Both Light Stimuli and Predation Risk Affect the Adult Behavior of a Stygobiont Crustacean

Round 1

Reviewer 1 Report

General comments

This manuscript provides results of a short experimental study into the potential effects of light stimulus and predation risk on the behavior of a stygobiont amphipod, Niphargus thuringius, which lives in both lightless groundwater habitats and surface spring waters. The manuscript is interesting and provides novel information on a poorly studied species. However, the experimental design is not fully explained and requires further expansion and explanation. Also, the entire manuscript requires moderate to extensive English editing, as some sentences, particularly in the Discussion, are difficult to interpret.

Specific comments

Line 53. Change “climatic” to “environmental”.

Line 56. “…can be particularly definite…” Do you mean “can be particularly defined”?

Lines 68-83. These two paragraphs have citations consisting of the surname of authors and year, instead of numbers in brackets. Therefore, their full references are not included in the References section. Please correct.

Lines 90-92. Odd sentence. Please rephrase.

Lines 130-140 (experimental design). I had a hard time trying to understand the experimental design. The authors state that they used 2 light treatments and 3 different conditions of predation risk, which yields 6 different treatment combinations (light x predation risk), but they do not specify how many replicate microcosmos they had for each combination. Further on (in Line 142) they mention that they collected 80 amphipods (Line 142) and that they assigned 5 amphipods to (each of) 16 microcosms (Line 151). Why 16 microcosms? 16 is not a multiple of 6. Was there a different number of replicate microcosms per treatment combination? If the design was unbalanced, the authors should clarify it because this could have consequences for the statistical analysis.

Lines 136-140. As this is the first time the dragonfly and salamander larvae are mentioned, please write here the scientific names for both species.

Lines 157-160. The dragonfly and salamander larvae were collected from different locations than the amphipods, but do all three species co-occur in nature?

Lines 162-165. How did you record the number of active individuals at night or in the no-light treatment? Did you use some kind of light source to observe the microcosms? Please explain.

Lines 173-176. How much time elapsed between the end of the 30-day experiment and the response to light stimulus tests? How long did each of the two behavioral tests conducted for each individual take and how much time elapsed between these two tests?

Line 202. Was the microcosm’s identity considered as a random factor nested in the corresponding treatment?

Lines 210-217. Here, the authors provide a hint into the number of replicates per treatment. If there were originally 30 amphipods in the 2-predators microcosms, that would mean there were 6 replicates for this predator treatment (5 amphipods per microcosm), which divided by 2 light regimes would yield 3 replicates per combination. This is also the case for the 1 mesopredator treatment. However, for the control (no predator) treatment, there were only 20 amphipods in total. At 5 amphipods per microcosm, this would mean there were only 4 replicates for this treatment, which divided by 2 light regimes, would yield 2 replicates per combination. If these numbers are correct, then please provide them in the Methods section and explain whether the unbalanced design is accounted for by the statistical analysis.

Throughout the Discussion, there are several odd sentences that are difficult to understand. Please rephrase them: Lines 261-267; Lines 278-279; Lines 292-295; Lines 301-303.

Figure 1: Please expand the figure caption, explaining what the different-colored (blue, white and brown) drawings stand for. Figure captions should be self-explanatory.

Table 1: What is DenDF? Please define initials in the table heading, which should also be self-explanatory.

References: I believe many references do not follow the format for Diversity.

Author Response

We thank the reviewer for the precious suggestions; we used them all to improve the quality of the manuscript.

Line 53. Change “climatic” to “environmental”.

Done

Line 56. “…can be particularly definite…” Do you mean “can be particularly defined”?

“definite” as in “distinct”, “precise”

Lines 68-83. These two paragraphs have citations consisting of the surname of authors and year, instead of numbers in brackets. Therefore, their full references are not included in the References section. Please correct.

Done, sorry for the mistake

Lines 90-92. Odd sentence. Please rephrase.

Done, we rephrased the sentence.

Lines 130-140 (experimental design). I had a hard time trying to understand the experimental design. The authors state that they used 2 light treatments and 3 different conditions of predation risk, which yields 6 different treatment combinations (light x predation risk), but they do not specify how many replicate microcosmos they had for each combination. Further on (in Line 142) they mention that they collected 80 amphipods (Line 142) and that they assigned 5 amphipods to (each of) 16 microcosms (Line 151). Why 16 microcosms? 16 is not a multiple of 6. Was there a different number of replicate microcosms per treatment combination? If the design was unbalanced, the authors should clarify it because this could have consequences for the statistical analysis.4

Following the suggestion we explained the different number of replicates and underlined the caveat. Initially it was planned to have 18 microcosms, such as three replicates for each condi-tion showed in Fig. 1. However, we were able to collect less animals that expected and we had only two replicates for each control condition, in which we expected an higher survival rate (and thus a final number of animals comparable to that of the predator treatments). Partially unbalanced final dataset was accounted in statistical analyses by using LMM modelling.

Lines 136-140. As this is the first time the dragonfly and salamander larvae are mentioned, please write here the scientific names for both species.

Done

Lines 157-160. The dragonfly and salamander larvae were collected from different locations than the amphipods, but do all three species co-occur in nature?

 Yes, they do, as we said at lines 93-96 with different references

Lines 162-165. How did you record the number of active individuals at night or in the no-light treatment? Did you use some kind of light source to observe the microcosms? Please explain.

Initially we explained it in the text (lines 177-178). However, thanks to the observation of the reviewer, e removed this part of the methods as not strictly related to the experiment and not described in the results.

Lines 173-176. How much time elapsed between the end of the 30-day experiment and the response to light stimulus tests? How long did each of the two behavioral tests conducted for each individual take and how much time elapsed between these two tests?

We thank the reviewer for the precious suggestion. We added these details in the text. We specififed also illuminance level and interval between trials.

Line 202. Was the microcosm’s identity considered as a random factor nested in the corresponding treatment?

Yes; it is specified at line 210.

Lines 210-217. Here, the authors provide a hint into the number of replicates per treatment. If there were originally 30 amphipods in the 2-predators microcosms, that would mean there were 6 replicates for this predator treatment (5 amphipods per microcosm), which divided by 2 light regimes would yield 3 replicates per combination. This is also the case for the 1 mesopredator treatment. However, for the control (no predator) treatment, there were only 20 amphipods in total. At 5 amphipods per microcosm, this would mean there were only 4 replicates for this treatment, which divided by 2 light regimes, would yield 2 replicates per combination. If these numbers are correct, then please provide them in the Methods section and explain whether the unbalanced design is accounted for by the statistical analysis.

We had pallend thrre replicates for each condition but at the end of the experiment two replicates for each control condition (light/darkness and total darkness) were available. We now explained it better in the methods; we underlined this aspect as a caveat in the discussion.

Throughout the Discussion, there are several odd sentences that are difficult to understand. Please rephrase them: Lines 261-267; Lines 278-279; Lines 292-295; Lines 301-303.

We rephrased the4 discussion.

Figure 1: Please expand the figure caption, explaining what the different-colored (blue, white and brown) drawings stand for. Figure captions should be self-explanatory.

 Done

Table 1: What is DenDF? Please define initials in the table heading, which should also be self-explanatory.

Added

Reviewer 2 Report

The article is devoted to the analysis of the behavioral reactions of cave amphipods, and is interesting both from the points of view on ecology and the formation of population behavior patterns.

I have only a few questions and remarks to the methodology of the study.

1. Write in the text, what illuminance intensity (lx) was used for illuminate microcosms with a varying photoperiod?

2. Indicate in the text, what illuminance (lx) intensity and light spectrum were by the flashlight when performing the behavioral test with amphipods?

3. Justify the use of salamander larvae as meso-predators for amphipods. Reference to publications indicating that juveniles of salamander eat individuals of Niphargus is required.

4. Why were two white plastic bowls placed in the microcosms? Explain this in the text.

5. Label all of the details on Figure 2: pierced transparent bottle, white bowls, stone as shelter. The text of the article contains information about the structure of a microcosm, but the caption to the figure should fully describe its content.

I recommend this article for publication in Diversity upon the minor revision.

Author Response

1. Write in the text, what illuminance intensity (lx) was used for illuminate microcosms with a varying photoperiod?

Illuminance intensity was of 1800 lux. We added it in the text.

2. Indicate in the text, what illuminance (lx) intensity and light spectrum were by the flashlight when performing the behavioral test with amphipods?

3. Justify the use of salamander larvae as meso-predators for amphipods. Reference to publications indicating that juveniles of salamander eat individuals of Niphargus is required.

We did refer to it at lines 93-96 and 140-142

4. Why were two white plastic bowls placed in the microcosms? Explain this in the text.

We added this explanation; plastic bowls were the place in which food for salamanders and amphipods was placed.

5. Label all of the details on Figure 2: pierced transparent bottle, white bowls, stone as shelter. The text of the article contains information about the structure of a microcosm, but the caption to the figure should fully describe its content.

Done

Reviewer 3 Report

The manuscript diversity-2171637 entitled “Both light stimuli and predation risk affect adult behaviour of a stygobiont crustacean” provides a comprehensive scientific information regarding the effect of light stimuli and predation on the behaviour of a stygobiont crustacean. In general, the topic is important for the readers of diversity journal. The manuscript was well written and well structured. I would like recommend for minor revision and my specific comments are as below:

Introduction:

The introduction is too long. Some unnecessarily parts must be removed.

Line 73: what is the different between “blind” and “eyeless”?

Materials and Methods

Line 129: For all statistical analysis two decimals is enough.

Line 147: “N. thuringius” must be in italic format.

Results

Line 212: “Cordulegaster boltonii” must be in italic format.

Author Response

Introduction:

The introduction is too long. Some unnecessarily parts must be removed.

We shortened the introduction according to this suggestion.

Line 73: what is the different between “blind” and “eyeless”?

Being eyeless means being blind, but one animal could be blind having non-functioning eyes

Materials and Methods

Line 129: For all statistical analysis two decimals is enough.

We used three decimals for the temperature as the first significant digit of the SE is the third decimal

Line 147: “N. thuringius” must be in italic format.

Done

Results

Line 212: “Cordulegaster boltonii” must be in italic format.

Done