Cowan, 1981; Holt et al., 1994). Ten days old female B. tau was collected from laboratory host (sweet gourd) reared population. Flies were dipped in 75% ethanol for disinfection and washed in sterile saline buffer (PBS) prior to dissection. The flies were then cold anaesthetized at 4°C in a refrigerator for 10 min prior to dissection. Gut of the flies was dissected aseptically under laminar flow hood and mid-gut were collected in sterile 4 mL vial containing 2 mL of normal physiological saline. The collected guts were opened with forceps and homogenized with vortex. Homogenized suspensions were serially diluted up to 10-2 CFU mL-1. Approximately 100 μL of diluted suspensions were inoculated onto nutrient agar (Oxoid, UK) and MacConey agar by spread plate technique and incubated at 37°C for 24-48 h. On the basis of different colony characteristics about 20 representative colonies were selected for identification. Selected colonies were purified as pure culture through repeated subculturing onto nutrient agar plate and stored at 4-8°C. Gram staining and microscopy were performed to study the morphology of the collected isolates. Isolates were then tested for different biochemical characteristics e.g., sugar fermentation, catalase, oxidate, methyl red, voges proskauer, indol production, citrate utilization etc. Based on overall cultural, morphological and biochemical characteristics, isolated bacteria were identified as per Bergey’s Manual of Determinative Bacteriology (Holt et al., 1994). Among identified gut bacterial species, Proteus rettgeri and Klebsiella oxytoca were randomly selected to observe their impact on the ovariole number and fecundity i.e., egg/female/day of B. tau fed on bacteria incorporated adult diets.
Preparation of bacteria incorporated of adult diets: Two different gut bacteria viz., P. rettgeri and K. oxytoca were sub cultured on nutrient agar from maintenance media and then inoculated in a 250 mL conical flask containing 100 mL nutrient broth and inocubated at 37°C for overnight. Cell suspension was centrifuged at 6000 rpm for 10 min and washed twice with 0.01 M sodium phosphate buffer. Finally the cell pellets were suspended with 0.8% NaCl solution and aseptically transferred in test tubes. Cell suspensions were then mixed with protein diet for feeding newly emerged B. tau. In the present study newly emerged B. tau were given access to six diet treatments viz., (i) Only protein diet (yeast extract:casin:sugar, 1:1:2), (ii) Isolates of K. oxytoca and protein diet (3.8x10-6 CFU g-1), (iii) Isolates of P. rettgeri and protein diet (3.8x10-6 CFU g-1), (iv) Only sugar diet (20% sugar solution), (v) Isolates of K. oxytoca and sugar diet (3.8x10-6 CFU mL-1) and (vi) Isolates of P. rettgeri and sugar diet (3.8x10-6 CFU mL-1).
Determination of ovariole number of B. tau fed on bacteria added protein and sugar diets: Newly emerged 30 male and 30 female adult B. tau were housed in small rearing cages (8x6x12 cm) and supplied with diet treatments mentioned above. Three replicates were maintained for each diet treatment. On day 14 of adult emergence females were collected in small vials (5 mL) and kept in refrigerator for 10 min. Both the ovary of B. tau was dissected under stereo microscope and total number of eggs per ovary were counted and recorded. The ovariole development of only sugar fed and bacteria added sugar fed 14 days old adult flies was also determined. Usually, in poly-phagous tephritid fruit flies the paired ovaries consist of about 30-40 polytrophic ovarioles (Fitt, 1990). In mature flies, there is normally only one mature oocyte (egg with shell) per ovariole that is ready for laying, although there may be oocytes in various earlier stag.
Determination of egg per female per day: Three sets of 50 pupae of B. tau were collected from stock culture and placed in 55 mm plastic Petri dishes. Each Petri dish was then placed individually inside three small rearing cages and provided with three different types of adult diets viz., only protein, P. rettgeri+protein and K. oxytoca+protein diet. A plastic container of water with cotton wick was provided inside each cage. Fourteen days after adult emergence, ten male and female B. tau from each rearing cage was again placed into small rearing cages (6x6x10 cm). The flies were provided with three types of diets as mentioned above and water was supplied via a cotton wick inserted into a plastic vial (5 mL). Plastic egging receptacles smeared with sweet gourd paste were placed inside the cage for egg collection for 24 h. Eggs were collected for 5 days. Fecundity was determined by counting total number of eggs produced by ten pairs of B. tau and divided by 5 days to determine mean eggs per female per day. The experiment was repeated for three times.
Statistical analysis: Data for the ovariole number and fecundity of B. tau fed on different bacteria treated diets were analyzed using Analysis of Variance (ANOVA) and Tukey’s family error rate was performed using Statistical Software-Minitab USA (version-15). Graphs were created in Microsoft Excel 2007.
RESULTS
Gut bacterial community isolated and identified from ten days old adult female B. tau shown in Table 1. Bacterial species identified were Proteus rettgeri, Proteus vulgaris, Klebsiella oxytoca, Streptobacillus moniliformis, Alcaligenes faecalis, Haemophilus ducreyi, Erwinia herbiloca, Chromobacterium lividum and Flavobacterium picketti. All bacterial species belongs to the family Enterobacteriaceae. Total viable counts in the gut of B. tau were 10-5 CFU mL-1.
The influence of bacteria supplemented adult diets on the ovariole number of B. tau presented in Fig. 1. No significant differences (p>0.05) were recorded among ovariole numbers of B. tau fed on P. rettgeri and K. oxytoca added protein diets and only protein diet. Mean ovariole number were 20.66±2.51, 20.56±3.53 and 22.41±3.75 per ovary of B. tau fed on P. rettgeri, K. oxytoca incorporated protein diets and only protein diet, respectively. Individually ovariole number ranged from 7-34 per ovary depending on diet treatments of adult B. tau. No ovariole development was recorded for only sugar and bacteria added sugar fed 14 days old B. tau in the present study.
| Table 1: | Gut bacterial community of adult female B. tau |
| Fig. 1: | Mean (±) No. of mature ovariloes of 14 days old B. tau fed on P. rettgeri, K. oxytoca added protein diets and only protein diets, respectively |
| Fig. 2: | Egg/female/day of B. tau fed on P. rettgeri, K. oxytoca added protein diets and only protein diet, respectively. Bars with same letter did not differ significantly (p>0.05) |
The effect of bacteria added adult diets on the fecundity of B. tau shown in Fig. 2. Total egg production per female B. tau per day was 16±0.9, 15.5±0.5 and 14.5±1 in number for those fed on only protein, P. rettgeri+protein and K. oxytoca+protein diets, respectively and did not differ significantly (p>0.05).
DISCUSSION
The bacteria associated with different species of Tephritidae have been studied by several authors (Behar et al., 2008a, b; Prabhakar et al., 2013; Sacchetti et al., 2008; Sood and Nath, 2002; Wang et al., 2011). Several bacterial genera viz., Proteous, Klebsiella identified from gut of B. tau in the present study (Table 1) were quiet common for other Tephtriitd fruit flies like, Bactrocera dorsalis (Hendel), B. tryoni, C. capiata (Behar et al., 2008a, b; Thaochan et al., 2009, 2010; Wang et al., 2011). Lauzon (2003) stated that the gut microbiota of these flies is relatively conserved in terms of species composition and comprised mainly various Enterobacteriaceae. Daser and Brandl (1992) also isolated and characterized the gut bacteria from adults of five fruit fly species and noted most of the bacterial strains were Enterobacteria. The uniformity across genera in morphology and in the composition of the microbiota suggested that bacteria are intimately associated with the life cycle of these flies (Ben-Yosef et al., 2008). Using molecular, biochemical and 16S rDNA (rrs gene) analysis (Prabhakar et al., 2009) characterize three bacterial symbionts of B. tau viz., K. oxytoca, Pantoea agglomerans and Staphylococcus sp. to determine their taxonomic position. Sood and Nath (2005) reported that antibiotic resistant strains of Pseudomonas putida (fruit fly symbiont), Bacillus subtilis (fruit fly pathogen) and Escheichia coli (non associated strain) could colonize in the gut of B. tau. Prabhakar et al. (2013) characterized five bacterial spesies of B. tau as Delftia acidovorans, Pseudomonas putida, Flavobacterium sp., Defluvibacter sp. and Ochrobactrum sp. of which four bacterial isolates viz., Delftia acidovorans, Flavobacterium sp., Defluvibacter sp. and Ochrobactrum sp. are reported new records from guts of B. tau. Out of eleven types of bacteria isolated from B. tau and B. cucurbitae infesting cucurbits five were noted common to both the species, viz., Pseudomonas putida, Erwinia herbicola (Pantoea agglomerans), Cedacea davisae (Cedecea davisae), Arthrobacter sp. and Xanthomonas maltophilia (Stenotrophomonas maltophilia) (Sood and Nath, 2002).
In the present study gut bacteria of natural host (sweet gourd) reared female B. tau was identified by conventional morphological and biochemical tests. We revealed the presence of eight bacterial genera among which four viz., Streptobacillus, Alcaligenes, Haemophilus and Chromobacterium were different from those reported by earlier mentioned authours worked on gut bacteria of B. tau. It appears that bacterial assemblages in the gut of adult tephritids can be varied and inconsistence (Murphy et al., 1994). For instance K. oxytoca and K. pneumonae have been found in both wild and laboratory B. tryoni but not but (so far) together (Drew and Lloyd, 1987; Fitt and O’Brien, 1985). Different environmental conditions and food supply could influence the diversity of the harboured bacterial communities and increase community variations of most of the bacteria species (Behar et al., 2008c; Sood and Nath, 2002; Wang et al., 2011).
In many Tephritid species proteinaceous component is required in the diet for sexual maturation and oogenesis of adult female fly (Carey et al., 2000; Drew and Lloyd, 1987; Meats and Leighton, 2004). Drew et al. (1983) established that bacteria of the gram negative family Enterobacteriaceae could serve as an attractant and proteinaceous food for adult B. tyoni from a long term laboratory culture. Diets of bacteria, sugar and water gave equal longevity and increased fecundity in B. tryoni compared with the conventional diet of autolyzed brewer’s yeast, sugar and water. In the present study B. tau fed on P. rettgeri and K. oxytoca added protein diet and only protein diet did not show significant influence of bacteria on mean ovariole number (Fig. 1). The present findings is partially in agreement with the findings of Meats et al. (2009) who noted that B. tryoni could not produce eggs or mature oocytes on a bacterial diet above the level attained with access to culture medium without bacteria. The present findings also in agreement with the findings of Halder et al. (2013) who reported that bacteria added protein diet had no effect on ovariole number of B. tau. However the authors used exogenous bacteria species e.g., E. coli and Lactobacillus lactis while we used original gut bacteria of B. tau, K. oxytoca and P. rettgeri as probiotics. Bacteria had no effect on the ovarian development and egg production also revealed by several observations. Howard and Bush (1989) noted that the absence of the bacterium did not positively or negatively affect most components of fitness of Rhagoletis pomonella (Walsh) and R. suavis (Loew) and suggested that larvae of Rhagoletis do not depend on gut microflora to provide essential nutrients to detoxify plant secondary components. The bacterial community in the sand fly (Lutzomiya longipalpis) larval habitat affects oviposition and larval development, although bacteria are not essential for successful development of L. longipalpis (Peterkova-Koci et al., 2012). Ben-Yosef et al. (2010) evaluate the presence of bacteria in female olive flies and monitored fecundity-an indirect measure of fitness. The authors reported that bacteria did not affect fecundity when females were fed a nutritionally poor diet of sucrose, or a protein-rich, nutritionally complete diet. However, when females were fed a diet containing non-essential amino acids as the sole source of amino nitrogen, egg production was significantly enhanced in the presence of bacteria. Niyazi et al. (2004) reported a significant benefit of probiotic postteneral diets on aspects of behavioural ecology in sterile male C. capitata. The inconsistency among the results of different investigations by different authours may be due to differences of the effects of different bacterial species or different strains of the same species on different insect hosts.
The present experimental result on egg/female/day of B. tau fed on different bacteria added adult diets and only protein diet partially in agreement with the findings of Singh et al. (2010) who reported B. tau lay an average 16 eggs day-1. Addition of bacteria in protein diet did not show any effect on the fecundity of B. tau in the present trail (Fig. 2). Hendrichs et al. (2010) reported that egg development was not sustained by host foliage leachate, Bird droppings, ahid honeydew and to a lesser extend hawthorn fruit leachate, contributed to moderate fly fecundity, whereas preparation of leaf surface bacteria, pollen, insect frass and uric acid did not support significant egg development of R. pomonella. Fecundity was greatest where flies were exposed to enzymatic yeast hydrolysate. In our study we also recorded higher number of egg/female/day of fly fed on protein diet but almost similar from those fed on bacteria added protein diet. Bacteria added sugar diet fed fly produce no egg indicate that bacteria had no significant effect on the egg production of B. tau and in agreement with the observation of Ben-Yosef et al. (2008) who noted that female C. capitata feeding on full diet produce significantly more eggs than females on the sugar diet, but the presence of bacteria does not affect numbers of egg produced.
In conclusion, in the present study, a bacterial community composed of P. rettgeri, P. vulgaris, K. oxytoca, S. moniliformis, A. faecalis, H. ducreyi, E. herbiloca, C. lividum and F. picketti was revealed in the female gut of B. tau. Enterobacteriaceae constituted the dominant population. Use of selected gut bacteria, P. rettgeri and K. oxytoca in protein diet did not exert significant influence on the ovariole number and egg/female/day of B. tau. Further investigation on the mating competitiveness, longevity and attractancy of B. tau to different bacteria supplemented adult diets can be evaluated in both laboratory and semi field-cage trials and may lead to new target for management of this pestiferous fruit fly. Moreover, studies related to approaches as comparative genomics and real-time PCR can be performed to know the molecular mechanisms that underpin symbiont-host interactions.