- Research article
- Open Access
LNA probes substantially improve the detection of bacterial endosymbionts in whole mount of insects by fluorescent in-situhybridization
© Priya et al.; licensee BioMed Central Ltd. 2012
Received: 31 October 2011
Accepted: 2 May 2012
Published: 24 May 2012
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.
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.
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.
Microscopic detection and localization of a specific DNA or RNA segment within single cells or a histological section has been made possible with the advent of the In-Situ Hybridization (ISH) technique. This technique relies principally on formation of Watson-Crick base pairing between the gene of interest and the applied complementary sequence to which the reporter molecule is attached . Fluorescent in-situ hybridization (FISH) is an extension of this technique in which a fluorophore tagged to the probe, acts as the reporter molecule. FISH is a widely used technique in clinical studies relating to diagnosis, prognosis and sometimes, even remission of diseases like cancer [2, 3]. In microbiology, studies pertaining to microbial ecology employ FISH in detection and identification of unculturable microbes in clinical and environmental samples as well as whole mount tissues [4, 5]. Some studies have employed FISH for revealing the distribution pattern of two very closely related (<3% difference in nucleotide sequence) species of marine cyanobacteria . DNA oligonucleotide probes are most commonly used when compared to ssDNA, dsDNA or RNA probes due to features like: stability, ease of availability and cost effectiveness.
A modification in the structure of nucleotides with methylene bridge to connect 2’ oxygen and 4’ carbon of the ribose ring, gives rise to Locked Nucleic Acid (LNA) . The extra bridge in an LNA structure makes the ribose moiety inaccessible, thereby locking the structure to high binding affinity conformation [8, 9]. Such LNA nucleotides can be mixed with DNA or RNA residues during synthesis of oligonucleotide to enhance the hybridization specificity, sensitivity and duplex stability [8, 10]. When compared to DNA only oligonucleotide probes, it is seen that LNA modified DNA oligonucleotide probes (hereinafter called LNA probes) are 10 fold more sensitive when applied in techniques like northern analysis . LNA probes have also been successfully used for FISH to identify individual E. coli cells . They have been used for temporal and spatial detection of miRNAs or mRNA by whole mount ISH and in tissue sections [13–16]. Some studies have used LNA in clinical studies for detection and differentiation between two fungal pathogens in tissue sections [17–19]. There are many reports that have identified and localized bacteria by targeting 16 S rRNA gene in whole mount or microtome section samples but till date there has been no report wherein LNA probes have been employed for bacterial detection by FISH in whole mount or microtome section of biological samples.
The insect Bemisia tabaci, commonly known as whitefly, is an agricultural pest with a wide host range. B. tabaci is a vector of a group of plant viruses known as Geminiviruses which significantly damage the host plant. Recent studies have linked the transmission of Tomato Yellow Leaf Curl virus (TYLCV), to the GroEL protein of a secondary endosymbiont of B. tabaci. Therefore, an extensive study of the type and nature of spread of B. tabaci endosymbionts is primary to understanding their functional role within the host insect. Two types of endosymbionts are reported to be present within the B. tabaci, namely the primary endosymbiont and the secondary endosymbiont . Whiteflies are one of the rare cases in which co-infection, of primary and secondary symbionts, occurs in the same cell . Therefore, in this study we have compared the efficiency of both DNA only and LNA modified DNA probes in the detection and localization of a primary endosymbiont that is present in abundance, as well as a secondary endosymbiont that is less abundant in nature.
We collected adult Bemisia tabaci from cotton leaves from fields of Indian Agricultural Research Institute (Pusa, New Delhi, India), washed them with ethanol and water, and stored in acetone at −20°C till further processing. The specimens were processed using standardized method of Gottlieb et al  for whitefly with slight modifications. B. tabaci specimens were stored overnight in Carnoy’s fixative (chloroform: ethanol: glacial acetic acid, 6:3:1) and decolorized with 6% H2O2 in ethanol for 24 hrs. Portiera and Arsenophonus detection was performed using FAM labeled probe bearing 5’ TGTCAGTGTCAGCCCAGAAG 3’ sequence and TYE-665 probe bearing of 5’ TCATGACCACAACCTCCAAA 3’ sequence respectively . The DNA probe and modified LNA were supplied by Exiqon A/S [the exact positions of the LNA modifications of Portiera (batch no. 5032716, containing 5 LNA) and Arsenophonus (batch no. 503274, containing 6 LNA), are not known to us]. The decolorized insects were hybridized at 40°C, with the DNA and LNA probes, in hybridization buffer (20 mM Tris-Cl [pH 8.0], 0.9 M NaCl, 0.01% sodium dodecyl sulfate) containing increasing amount of formamide (0%-80%). Probe concentrations of 0.6 pmoles for Portiera and 1.0 pmoles for Arsenophonus were kept identical for LNA and DNA. After the overnight incubation, the samples were thoroughly washed in a washing buffer (0.3 M NaCl, 0.03 M sodium citrate, 0.01% sodium dodecyl sulfate) for 5 minutes and mounted using Vectashield (Vector Labs). Each of the endosymbiont was detected at 9 different formamide concentrations (0% - 80%) separately, with DNA as well as LNA probes. Replicates consisted of 10 insects for each condition. Specificity of detection was confirmed using no probe staining and RNase- digested specimen staining. All the images were acquired at fixed camera and microscope settings for DNA and LNA with Nikon A1 confocal microscope. The fluorescence intensities were quantified by NIS elements (V 3.21.02) image analysis software (Nikon).
Results and discussion
The primary endosymbiont of Bemisia tabaci is Portiera. This symbiont is housed exclusively in specialized structures called bacteriocytes . Since this insect cannot survive without its obligate primary endosymbiont, these symbionts are present in higher proportion or abundance than other secondary endosymbionts. FISH studies pertaining to localization of Portiera using confocal microscope has been described earlier . Arsenophonus is a secondary endosymbiont whose exact role is yet to be ascertained and whose population within the insect is lower than that of Portiera. Location of Arsenophonus is reported to be in the same cell as Portiera i.e. the bacteriocytes .
Comparing LNA and DNA probes to detect Portiera the primary bacterial endosymbiont of Bemisia tabaci
Comparing LNA and DNA probes to detect Arsenophonus the secondary bacterial endosymbiont of Bemisia tabaci
The results presented here show that apart from many other applications reported so far [11–19], modified LNA probes are more effective for detecting bacteria in whole mounts of insect tissue than the conventional DNA oligonucleotide probes. This is because LNA probes are stable against 3'-exonucleolytic degradation and possess excellent aqueous solubility . Additionally, the charged phosphate backbone of LNA oligonucleotides allows them to be transfected and taken up by cells just like DNA, thus finding use in many biological applications [7, 15, 26]. LNA modification of oligonucleotides reduces flexibility and results in more stable duplex structures . The integration of 2–4 LNAs with oligonucleotides increases their binding to 16 S ribosomal RNA by up to 22-fold . The improvement in detecting the endosymbionts of interest by LNA probes, when compared to DNA counterpart, is due to their increased thermodynamic stability and improved discrimination between perfectly matched and mismatched target nucleic acids . It can be suggested that the features like higher melting temperature, better tissue penetrability and target accessibility  are the reasons why LNA outperforms DNA at nearly all formamide concentrations.
Detection of bacteriocytes in male B. Tabaci
Further studies using LNA probes for whole mount FISH can give us a better idea about the spread of endosymbionts and the various niches occupied by them within a tissue sample. In B. tabaci the use of LNA probes for detection of other endosymbionts will provide better understanding about the fly. Use of LNA can also be extended to the level of visualizing the existing interaction between the virus and the endosymbionts.
We are grateful to NAIP, Indian Council for Agricultural Research, Govt. of India for financing this work. NP acknowledges the JRF support received from CSIR, Govt. of India. We would like to thank Mr. Ashok for his valuable laboratory assistance.
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