Effect of Light–Dark Cycle on Skin Mucosal Immune Activities of Gilthead Seabream (Sparus aurata) and European Sea Bass (Dicentrarchus labrax)

Round 1

Reviewer 1 Report

This is a very well written manuscript with interesting implications for fish immunology that are pertinent both for fish industry and immunologists at large. The experiments are well designed and the data is interpreted appropriately. We have no major comments and propose to accept the manuscript for the publication. The only two very minor comments concern

1. The description and portrayal in figures of the significance values as it is unclear which time-points are compared specifically.   

2.  It would have been interesting to also assess the levels of IgT/Z which is considered to be the most important immunoglobulin of mucosal surfaces. 

Author Response

This is a very well written manuscript with interesting implications for fish immunology that are pertinent both for fish industry and immunologists at large. The experiments are well designed and the data is interpreted appropriately. We have no major comments and propose to accept the manuscript for the publication. 

Thank you very much for your interest in our work.

The only two very minor comments concern.

The description and portrayal in figures of the significance values as it is unclear which time-points are compared specifically.

Statistical analysis (ANOVA) was performed by comparing the levels of each immune activity at different sampling times. In addition, the Ritme software was used to evaluate whether these activities showed rhythms over 24 hours. In both cases, the significance was set as p < 0.05. The figure legends have been corrected in the revised version of the manuscript accordingly to this explanation.

It would have been interesting to also assess the levels of IgT/Z which is considered to be the most important immunoglobulin of mucosal surfaces.

We agree with the review. We analyzed the IgM because, at present, it is the only IgM antibody available for these two fish species. However, the interest in studying other Ig is indicated in the revised version of the paper. 

Reviewer 2 Report

This study investigates the effects of the light-dark cycle on the immune system of two fish species. The relationship between the light condition and the fish immune system is very interesting.

In this study, however, the photocycle of the investigation was only one pattern; 12L-12D. Although immune systems have changed with the progress of time, it is not clear whether these changes are due to photoperiod or circadian rhythm. Further, there are no experiments to clarify the biological significance of changes in the immune system shown in this MS. Therefore, it is difficult to understand the biological mean of this immunological change.

Author Response

This study investigates the effects of the light-dark cycle on the immune system of two fish species. The relationship between the light condition and the fish immune system is very interesting. In this study, however, the photocycle of the investigation was only one pattern; 12L-12D.

Thank you for your interest in our work. We wanted to study the possible variations in the immune parameters over 24 hours. In this work, fish were maintained exposed to an artificial 12L-12D cycle because this unique pattern of photoperiod has been previously established in several works (Bayarri et al., 2004; Esteban et al., 2006; Montoya et al., 2010; Lazado et al., 2015). By using this pattern, present results can be compared with some other obtained in similar conditions. It could be very interesting to study the influence of other light-dark patterns in the next future. This idea is now discussed in the new version of the manuscript.

Although immune systems have changed with the progress of time, it is not clear whether these changes are due to photoperiod or circadian rhythm.

In our study, the experimentation was performed with fish maintained in some light-dark conditions subjected to a constant photoperiod for a long time. Therefore, it can be assumed that the photoperiod and the circadian rhythm were synchronized since light is the primary exogenous factor used to synchronize endogenous circadian clocks that integrate physiological processes (Pittendrigh, 1981). It is a circadian rhythm because the duration was 24 h but the light, the photoperiod, was the real factor affecting fish. However, some other factors should be also affecting. For example, the pineal gland via its secretory product, melatonin, which influences the light-dark rhythm in most vertebrates including fish. Future studies will serve to shed some light on the biorhythms in fish. All these ideas are discussed in paragraphs one and two of the discussion.

Further, there are no experiments to clarify the biological significance of changes in the immune system shown in this MS. Therefore, it is difficult to understand the biological mean of this immunological change.

The biological significance of this work focuses on the evidence that immune status is influenced by the photoperiod. There are hours at which the immune parameters are increased and other hours at which the same parameters are reduced. In other words, the fish immunity is not constant. Therefore, if fish in farms need to face some adverse situation it should better to do it when fish are better defended and vice versa, avoid negative situations when fish are worst defended, if possible. It can be expected that fish will have a better response to any adverse situation (e.g. stress, presence of pathogens, physical-chemical changes in the water, transportation) in the hours with high immunity. This could be very important by farmers knowing when fish are more or less sensitive to such factors, which are very common in aquaculture, in order to decrease the negative impact caused by them. These considerations are in the third paragraph of the discussion: "A deeper knowledge of the daily rhythm of bactericidal activity (not only in fish skin mucus but also in serum) would help us to anticipate the normal response of farmed fish in order to improve their welfare, to increase immune system robustness or to prevent and/or treat possible diseases. Similarly, knowing the periodic changes that occur in fish immunocompetence could throw light on seasonal changes in disease incidence and severity in nature, and provide a useful framework for understanding brain-immune interactions".

In addition, the manipulation of photoperiod could be also considered as a very useful tool to enhance fish immune status or to decrease the negative impact of foreseeable stressful situations. These considerations were underlined in the conclusions.

Reviewer 3 Report

Examining additional immune parameters, also in serum, will be very valuable.

Author Response

 

Examining additional immune parameters, also in serum, will be very valuable.

In the present work, our aim was to carry out a non-invasive study and that was the main reason for selecting skin mucus to test innate immunity. We want to collaborate, according to the 3Rs concept (Replacement, Reduction, Refinement) to minimize animal use and suffering. Besides that, the fish mucosal immunity is a hot topic at present. 

In a previous study, we evaluated the changes in the humoral innate immune parameters in the serum of gilthead seabream and sea bass specimens exposed to the same constant light-dark photoperiod used in this study (12 hr L:12 hr D) in (Esteban et al., 2006, reference number 8 of the manuscript). 

Round 2

Reviewer 2 Report

I understand the authors' answers. Although the question of changes in immuno activity due to different photoperiods did not disappear, I hope this problem will be resolved in the new study.