Sewage Pipe Waters Affect Colour Composition in Palaemon Shrimp from the Intertidal in the Canary Islands: A New Non-lethal Bioindicator of Anthropogenic Pollution

Round 1

Reviewer 1 Report

Dear authors:

Your manuscript is very good, I suggest add or complement with more recent references. 

In methodology, you explain very good the data obtention, but I suggest add references as support (lines 80-92). On this context, I don´t understand how you convert coloration to numbers. Is it suggested by references?

Many success and blessings !!

Author Response

Thank you for your kind words. We have specified that ImageJ uses colour histogram and colour measurement tools to extract color data as numbers (RGB coordinates) from the selected region of the image.

Reviewer 2 Report

Review

Paper title: Sewage pipe waters affect color composition in Palaemon shrimps from the intertidal in the Canary Islands: a new non-lethal bioindicator of anthropogenic pollution

 

The authors conducted an analysis of color data for Palaemon elegans from the Canary Islands at reference and polluted sites and found specimens from more polluted areas to be darker in color. The authors concluded that these results are promising for monitoring and further studies.

 

All these reasons explain the relevance of the paper by Enrique Lozano-Bilbao and Jesús Alcázar-Treviño submitted to "Diversity".

 

General scores.

 

The data presented by the authors are original and significant. The study is correctly designed and the authors used appropriate sampling methods. In general, statistical analyses are performed with good technical standards. The authors conducted careful work that may attract the attention of a wide range of specialists focused on biological indication.

 

Recommendations.

Figure 2. The authors stated that they used the abdomen for RGB measurement but in the photo, we can see the dorsal part. Also it is difficult to distinguish true color differences from these photos. Can the authors provide more visual images?

Table 2 has only one line. The authors should include F-values for PERMANOVA in addition to p-values.

The authors should discuss their results in more detail. For example, darker coloration may be due to the accumulation of pollutants in the body. Also, the authors should consider the possibility that coloration patterns may reflect the role of other factors, for example, food habits. The authors did not provide data on the sex of their specimens. Are males and females in this species the same in color? Without these data, it is difficult to evaluate the true pattern.

The authors stated that their data may be used in citizen science projects. They should discuss difficulties in the application of their method because a detailed guide and perfect color scale are required to avoid incorrect results by non-professionals. Moreover, the existing data indicated an overlap between different sites.

 

Specific remarks.

L 19. Consider replacing “its RGB” with “their RGB”

L 21. Consider replacing “between control” with “between the control”

L 22. Consider replacing “hypothesis of pollution affecting” with “hypothesis that pollution affects”

L 56. Consider replacing “its tissues” with “their tissues”

L 112. Consider replacing “data was extracted” with “data were extracted”

L 162. Consider replacing “shows that” with “showed that”

L 166. Consider replacing “so that the areas” with “so the areas”

L 170. Consider replacing “tend to white” with “tend to be white”

L 188. Consider replacing “associated to” with “associated with”

L 192. Consider replacing “a monitoring of the pollutants presence” with “monitoring of pollutants’ presence”

L 202. Consider replacing “in all the coasts” with “on all the coasts”

Author Response

Thank you for your review. We have carefully followed the recommendations. We have added in the methods section which specific part of the body of the shrimp was used for the colourimetry analyses. The first abdominal segment of the shrimp is the first section of the body of this animal posterior to its cephalothorax section, and its left part was photographed for the analyses. The F-values have been added to Table 2. More information has been added to the discussion. We note that the results were  not separated by sex of the shrimps because previous studies have shown that the contamination present in these shrimp in the same area does not depend on sex. We believe that the additions to the methods make it possible to replicate the experiment as citizen science, since we specify the position of the shrimp, the color of the surface to place it, and the image processing analysis.

All specific remarks have been accepted and modified in the manuscript.

Reviewer 3 Report

The authors have provided a new and simple method of indicating human impact on the sea. They used the colouration of a shrimp (Palameon elegans) to indicate sewage discharge into the sea. 

This darker colouration reminds me of the textbook example of adaptive industrial colouration in British peppered moths. The colour of sewage is generally much darker brown or blackish than the sea, so the whiter individuals are more visible to their visual predators, so darkness is an adaptive strategy to reduce predation risk. The citizen science approach enhances the value of this ms.

Methods:

Shrimps were photographed, and the colouration was measured in the RGB additive colour space using the value of the three primary colours (red, green, blue) of the shrimp's abdomen. They found that the shrimp in the sewage-affected sites were darker than those in the control (no sewage) sites.

It would be useful to have more details about the image processing: how did they obtain RGB data from the colour of the post-abdomen? Did they assess the whole post-abdomen or just a darker section of it?

It would also be useful to know how the background colour or transparency of the exoskeleton affects the values of RGB colours.

Authors must pay attention to English and typing (colour -> colour).

Comments for author File: Comments.pdf

Author Response

Thank you for your review. We have carefully followed the recommendations. We have added more information in the discussion and the sampling information regarding image processing, as suggested.

Reviewer 4 Report

This article is interesting and well-written, it can be published in present form.

Author Response

Thank you for your kind words.

Round 2

Reviewer 2 Report

The authors have revised the paper according to my comments. 

Author Response

Thank you again