Name: visualization of microbiota in tick guts by whole-mount in situ hybridization
Uploaded: 2018-06-01T14:45:00
Description: Watch this Scientific Journal Video about Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization at JoVE.com

We sincerely thank Dr. Mustafa Khokha, Yale University, for providing the use of his laboratory resources. We are grateful to Mr. Ming-Jie Wu for excellent technical assistance. EF is an HHMI investigator.This work was supported by a gift from the John Monsky and Jennifer Weis Monsky Lyme Disease Research Fund.

1. Preparation of DNA Templates
<ol>
	<li>Download the 16S rRNA sequence specific to each bacterial genera from the NCBI database and design polymerase chain reaction (PCR) compatible forward and reverse primers that will amplify genera-specific regions (~200 - 250 base-pair long) as described earlier19. Also refer to the open source databases SILVA20,21 and probeBase22 online resources for rRNA-targeted oligonucl...

<ol>
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This is the first use of a whole-mount in situ hybridization (WMISH) technique to study the microbiota of an arthropod vector of pathogens. Our protocol was adapted from one used to study development in Drosophila and in frog embryos25,26. Whole-mount RNA in situ hybridization has been routinely used to localize gene transcripts spatially and temporally27 and visualization of transcripts can be done by bright-fie...

Human and livestock pathogens transmitted by arthropod vectors are found worldwide and account for about 20% of infectious diseases globally1, but effective and safe vaccines against most of these pathogens are not available. Our understanding of the important role of commensal, symbiotic and pathogenic microorganisms, collectively known as the microbiome2, in modulating and shaping the health of almost all metazoans3 is expanding. It is now evident that arthropod vectors of pathogens also harbor gut microbiota and these vector-associated microbiota have been shown to influence diverse vector-borne pathogens4,5. The arthropod microbiome is composed of eubacteria, archaea, viruses, and eukaryotic microbes such as protozoa, nematodes, and fungi6. However, the predominant research focus has been on eubacteria due, in part, to the availability of marker genes and reference databases to identify specific bacterial members.
With a focus on Ixodes scapularis, the tick vector of multiple human pathogens including Borrelia burgdorferi7, the causative agent of Lyme disease, the optimization of a microbial visualization technique was aimed at improving our understanding of tick gut microbiota in the context of vector-pathogen interactions. Several questions remain to be answered in the tick microbiome field. The gut is the site of the first extended encounter between the tick and the incoming pathogen in the context of horizontally transferred pathogens; therefore, understanding the role of vector gut microbiota in modulating vector-pathogen interactions will reveal meaningful insights. Ticks have a unique mode of blood meal digestion, where processing of blood meal components takes place intracellularly8. The gut lumen seemingly serves as a vessel to contain the blood meal as the tick feeds, and nutrient digestion and assimilation ensue throughout the several days of feeding and continue post-repletion. The pathogens acquired by the tick during feeding enter the gut lumen along with the bloodmeal and thus the lumen becomes a primary site of interactions among the tick, pathogen, and resident microbiota. As digestion proceeds through the repletion and Ixodid tick molting, the gut undergoes structural and functional changes9. The composition and the spatial organization of gut bacteria is also likely to vary in concert with the changing gut milieu. It is, therefore, important to understand the architecture of resident bacteria in the tick gut to fully understand the interplay of tick, pathogen, and gut microbiota.
Molecular techniques to describe host-associated microbiota routinely utilize high-throughput parallel sequencing strategies10 to amplify and sequence bacterial 16S ribosomal DNA (rDNA). These sequencing strategies circumvent the need to obtain axenic cultures of specific bacteria and provide an in-depth description of all bacterial members represented in the sample. Nevertheless, such strategies are confounded by the inability to distinguish environmental contaminations from bona fide residents. Further, when assessing samples, such as ticks, that are small in size and hence contain low microbiota-specific DNA yields, the likelihood of amplification of environmental contaminants is increased11 and results in the ambiguous interpretation of microbiome composition. Functional characterization in conjunction with the visualization of specific viable bacteria will, therefore, be critical to define and discern the microbiome of the tick temporally and spatially. Towards this goal, we took advantage of the whole-mount RNA in situ hybridization. This technique is routinely used to assess gene expression patterns in organs and embryos12,13,14 and allows semiquantitative analysis of expression over the entire sample of interest. This differs from traditional in situ hybridization techniques which utilize tissue sections and often require extensive analysis of sectioned material with a computational assembly to predict expression in whole organs15. While whole-mount generally refers to whole organisms12, here whole-mount refers to whole guts or organs. The advantages of using the whole-mount RNA in situ hybridization approach to assess the architecture of tick gut microbiota are multifold. The tick gut is composed of 7 pairs of diverticula, each pair varying in size16. The functional differences, if any, among these diverticula, are not understood in the context of tick biology, tick microbiota or tick-pathogen interactions. Manipulations of the gut that rupture the gut diverticula would displace microbiota present in the gut lumen or those associated loosely with the gut and result in misinterpretation of the spatial localization of microbiota. Fluorescence-labeled RNA in situ hybridization has been utilized earlier to examine tick gut transcripts17 by fixing and opening individual gut diverticula to ensure probe hybridization and to localize B. burgdorferi transcripts by sectioning paraffin-embedded whole ticks18. Both these approaches require manipulations of the tick tissues prior to hybridization that would affect the gut microbiota architecture.
In this report, we describe in detail the protocol to examine viable tick gut microbiota using whole-mount in situ hybridization (WMISH). The use of whole-mount RNA in situ hybridization enables a global understanding of the presence and abundance of specific gut bacteria in the different regions of the gut and may spur new insights into tick gut biology in the context of pathogen colonization and transmission. Further, the use of RNA probes directed against specific bacterial RNA allows detection of viable bacteria in the tick gut.

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			<td height="21" style="height:21px;width:320px;">pGEM-T Easy Vector System</td>
			<td style="width:220px;">Promega</td>
			<td style="width:348px;">A1360</td>
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			<td height="21" style="height:21px;width:320px;">Digoxygenin-11-UTP</td>
			<td style="width:220px;">Roche</td>
			<td align="right" style="width:348px;">1209256910</td>
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			<td height="21" style="height:21px;width:320px;">dNTP</td>
			<td style="width:220px;">New England Biolabs&#160;</td>
			<td style="width:348px;">N0447S</td>
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			<td height="21" style="height:21px;width:320px;">DNAse I(RNAse-free)</td>
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			<td height="21" style="height:21px;width:320px;">HiScribe SP6 RNA synthesis kit</td>
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			<td height="21" style="height:21px;width:320px;">HiScribe T7 High Yield RNA Synthesis Kit</td>
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			<td height="21" style="height:21px;width:320px;">Water, RNase-free, DEPC-treated</td>
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			<td style="width:348px;">AB02128-00500</td>
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			<td height="21" style="height:21px;width:320px;">EDTA, 0.5M, pH 8.0</td>
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			<td height="21" style="height:21px;width:320px;">Formaldehyde, 37%</td>
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			<td height="21" style="height:21px;width:320px;">Formamide</td>
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			<td height="21" style="height:21px;width:320px;">EGTA</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td style="width:348px;">E-4378</td>
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			<td height="21" style="height:21px;width:320px;">RNA from torula yeast</td>
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			<td height="21" style="height:21px;width:320px;">Heparin, sodium salt</td>
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			<td height="21" style="height:21px;width:320px;">Denhardt&#39;s Solution, 50X</td>
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			<td height="21" style="height:21px;width:320px;">RNase A</td>
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			<td style="width:220px;">Sigma Aldrich</td>
			<td style="width:348px;">M0375-100G</td>
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			<td height="21" style="height:21px;width:320px;">Blocking reagent</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td align="right" style="width:348px;">11096176001</td>
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			<td height="21" style="height:21px;width:320px;">Anti-Digoxigenin-AP, Fab fragments</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td align="right" style="width:348px;">11093274910</td>
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			<td height="21" style="height:21px;width:320px;">Levamisol hydrochloride</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td style="width:348px;">31742-250MG</td>
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			<td height="43" style="height:43px;width:320px;">Chromogenic substrate for alkaline phosphatase</td>
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			<td align="right" style="width:348px;">11442074001</td>
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		</tr>
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			<td height="21" style="height:21px;width:320px;">Bouin&#39;s solution</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td style="width:348px;">HT10132-1L</td>
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			<td height="21" style="height:21px;width:320px;">Hydrogen peroxide</td>
			<td style="width:220px;">Mallinkrodt Baker, Inc</td>
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			<td height="21" style="height:21px;width:320px;">&#160;#11 High-Carbon steel blades&#160;</td>
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			<td height="21" style="height:21px;width:320px;">Spectrophotometer</td>
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			<td height="21" style="height:21px;width:320px;">Shaking water bath</td>
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			<td style="width:348px;">Gel Doc XR+ Gel documentation system</td>
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			<td height="21" style="height:21px;width:320px;">Bright-field Microscope&#160;</td>
			<td style="width:220px;">Nikon</td>
			<td style="width:348px;">NikonSM2745T</td>
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			<td height="21" style="height:21px;width:320px;">Bright-field Microscope&#160;</td>
			<td style="width:220px;">Zeiss</td>
			<td style="width:348px;">AXIO Scope.A1</td>
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			<td height="21" style="height:21px;width:320px;">Dissection microscope&#160;</td>
			<td style="width:220px;">Zeiss</td>
			<td style="width:348px;">STEMI 2000-C</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:320px;">&#160;Light box</td>
			<td style="width:220px;">VWR</td>
			<td style="width:348px;">102097-658</td>
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			<td height="21" style="height:21px;width:320px;">PCR purification kit&#160;</td>
			<td style="width:220px;">Qiagen</td>
			<td align="right" style="width:348px;">28104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Image capture software</td>
			<td style="width:220px;">Zeiss</td>
			<td style="width:348px;">Zen lite</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Image editing software&#160;</td>
			<td style="width:220px;">Adobe&#160;</td>
			<td style="width:348px;">Adobe Photoshop CS4 version 11.0</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:320px;">Image analysis software</td>
			<td style="width:220px;">National Institutes of Health</td>
			<td>ImageJ-NIH /imagej.nih.gov/ij/</td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;">Automation compatible instrumentation</td>
			<td style="width:220px;">Intavis Bioanalytical Instruments, Tubingen, Germany).&#160;</td>
			<td style="width:348px;">Intavis, Biolane HT1.16v</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of microbiota in tick guts by whole-mount in situ hybridization

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these diseases, do not exist in isolation when they colonize the vector; rather, they likely engage in interactions with resident microorganisms in the gut lumen. The vector microbiota has been demonstrated to play an important role in pathogen transmission for several vector-borne diseases. Whether resident bacteria in the gut of the Ixodes scapularis tick, the vector of several human pathogens including Borrelia burgdorferi, influence tick transmission of pathogens is not determined. We require methods for characterizing the composition of the bacteria associated with the tick gut to facilitate a better understanding of potential interspecies interactions in the tick gut. Using whole-mount in situ hybridization to visualize RNA transcripts associated with particular bacterial species allows for the collection of qualitative data regarding the abundance and distribution of the microbiota in intact tissue. This technique can be used to examine changes in the gut microbiota milieu over the course of tick feeding and can also be applied to analyze expression of tick genes. Staining of whole tick guts yield information about the gross spatial distribution of target RNA in the tissue without the need for three-dimensional reconstruction and is less affected by environmental contamination, which often confounds the sequencing-based methods frequently used to study complex microbial communities. Overall, this technique is a valuable tool that can be used to better understand vector-pathogen-microbiota interactions and their role in disease transmission.

The measurement and estimation of the quality of the RNA probes are critical prior to beginning the staining. In vitro transcription efficiency depends highly on the amount and quality of the DNA template. We routinely visualized the RNA probes on a formaldehyde gel to verify the purity and amount of probe generated by the transcription reactions. The probes should appear as bright, discrete bands (Figure 2). Spectrophotometric measurements of RNA co...

Watch this Scientific Journal Video about Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization at JoVE.com

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Here, we present a protocol to spatially and temporally assess the presence of viable microbiota in tick guts using a modified whole-mount in situ hybridization approach.

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these ...

This method can help to answer key questions about tick entomology, such as assessing the presence of viable bacteria in the tick spatially and temporally. The main advantages of this technique are that it does not require sectioning of the ticks or expensive equipments, and that it allows spacial and temporal visualization of gene transcripts. Generally, individuals new to this technique will struggle because it involves multiple steps and reagents.However, this technique is not laborious. Although it can take about three days to complete, hands on time is only a few hours each day. In preparation, feed the collected ticks for 48 to 72 hours before the gut collection.To dissect the gut from the tick add 20 to 30 microliters of MEMFA solution to a clean glass slide. Then place the tick into the MEMFA, and adjust focus to the tick's head. Now, using a pair of sterile high carbon steel razor blades slice off the head region at the scutum leaving the body of the tick in tact.Next, gently press the body to push the gut diverticula and salivary glands out of the body cuticle. Then without damaging the guts, carefully separate them from the salivary glands and carefully scoop the cuts onto a razor blade. Collect all of the harvested guts in a 1.5 milliliter tube in 500 micrometers of MEMFA.Then incubate the tube at room temperature with gentle rocking for one hour. The next day dehydrate the tissue as indicated in the text protocol and store it at minus 20 degrees Celsius. To prepare a mesh basket, first, use a heated single edge blade on a scalpel to cut the bottoms off of a two milliliter or five milliliter snap top centrifuge tube.The blade may need to be reheated several times before the cut is completed. Next, cut a square of a 110 micron nylon mesh that is slightly larger than the new openings. Then, bond the mesh to the opening by lightly pressing the tube into the mesh on a hotplate set to medium/low heat until a good seel is made.After it cools, trim off the excess mesh, and make sure there are not gaps. Make 24 of these baskets to match a 24 well plate. Next, cut holes into the cover of a 24 well plate, such that the lip of the sample basket will grip the hole and not fall through.When completed, the assembled sample baskets should sit all the way into the wells. Now, use this fitted basket holder to transfer baskets from one plate to another with relative ease all through the in situ hybridization. Use two such plates so that while the sample is incubating in one, the other can get prepared for the next wash.This section covers certain steps of the in situ hybridization. See the text protocol for a complete description. To begin, transfer each fixed tissue sample to a labeled sample basket.Organize the plate by experimental conditions, and stain at least five guts per condition. First, gently rehydrate the samples. Avoid introducing air bubbles by gradually increasing the ratio of PBS-T to ethanol in each wash.During the first day, after the two hour pre-hybridization step, instead of discarding the hybridization buffer, store it for future use during the assay. Later, when treating the samples with probe hybridization solution, cover the plate snugly with aluminum foil to prevent evaporation of the solution. On the second day, load the reserved hybridization buffer into the 24 well plate, and warm the plate up to 60 degrees Celsius.Later that day, to remove all traces of probe and hybridization buffer, wash the samples twice with 2x SSC for three minutes per wash. Then wash the samples three times with 2x SSC for 20 minutes per wash at 60 degrees Celsius. Then prepare the primary antibody in blocking buffer.Load 500 microliters of this mixture into each well. After the blocking step, incubate at room temperature for about four hours or at four degrees Celsius overnight. On the third day, run the color reaction in chromogenic substrate.Cover the sample plate with aluminum foil. And during the reaction, periodically check for over staining with the dissection microscope. On the fourth day it is important to incubate and bleach on a light box as this allows for any pigmentation in the samples or coloration from Bouin's solution to be removed, and enhances the probe signal for clear visualization.Then after washing with SSC, carefully transfer the gut samples by inverting the basket into a new 24 well plate. While in wells, thoroughly rinse off the baskets using 70%ethanol. Then precede with imaging the samples.It is critical to estimate the quality of the RNA probes before using them. The probes can be checked on a formaldehyde gel. If they appear as bright, discrete bands they are of good quality.When staining some variation in distribution is normal among biological replicates as well as among the seven pairs of diverticula of individual guts. Therefore, it is best to stain at least five guts per condition to get an idea of average staining pattern. Overall success is best gaged by comparing the staining of sense and antisense samples for each condition.Both must be developed for the same amount of time. The sense samples should have minimal to no purple color, while the antisense samples should display robust staining with minimal background. Critically, the tissues must be fully emersed in the stain for an even exposure.It is important to avoid over development of the color reaction. This can result in a high amount of background staining, and the sense samples may begin to look like antisense samples. Another factor that affects results is how much the ticks are fed beforehand.Ticks that are unfed, or fed for only 24 hours, are difficult to work with. Their guts are more easily damaged and do not stain well. Once mastered, this technique can be done in two and half to three days if it is performed properly.While attempting this procedure, it is important to remember to keep all of the supplies listed in the table of reagents and materials ready before beginning your experiment. Thanks for watching, and good luck with your experiments.

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