The Association of Waminoa with Reef Corals in Singapore and Its Impact on Putative Immune- and Stress-Response Genes

Round 1

Reviewer 1 Report

This study integrates the ecological survey of the prevalence of Waminoa and stress-responses gene of host in Singapore. The lower transcript levels of selected responses genes (actin, c-type lectin C3 and Hsp70) in infected tissues were consistently lower than the un-infected tissues. In my opinion, the study can be published but require providing more information, especially in the Material and method section. Here are some detailed remarks and suggestions.

Introduction:

1. Line 464: In [89] the authors should change to Mohamed et al. [89]

Material and method:

2. line 118: “Fringing reefs of two island south of mainland Singapore” should be “Fringing reefs of two islands in the south of mainland Singapore”
3. Line 131: identified to genus level, according to [59,60] should change to “identified to genus level, according to Huang et al. [59] and Wong et al. [60]”.
4. provide ecological survey period.
5. Provide approaches or software to define the percentage of the coverage.

Coral collection section:

6. provide the information about location and time for the sampling collection
7. Provide the information about the degree of infection of worms in the five collected samples for the gene expression. Low, moderate, severe and extreme as you mention in line 135-136.
8. Provide housekeeping genes for the qPCR analysis
9. Should include the healthy colonies of L. radians for the gene expression analysis.
10. How did you standardize the concentration of cDNA. Please provide information.

Discussion:

11. Between line 377 and 388: comparison the prevalence of Waminoa to other studied locations. Please considered if those studies used the same survey approaches.

 

 

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

The authors examined the association frequency of Waminoa on coral in Singapore and their preference for the host corals, as well as the impacts of Waminoa on corals. Their results showed a higher association of Waminoa in corals in Kusu island, and Waminoa preferred massive or laminar coral morphologies.

Their also showed that Waminoa association affect on the expression of immune related genes.

Its interesting research because I was wondering that the Waminoa could be threat to corals. However, before accepting, it is necessary to add detailed experimental information of RT-PCR.  Depending on Sequence information, the credibility of the result of RT-PCR is diminished, so I will use it as a major revision.

 

Major comment, 

 

The fact that Waminoa is often seen in Kusu island corals gives the impression that Waminoa itself prefers a clean environment and is vulnerable to environmental disturbances. In addition, if Waminoa prefers to inhabit some corals, I don't think it's a big threat to the entire reef. In fact, as a result, I wasn't sure if Waminoa was harmful or not, but what about it?

 

The polyp without Waminoa was described as being 5 cm away from the polyp with Waminoa attached, but isn't Waminoa itself moving around frequently on the coral polyp? As far as I can see, the distribution of Waminoa on the polyp surface changed between day and night. Did the Waminoa in this study stay in a fixed position in the coral colony, at least during the day, and hardly move?

In the prevalence of the association test (fig. 4), is it okay to not include corals, Acropora, and Stylophora that did not have Waminoa attached?

 

In the analysis using the MCMC.qpcr package, it was stated that the method without using the control gene was applicable, but under both W and W0 conditions, the expression of all five RNAs was reduced. This result raises the suspicion that W0 has a lower overall RNA expression because the number of target genes is also small. If possible, it is best to perform real-time PCR on sequences whose expression level does not change under any conditions and to obtain results as reference values.

 

 

Minor comment,

 

1. 29-51 It is recommended to cite the following paper.

Hikosaka-Katayama et al., 2012, Mechanisms of Maternal Inheritance of Dinoflagellate Symbionts in the Acoelomorph Worm Waminoa litus

 

1. 214, Did you homogenize the coral sample? If so, please describe how to homogenize corals.

 

1. 229, Table S1 → Table S4?

 

Supplementary Materials

Primer design and validation,

“L. radians transcriptome is previously sequenced” Please insert the transcriptome accession number, or Please register the sequence from which primer the created.

 

Figure 2, Please check the description of the scale bar for Figure D.

 

Figure 3, I couldn't understand the meaning of the Letter above the bar graph.

 

Figure 5 and Figure 6 have the same Figure legend. Figure legend for Figure 5 needs to be changed.

 

Figure 6 should show the results of gene downregulation. It is easier to understand if the result of log2 times change shows that gene expression is decreased in W (the vertical axis extends in the negative direction).

 

Table 1 Please include the sequence information such as the Accession Number of each sequence or the sequence result of the PCR product. Also include the blast search results for that sequence.

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The manuscript is much improved and I have no further questions and problems for this research. I agree to accept this research. 

Author Response

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Author Response File: Author Response.doc

Reviewer 2 Report

Minor comments,

 

I think the source of the used L. radians sequence should be clearly stated.

 

Author state “The transcriptome for L. radians (Quek and Huang 2019) is available for download at reefgenomics.org (Liew et al. 2016)” in reply.

But, It was unclear where this was described in the main text. Please check main text and insert explanation. Or it's better to register the used sequence in DDBJ or NCBI etc. and get accession Number.

 

 

 

Did you homogenize the coral sample?

Means

Did you crush the coral sample, before RNA extraction

please describe it in detail.

Author Response

Please see the attachment.

Author Response File: Author Response.doc