I1536 2442 10 131of Agricultural Technology, Walailak University, Nakhon Si Thammarat, 80161, Thailand 2Center for Agricultural Biotechnology (AG-BIO/PERDO-CHE), Thailand 3International Centre for the Management of Pest Fruit Flies, Griffith School of Environment, Griffith University, Nathan campus, Nathan, Queensland, 4111, Australia 4Griffith School of Environment, Griffith University, Nathan campus, Nathan, Queensland, 4111, Australia
Abstract Bacteria were isolated from the crop and midgut of field collected Bactrocera cacuminata Klebsiella oxytoca.Providencia rettgeri, Klebsiella pneumoniae ssp ozaenae and Serratia marcescens were isolated from B.tryoni only.
Using the molecular cloning technique that is based on 16S rRNA gene sequences, five bacteria classes were dignosed
Alpha-, Beta-, Gamma- and Delta- Proteobacteria and Firmicutes
including five families, Leuconostocaceae, Enterococcaceae, Acetobacteriaceae, Comamonadaceae and Enterobacteriaceae.The bacteria affiliated with Firmicutes were found mainly in the crop while the Gammaproteobacteria, especially the family Enterobacteriaceae, was dominant in the midgut.This paper presents results from the first known application of molecular cloning techniques to study bacteria within tephritid species and the first record of Firmicutes1983; Drew and Lloyd 1987; Fitt and OBrien 1985; Howard et al.1985; Kuzina et al.2001; Lloyd et al.
1986; Murphy et al.1994).
However, besides the Enterobacteriaceae, little is known of other species of bacteria in other families that may also inhabit the alimentary tract of fruit flies.Molecular approaches for the detection and characterization of microbes in other insect species have revealed considerable bacterial diversity (Friedrich et al.2001).For example, considerable research using both culture-dependent and culture-independent techniques has been conducted on diagnosing the gut bacteria of Coleoptera (Delalibera et al.2007; Vasanthakumar et al.2006, 2008).
Studies on the adult southern pine beetle,Dendroctonus frontalis, and the adult pine engraver, Ips pini, revealed that their bacterial gut communities have a relatively low species richness.
In the adult emerald ash borer, a Agrilus planipennis, more diverse bacterial community was detected and, in all three cases, a higher diversity of bacteria was detected by the analysis of 16S rRNA gene sequences of gut isolates.To date, recent molecular diagnostic techniques have not been employed to identify the alimentary tract bacteria of fruit flies.
This paper presents results of research carried out on alimentary tract bacteria of two Australian fruit fly species, Bactrocera cacuminata (Hering) and Bactrocera tryoni (Froggatt) (Diptera: Tephritidae), identifying the bacteria using both the API-20E system and 16S rRNA gene molecular analyses, and based on these results, an analysis of bacteria species diversity and community similarity in these two species of fruit flies.
Methods and Materials
Fruit fly collecting and handling Adult flies of Dissection and isolation of bacteria from the alimentary tract of fruit flies Five males and five females of each species of the field-collected fruit flies were killed immediately on return to the laboratory by freezing at -20 C for 3 min.
Flies were then surface sterilized by immersing in 70% ethanol for 1 min, 0.5% sodium hypochlorite for 1 min and then washed twice in sterile distilled water (modified from Lloyd 1991).The surface-sterilized flies were individually dissected under sterile distilled water in a sterile glass cavity block.
Before dissecting, the water in each glass cavity box was sampled and spread onto tryptone soya agar (TSA) (Oxoid) and peptone yeast extract agar (PYEA) (Oxoid, www.oxiod.com) and incubated at 35 C for 24-48 h to determine whether any contaminant bacteria were present.The fruit fly sample was discarded if contamination occurred on the media.Although PYEA and TSA are recognised as media that grow similar groups of microorganisms, it was decided to use both in this study to maximise the chance of isolating most of the bacteria species in the fly.
The crop and midgut of each fly were aseptically removed following the method described by Drew et al.(1983) and Lloyd (1991), and placed in a sterile 1.5 ml microcentrifuge tube and homogenized with a sterile inoculation loop.
These contents were spread onto TSA and PYEA and incubated at 35 C for 2448 h.All steps in the isolation procedure were performed in a laminar flow sterile 1.5 ml centrifuge tubes.DNA was extracted using a modification of the CTAB/phenol-chloroform DNA extraction protocol (Doyle and Doyle 1987).
Extracted DNA from the crop and midgut were combined and the fragments were amplified in a polymerase chain reaction (PCR) using the universal primers for bacteria, forward primer Y1 (5-TGGCTCAG AACGAACGCTGGCGGC-3) (Sigma, www.sigmaaldrich.com) and reverse primer Y2 (5-CCCACTGCTGCCTCCCGTAGGAG T-3) (Sigma) (Young, Downer & Eardly, 1991).The reactions were carried out in a 100 !l volume containing 2 !l of template DNA solution, 2 !M of the primer, 200 M of deoxynucleosidetriphosphate (Astral Scientific, www.astralscientific.com; Bioline, www.bioline.com) and 2 U of Tag DNA polymerase (Astral scientific, Bioline).The amplifications were performed using the following protocol: initial denaturation at 94 C for 5 min; 30 cycles of 45 s at 94 C, 40 s at 62 C and 2 min at 72 C, and final extension at 72 C for 10 min.After the reaction, 5 !l aliquots of PCR products were examined by electrophoresis in 1% agarose gel.
The PCR products were extracted with QIA quick gel extraction kit (Qiagen, www.qiagen) for ligation.
The ligations were performed in 10 !l containing 1 !l pDrive cloning vector (50 while on both media more bacteria species were recovered from the fly midgut than the crop
.A total of 125 bacteria
800014pricelistBioMerieux Pricelist 8 5 2009 to 8 4 2010Identification Products - Automated VITEKItem NoDescriptionUnits/PkgCatalogue PriceState of Ohio Price21341Gram Negative Card, VITEK 2 Compact20$193.20$140.7621342Gram Positive Card, VITEK 2 Compact20$193.20$140.76Yeast Card, VITEK 2 Compact20$193.20$140.76Bacillus Card, VITEK 2 Compact20$266.70$194.31Neiserria Heamophilus Card, VITEK 2 Compact20$266.70$194.31Anaerobe Card, VITEK 2 Compact20$266.70$194.31V1304BAC Bacillus Test Kit, VITEK Legacy 20$249.48$164.48V1305GPI Gram Positive Test Kit, VITEK Negative Multi Lang Test Kit, VITEK Legacy20$149.21$89.51 .45% Saline, 500ml, VITEK18$133.35$94.46 .45% Saline, 1000ml, VITEK12$127.05$90.10Test Tubes, Products - Manual APIItem NoDescriptionUnits/PkgCatalogue PriceState of Ohio Price20300API 20A, Anaerobe Idenification strips25$270.90$186.4120100api 20E,
Identification of Enterobacteriaceae and non-fermenting Gram-negative bacteria25$216.30$148.8420160api 20E, Identification of Enterobacteriaceae and non-fermenting Gram-negative bacteria100$811.65$558.4920701api Rapid 20E, 4-hour identification of CAMPY, 24-hour identification of Campylobacter species12$187.95$129.3310300api LISTERIA, 24-hour identification of all Listeria species10$130.20$89.5920300api 20A, 24-hour identification of Reagent, 5 ml2$26.25$18.0670422VP-1 Reagent, 5 ml + VP-2 Reagent, 5 ml1$50.40$34.6870402TDA, 5 ml2$26.25$18.0670520ERH, 5 ml1$12.60$8.6770510BCP, 5 ml1$13.65$9.3970380Zinc Dust, 10 g2$21.00$14.4570100Mineral Oil, 125 ml1$21.00$14.4570530XYL, 5 ml1$12.60$8.6720230NaCl 0.85%, 5 ml100$123.90$85.2670442NIT 1 + NIT 2 Reagent, 5ml4$47.25$32.5170900McFarland Kit1$61.95$42.63bioMerieux Pricelist 8 5 2009 to 8 4 2010VIDAS Assays and AccessoriesItem NoDescriptionUnits/PkgCatalogue PriceState of Ohio Price30700VIDAS LIS, Listeria assay60$784.25$372.4730704VIDAS LMO2, Listeria monocytogenes assay60$867.41$427.6330702VIDAS SLM, Salmonella assay60$477.02$211.0730435VIDAS ICS, Immunoconcentration Salmonella assay60$382.31$146.2342610VIDAS ICS Broth, Use with ICS Salmonella assay, 2 ml100$112.04$80.9530112VIDAS ECO, E.coli O157 assay30$435.44$203.5730122VIDAS UP, E.coli O157 H7 Rapid assay30$441.00$206.1730705VIDAS SET2, Staph Enterotoxin assay30$462.00$215.9930111VIDAS CAM, Campylobacter assay30$435.44$203.5730526VIDAS ICE, Immunoconcentration E.coli O157 assay30$294.53$137.6930224VIDAS LSX, Listeria Express Next Day Xpress Broth, 225 ml, for LSX assays 6$46.86$33.8642117Listeria Xpress Broth, 6ml, for LSX assays20$32.13$23.2133877Listeria Xpress Broth (for Swabs), 10ml, for LSX assays20$34.65$25.0333878Listeria Xpress Broth (for Sponges), 100ml, for LSX assays6$38.12$27.5430706VIDAS QCV Quality Control test60$123.59$90.04TEMPO Assays and AccessoriesItem NoDescriptionUnits/PkgCatalogue PriceState of Ohio Price80000TEMPO Control Kit cards, vials5$69.93$53.5580004TEMPO E.coli Enumeration, EC48$186.48$112.2080007TEMPO Total Viable Count, TVC48$186.48$112.2080002TEMPO Lactic Acid Bacteria Enumeration, LAB48$186.48$112.2080044TEMPO Total Coliform Count, CC48$186.48$112.2080003TEMPO Enterobacteriaceae Enumeration, EB48$186.48$112.20800071TEMPO Staph aureus Enumeration 48$186.48$112.2080013TEMPO Reader Tray2$33.30$22.6480011TEMPO Filler Tray1$75.48$64.1680015TEMPO Stomaching Bag500$222.00$153.00bioMerieux Pricelist 8 5 2009 to 8 4 2010Culture Media and AccessoriesItem NoDescriptionUnits/PkgCatalogue PriceState of Ohio Price43741chromID Vibrio20$60.05$44.9243531Baird Parker + RPF plates20$60.05$44.9243641chromID Ottaviani Agosti Agar (O2A) plates20$60.05$44.92P3070Buffered Listeria Enrichment Broth, 225ml10$100.44$75.13T8082Fraser Broth w/o Ferric Amm.
Citrate, 10ml20$28.58$21.3842046Fraser Broth (bioMerieux Formula), 225 ml6$56.54$42.2942072Fraser Broth (bioMerieux Formula), 10ml20$30.52$22.8342615Half Fraser Broth (bioMerieux Formula), 225ml4$37.24$27.8642048Half Fraser Broth (bioMerieux Formula), 225ml6$45.52$34.05S1210-10UVM Modified LEB, 10ml20$28.58$21.38CV1050UVM Modified LEB, 10ml25$42.32$31.66M1091Oxford Agar, Modified (MOX) plates10$19.56$14.6343621chromID Salmonella Agar, plates20$60.05$44.9242121VIDAS SX2 Broth, Salmonella Easy SLM method, 10 ml20$34.30$25.6642111Buffered Peptone Water, 9 ml100$78.95$59.0542042Buffered Peptone Water, 90 ml6$17.67$13.2242043Buffered Peptone Water, 225 ml6$22.13$16.56P1200Lactose Broth, 90 ml25$117.53$87.91P2280Lactose Broth, 90 ml25$125.37$93.78P2290Lactose Broth, 100 ml25$130.09$97.31P1750Lactose Broth (Pour, Screw Cap), 90 Broth Base, 10 ml20$25.83$19.32T8034 - CTetrathionate Broth Base, 10 ml100$129.13$96.59T7079Selenite Cystine Broth, 10 ml20$25.83$19.32T7079 - CSelenite Cystine Broth, 10 ml100$129.13$96.5942073Rappaport Vassiliadis Broth, 10 ml20$34.80$26.03T8080M Broth, 20 ml10$23.58$17.64M1016Hektoen Enteric Agar Plates10$10.03$7.50B3045Hektoen Enteric Agar / XLD (bi-plate)10$16.94$12.67M1031XLD plates10$10.03$7.50T7087Triple Sugar Iron Agar Slant, 10 ml20$20.05$15.00T7071Lysine Iron Agar Slant, 10 ml20$21.32$15.9543471Campyfood ID Agar Pack20$63.28$47.3420-05Microaero Jar, 2.5L1$82.84$61.9750-25LIDJar Lid, for 2.5 L1$45.63$34.1350-70Rectangular Jar, 7L1$135.58$101.4150-70LIDJar Lid, for 7L1$76.04$56.8860-01AnaeroPouch Bag20$12.03$9.0060-05AnaeroPouch Clip10$12.14$9.0842605chromID O157:H7 ID Agar, 200ml6$180.15$134.7542630chromID O157:H7 ID Agar, 20ml5$15.02$11.2342606Cefixime Tellurite Sorbitol plates10$11.78$8.81bioMerieux Pricelist 8 5 2009 to 8 4 2010Instruments - VITEK 2 Compact and VITEK LegacyItem NoDescriptionUnits/PkgCatalogue PriceState of Ohio Price27672VITEK 2 Compact
30 card capacity instrument, includes incubator, computer, printer, software and 2 Compact
60 card capacity kit, includes incubator, for 60 cards, computer, printer, software and all other 2 Compact Validation 2 Compact Networking Configuration 2 Compact DensiChek 21$1,103.13$825.1493059VITEK 2 Compact DensiChek 2 Compact DensiChek Power Adaptor1$67.98$50.85W0441VITEK 2 Com