title: LNA probes substantially improve the detection of bacterial endosymbionts in whole mount of insects by fluorescent in-situ hybridization.
abstract: BACKGROUND
  Detection of unculturable bacteria and their localization in the host, by fluorescent in-situ hybridization (FISH), is a powerful technique in the study of host-bacteria interaction. FISH probes are designed to target the 16 s rRNA region of the bacteria to be detected. LNA probes have recently been used in FISH studies and proven to be more efficient. To date no report has employed LNA probes for FISH detection of bacterial endosymbiont in the whole mount tissues. Further, though speculated, bacteriocytes have not been reported from males of Bemisia tabaci.
  
  
  RESULTS
  In this study, we compared the efficiency in detecting bacteria by fluorescent DNA oligonucleotides versus modified probes containing Locked Nucleic Acid (LNA) substitution in their structure. We used the insect Bemisia tabaci as the experimental material since it carried simultaneous infection by two bacteria: one a primary endosymbiont, Portiera (and present in more numbers) while the other a secondary endosymbiont Arsenophonus (and present in less numbers). Thus a variation in the abundance of bacteria was expected. While detecting both the bacteria, we found a significant increase in the signal whenever LNA probes were used. However, the difference was more pronounced in detecting the secondary endosymbiont, wherein DNA probes gave weak signals when compared to LNA probes. Also, signal to noise ratio for LNA probes was higher than DNA probes. We found that LNA considerably improved sensitivity of FISH, as compared to the commonly used DNA oligonucleotide probe.
  
  
  CONCLUSIONS
  By employing LNA probes we could detect endosymbiotic bacteria in males, which have never been reported previously. We were able to detect bacteriocytes containing Portiera and Arsenophonus in the males of B. tabaci. Thus, employing LNA probes at optimized conditions will help to significantly improve detection of bacteria at the lowest concentration and may give a comprehensible depiction about their specific distribution within samples.
authors.affiliation: Gut Biology Lab, Department of Zoology, University of Delhi, Room No 117, Delhi 110007, India.
authors.forename: Natarajan Gayatri
authors.initials: NG
authors.lastname: Priya
authors.forename: Neeti
authors.initials: N
authors.lastname: Pandey
authors.forename: Raman
authors.initials: R
authors.lastname: Rajagopal
mesh_terms: D000818:Animals; D001431:Bacteriological Techniques / Q000379:methods*; D004755:Enterobacteriaceae / Q000235:genetics / Q000302:isolation & purification*; D044129:Halomonadaceae / Q000235:genetics / Q000302:isolation & purification*; D006430:Hemiptera / Q000382:microbiology*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008297:Male; D015345:Oligonucleotide Probes / Q000235:genetics; D009841:Oligonucleotides / Q000235:genetics; D012680:Sensitivity and Specificity
chemical_list: D015345:Oligonucleotide Probes; D009841:Oligonucleotides; C477371:locked nucleic acid