FISHing for Rickettsia in tardigrades: additional evidence for tardigrade endosymbionts
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Abstract
Many ecdysozoans harbour endosymbiotic bacteria within their microbiota, and these endosymbionts can have a range of positive and negative effects on their hosts. Recent 16S rRNA gene amplicon sequencing studies have provided evidence for endosymbionts within the tardigrade microbiota. In a previous amplicon study, we determined that sequences corresponding to the endosymbiotic genus Rickettsia were significantly more associated with tardigrades than with the substrate from which they were isolated. Here, we performed fluorescence in situ hybridization (FISH) using a Rickettsia-specific probe, RickB1, to determine if Rickettsia could be found in tardigrades. RickB1 and a probe targeting most bacteria, EUB338, colocalized within tardigrade tissues, indicating the presence of Rickettsia. We also performed FISH using RickB1 and a nonsense probe, which allowed us to distinguish between false-positives and true positives. This method revealed RickB1 signals in tardigrades that were not due to erroneous probe binding, providing further evidence that Rickettsia is present in tardigrades. Future research will be necessary to determine the effects, if any, of these endosymbionts on their tardigrade hosts.
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Additionally, no features similar to the points displayed in Figure 1 were observed in the no-probe controls. Although other specimens were stained with the two probes, the strong levels of autofluorescence made it difficult to examine these other specimens for RickB1 signals.
Would the authors be able to provide images of the no-probe controls, perhaps as a supplement? Given the high levels of background in the epifluorescence images, it is indeed challenging to distinguish what is true signal from autofluorescence or debris.
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OUT 180.
OTU 180?
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Epifluorescence microscopy reveals that RickB1 and EUB338 colocalize at points within the body cavity of a specimen of Paramactobiotus tonollii. The viewing field is focused on the dorsal side of the animal between legs III and IV. Blue arrows point to features with colocalization of EUB338 and RickB1, likely Rickettsia. White arrows point to features with only an EUB338 signal, possibly another bacterium not belonging to Rickettsia. Scale bars = 20 μm. (A) Green channel for detection of EUB338 (B) Red channel for detection of RickB1 (C) Phase contrast (D) Overlay of green and red channels. Overlap of green and red indicates colocalization of EUB338 and RickB1 (orange) (E) Overlay of fluorescence channels and phase contrast.
The images for this figure in both the web-based full text and .PDF document appear to have compression …
Epifluorescence microscopy reveals that RickB1 and EUB338 colocalize at points within the body cavity of a specimen of Paramactobiotus tonollii. The viewing field is focused on the dorsal side of the animal between legs III and IV. Blue arrows point to features with colocalization of EUB338 and RickB1, likely Rickettsia. White arrows point to features with only an EUB338 signal, possibly another bacterium not belonging to Rickettsia. Scale bars = 20 μm. (A) Green channel for detection of EUB338 (B) Red channel for detection of RickB1 (C) Phase contrast (D) Overlay of green and red channels. Overlap of green and red indicates colocalization of EUB338 and RickB1 (orange) (E) Overlay of fluorescence channels and phase contrast.
The images for this figure in both the web-based full text and .PDF document appear to have compression artifacts. This might be a problem with how BioRxiv image compression works. However, having clearer images would make it much easier to determine if the co-localization indicated by the blue arrows is genuine. Perhaps the authors could also add cropped, zoomed versions of the specific colocalization spots to make the images easier to interpret.
For ease of comprehension, it would also be helpful to have text labels for each channel, perhaps overlaid on the bottom left corner of each image. This change could be made to each of the figures in the manuscript.
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This manuscript appears to be a follow-up study of previous metagenomic analysis. The authors use FISH to assess whether members of the genus Rickettsia might be found in tardigrades. The authors use three probes: a Rickettsia-specific probe, a probe that generally labels the 16S ribosomal RNA of most bacteria, and a nonsense probe to assess whether RickB1 signal is genuine. The authors report a single sample with apparent Rickettsia staining.
This is intriguing, but additional images and information about the number of samples examined and image processing methods would be helpful to evaluate the strength of the evidence presented in this manuscript. I've annotated particular places where additional information might be helpful.
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one specimen
Would the authors be able to provide some statistics about the number of samples of each species examined? Perhaps a table of the number of individual animals imaged for each species, and a categorical description of how many animals appeared to have "true positive" puncta.
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Confocal microscopy
As the images in Figs 2 and 3 were taken using a confocal microscope, could the authors provide a rendering of the Z-axis of each image for each channel? Could the authors also describe how the images were processed? Are the images maximum intensity projections, or a single slice from a Z-stack? If the images are maximum intensity projections, for example, it is possible that some foci that appear to be fluorescent in the RickB1 channel but not the NONEUB channel could be obscured.
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This manuscript appears to be a follow-up study of previous metagenomic analysis. The authors use FISH to assess whether members of the genus Rickettsia might be found in tardigrades. The authors use three probes: a Rickettsia-specific probe, a probe that generally labels the 16S ribosomal RNA of most bacteria, and a nonsense probe to assess whether RickB1 signal is genuine. The authors report a single sample with apparent Rickettsia staining.
This is intriguing, but additional images and information about the number of samples examined and image processing methods would be helpful to evaluate the strength of the evidence presented in this manuscript. I've annotated particular places where additional information might be helpful.
-
Confocal microscopy
As the images in Figs 2 and 3 were taken using a confocal microscope, could the authors provide a rendering of the Z-axis of each image for each channel? Could the authors also describe how the images were processed? Are the images maximum intensity projections, or a single slice from a Z-stack? If the images are maximum intensity projections, for example, it is possible that some foci that appear to be fluorescent in the RickB1 channel but not the NONEUB channel could be obscured.
-
OUT 180.
OTU 180?
-
one specimen
Would the authors be able to provide some statistics about the number of samples of each species examined? Perhaps a table of the number of individual animals imaged for each species, and a categorical description of how many animals appeared to have "true positive" puncta.
-
Additionally, no features similar to the points displayed in Figure 1 were observed in the no-probe controls. Although other specimens were stained with the two probes, the strong levels of autofluorescence made it difficult to examine these other specimens for RickB1 signals.
Would the authors be able to provide images of the no-probe controls, perhaps as a supplement? Given the high levels of background in the epifluorescence images, it is indeed challenging to distinguish what is true signal from autofluorescence or debris.
-
Epifluorescence microscopy reveals that RickB1 and EUB338 colocalize at points within the body cavity of a specimen of Paramactobiotus tonollii. The viewing field is focused on the dorsal side of the animal between legs III and IV. Blue arrows point to features with colocalization of EUB338 and RickB1, likely Rickettsia. White arrows point to features with only an EUB338 signal, possibly another bacterium not belonging to Rickettsia. Scale bars = 20 μm. (A) Green channel for detection of EUB338 (B) Red channel for detection of RickB1 (C) Phase contrast (D) Overlay of green and red channels. Overlap of green and red indicates colocalization of EUB338 and RickB1 (orange) (E) Overlay of fluorescence channels and phase contrast.
The images for this figure in both the web-based full text and .PDF document appear to have compression …
Epifluorescence microscopy reveals that RickB1 and EUB338 colocalize at points within the body cavity of a specimen of Paramactobiotus tonollii. The viewing field is focused on the dorsal side of the animal between legs III and IV. Blue arrows point to features with colocalization of EUB338 and RickB1, likely Rickettsia. White arrows point to features with only an EUB338 signal, possibly another bacterium not belonging to Rickettsia. Scale bars = 20 μm. (A) Green channel for detection of EUB338 (B) Red channel for detection of RickB1 (C) Phase contrast (D) Overlay of green and red channels. Overlap of green and red indicates colocalization of EUB338 and RickB1 (orange) (E) Overlay of fluorescence channels and phase contrast.
The images for this figure in both the web-based full text and .PDF document appear to have compression artifacts. This might be a problem with how BioRxiv image compression works. However, having clearer images would make it much easier to determine if the co-localization indicated by the blue arrows is genuine. Perhaps the authors could also add cropped, zoomed versions of the specific colocalization spots to make the images easier to interpret.
For ease of comprehension, it would also be helpful to have text labels for each channel, perhaps overlaid on the bottom left corner of each image. This change could be made to each of the figures in the manuscript.
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