Biomerieux Api 20e
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Biomerieux Api 20e

I1536 2442 10 131
of 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
biomerieux api 20e
.A total of 125 bacteria
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