Biological Sunglasses in a Deep-Sea Squid: Pigment Migration in the Retina of Gonatus onyx
Round 1
Reviewer 1 Report (New Reviewer)
Comments and Suggestions for AuthorsI really appreciate the work and learned a lot reading it. I therefore support its publication in Vision.
The experimental scheme is solid and conclusion are well supported. Nevertheless, I have few comments and suggestion to improve it before publication.
I here suggest minor changes.
I didn’t get the reason why the control specimen, differently from those fixed in PFA 4%, was instead fixed in Glutaraldehyde 2,5% (lines 127-136), considering also the fact that it makes less visible the pigment granules. Please clarify this point in the text.
I got track changes at lines 156-157, that need to be fixed.
Figure 2
- Should “outer photoreceptor segment (osp)” be “outer segment of photoreceptor (osp)”? Otherwise, the acronym would result wrong.
- Is the “cartilaginous part of the sclera” that visible in pink? If yes please add (pink) to facilitate the figure comprehension.
- I think that in Figure 2 the sentence “In the dark-adapted eye, the ommin layer (ol) would be expected to be 201 concentrated at the base of the outer photoreceptor segment (osp)”, create confusion to the reader. This because what they state is not what you see in the figure. I would not anticipate this to avoid that the readers need to go forward and back to get the sense of it. According to me the sentence is useful somewhere in the text, but not in Figure 2. It is very well visible afterwards, in fact, in Figure 5 and 6.
- It would be very useful to maintain fixed the section planes in all figure of the paper to avoid misinterpretations. In other words, I suggest the authors to keep the coordinates constant: dorsal (do), ventral (ve), posterior (po), anterior (an) as in Figure 2. Therefore, they should turn 180° the Figure 5 and Figure 6, to have the proximal part of the eye on the left and the distal one on the right, as in Figure 2.
Lastly, I appreciated the immunogold labeling of glutamate in the retina, that remind me old times, but I am missing something here. I do not get the reason why in Figure 9 A and B they showed a duplicate of the experiment. I have nothing against it if properly stated what, but again it leads to confusion the fact that the top 9A and bottom 9A (similarly the top 9B and bottom 9B) are not adequately explained in the text (in case they reinforce each other or differences are present) and indicated as well.
Author Response
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Reviewer 2 Report (Previous Reviewer 3)
Comments and Suggestions for AuthorsThe article describes an interesting feature of the retina of deep-sea squids - the presence of a pigment layer in the retina, the structure of which changes depending on the state of light/dark adaptation. This phenomenon has been known for a long time, but was studied mainly in the octopus and in a few species of cephalopods that live at a sufficient level of illumination. The authors used rather simple histological methods to visualize the effect in live juvenile quids kept under different light conditions. In addition, immunochemical studies were carried out to determine the presence of glycine in the retina. The very fact of the presence and detection of the movement of screening pigments in a deep-sea species is interesting and deserves further detailed studies, especially since it is believed that this effect, expressed in young individuals, will change in ontogeny. All presented data are qualitative, but could be published as first approach to study deep-see squid juveniles. The manuscript has some drawbacks and not clear points which are noted below.
METHODS: p. 2.3. – Were the results of adaptation of only one or both eyes studied?
RESULTS.
The state of adaptation immediately after light adaptation is not presented.
LINES
233 “small amount” – unsuccessful assessment. There's actually quite a lot of pigment.
234, 250 “eccentricities” ------ regions, areas?
246-250 There is no quantitative assessment of the amount of pigment, it is only visually noticeable that after 75 minutes more pigment had migrated.
249 – Not “slightly”, the picture shows that there is significantly more pigment left.
DISCUSSION
Only qualitative results are discussed, which do not allow for a deeper analysis, so the results of the work can be considered preliminary, although interesting. It is unclear whether material was used from two eyes or one from each specimen. In the first case, it would be possible to obtain more representative material. The limited number of specimens obviously did not allow to demonstrate the result of complete light adaptation, that is, the “starting point” of the dark adaptation effect.
Lines
339 – Why exactly 10%? For this species, a similar mechanism can be assumed for other parameters of excessive isomerization.
340-351 – It is most likely that the light stimulation was inadequate in combination with housing conditions in which the animal experiences stress or begins to gradually die approximately 1 hour after the onset of light adaptation. The assumption of a longer period of dark adaptation has not been confirmed by experimental material, since in at least 4 individuals the “normal” expected dynamics could be fully explained. Moreover, this assumption should not be put into the abstract (lines 25-27: ”extended time” compared to what? Light adaptation?).
Author Response
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Round 2
Reviewer 1 Report (New Reviewer)
Comments and Suggestions for AuthorsAll the changes made are ok for me
Reviewer 2 Report (Previous Reviewer 3)
Comments and Suggestions for AuthorsAll important comments of the reviewers were considered.
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
The authors analyzed ommin migration in the retina of Gonatus onyx, a deep-sea squid. They showed the migratory pattern of ommin granules after 2 different experimental paradigms – 1. exposure to light for 20 min and then incremental adaptation to darkness during multiple 15 min periods up to 75 min; 2. exposure to light for 30 min and incremental adaptation to darkness during multiple 10 min periods for up to 60 min.
The paper is interesting and merits publication. However, I have several concerns that should be addressed in order to improve the quality of the paper.
The authors introduce two complex experimental paradigms in which to study ommin migration. They should explain why are these paradigms chosen. What would be the expected outcome from the light exposure lasting 20 min in comparison to 30 min? Why are increments of dark adaptation different- 10 versus 15 min? In addition, the study could benefit from the analyses of retinas that has been exposed to light without the adaptations to darkness and the analyses of retinas from squids that have been only in the darkness. In other words, there is a need for negative and positive control as reference points. I strongly suggest the elimination of the specimen that were dead or in other way unsuitable for further analyses. They will only make the interpretation of results more difficult and uncertain.
Although the introduction is quite informative about the eye’s adaptations for the life in the deep sea it is missing the information relevant for the present study. The authors study the migration of ommin pigment granules in the retina and it would be of interest to the readers to get some information about the origin of these granules, which cells are producing them and how is that production regulated. Is there any possible comparison with the retinal pigmented epithelium of vertebrate eyes? What are the mechanisms involved in the regulation of ommin granule migration? The introduction should also have an overview of current literature about the role of glutamate, glycine, and GABA in the retinas of deep-sea squid (or other mollusks) in order to justify the performed analyses.
Figure 4 and 5. It appears that the migration of ommin granules is stronger after 30 min of light than after 20 min of light exposure. The authors should comment on that and propose the possible reasons for this phenomenon in the Discussion. It also appears that 20 min of light exposure did not have any effect on granules during the process of dark adaptation. Could we assume that the distribution of ommin granules in Fig.5 at 60 min would be the most similar to the situation in the total darkness?
Figure 6: It is unclear if the specimen analyzed for the presence of neurotransmitters were exposed to light or not, if so for how long? Why are these specific conditions of dark exposure chosen particularly for the analyses of the neurotransmitter distribution. Why are not these specimen analyzed for the location of ommin granules? The micrographs need to be labelled for the specific layers. The authors should comment on the specific location of neurotransmitters. If these specimen were already adapted to darkness for 60 and 90 min is the distribution of neurotransmitters in any way comparable to the location of ommin granules in the Figs. 4. and 5.
It is not clear if there are more or less glutamate granules in fig.6.b when compared to fig.6a. in general, it is very difficult to quantify with certainty anything with n=1. How does authors justify that.
Importantly, is there a different distribution of neurotransmitters in the retinas exposed to light.
Discussion: the authors should focus their discussion on their own results (see above). Any putative mechanisms should be suggested.
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Reviewer 2 Report
Comments and Suggestions for AuthorsPlease see comments and suggestions on the attached document.
Comments for author File: Comments.pdf
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Reviewer 3 Report
Comments and Suggestions for AuthorsThe article describes an interesting feature of the retina of deep-sea squids - the presence of a pigment layer in the retina, the structure of which changes depending on the state of light/dark adaptation. This phenomenon has been known for a long time, but was studied mainly in the octopus and in a few species of cephalopods that live at a sufficient level of illumination. The authors used rather simple histological methods of material processing to visualize the effect in live squids kept under different lighting conditions. In addition, immunochemical studies were carried out to determine the presence of neurotransmitters in the retina. The very fact of the presence and detection of the movement of screening pigments in a deep-sea species is interesting and deserves further detailed studies, especially since the authors gave reasons to believe that this effect, expressed in young individuals, will change in ontogeny. The results of the histological studies and of the immunological analysis are presented and discussed in the manuscript, however have many substantial shortcomings that do not allow its positive evaluation. Most data are descriptive, without any statistical evaluation, some figures are of low quality and do not clearly show detailes described in the text. Besides this, the manuscript has many obscure points and other significant drawbacks, which are noted below.
ABSTRACT. Lines 22-23. The authors erroneously write about the levels of transmitters,since quantitative studies have not been conducted. INTRODUCTION Lines 45-46. Information about the propagation of light in sea water is given without reference. In addition, the upper layers of water absorb not only short-wave radiation, as the authors point out, but also long-wave light. Lines 93-92. It is erroneously stated that the pigment moves ”through the
supporting cells”. The pigment moves independently in photoreceptors and
supporting cells, and mainly in photoreceptors, reaching the most distal end
(Hanke, Kelber, 2020, Front. Physiol., 10:1637). Lines 109-112. The claim that "photoreceptive cells trigger neurotransmitter
release" is confusing because dopamine actually acts on the retina and
photoreceptors as a result of the activity of efferent nerve fibers. Lines 112-115. Obviously, here the authors tried to formulate the purpose of
the work, but it is not clear what mechanism they are talking about. About the
mechanism of influence of neurotransmitter levels on pigment movement?
What kind of mediators? Only dopamine was mentioned above. Lines 120-133. This paragraph should be shortened because it contains
information that is not directly related to the topic of the study, in particular,
discussions about the role of this squid in food chains. It is not clear what the
authors meant by using the term "ontogenetic descent" (line 129). If this meant
the ontogenetic stages of squid development, which have a different
distribution in the ocean, then why is it further said about “similarly sized
individuals”, and not about individuals of the same age (line 133)? MATERIALS AND METHODS Lines 140-141. What was the objective of dividing individuals into 2
experimental groups? Line 147. Who performed barcoding and where? A reference to the standard
method description is also required. Line 152. Why were preparations stained with DAPI? It should be noted right
away that the figure shown in the Appendix, showing a transverse section of
the eye stained with DAPI, is superfluous. It shows the distribution of the
nuclei of many cells, but does not provide any useful information in
connection with the purposes of this work. Line 159. What kind of immunolabeling is noted here? Which retinas from
which individuals were used and where are the results presented? The next
paragraph also contains a description of immunological procedures, so the
sequence of material processing is completely unclear. Lines 161-171. The choice of specific neurotransmitters for research is not
justified. There is no detailed description of the control for non-specificity of
antibodies. Why was the localization of glutamate, glycine and GABA studied?
By the way, the choice of the latter is especially questionable, since GABA
was not previously found in the retina of 4 cephalopod species
(Lam et al., Brain Res., 1974, 82: 365-368). Lines 193-194. See comment to line 152 above. It’s clear that all cells contain
nuclei. RESULTS Section 3.1. The work is devoted to the study of the dynamics of the pigment layer of the
retina, however, in Figs. 2 and 3, this layer is absent and the place of its
supposed location is not even shown. In addition, unsuccessful sections
(oblique?) were chosen to demonstrate the structure of the retina, in which the
layer of photoreceptors was cut across the rhabdoms, which does not allow us
to evaluate the ratio of different layers. The scale line is missing in Fig. 2. Figures 4 and 5. Figure captions do not match the images. For example, if,
judging by the description, the first strip in Fig. 4 corresponds to the position
of the pigment granules 15 min after keeping the squid in the light, then a
significant amount of pigment should have been in the apical part of the cells
(on the right side of the strip). However, the pigment is missing. The same can
be noted about the inconsistencies in Figure 5. In addition, it is obvious that the
upper band in this figure was photographed at a higher magnification than the
rest, judging by the thickness of the pigment bands in the rhabdom zone.
By the way, scale bars are missing in both figures; an indication of the
magnification factor is not enough. Section 3.1. See comments to Lines 161-171 regarding neurotransmitters. Quantitative
data are not available, so it is impossible to judge the level of neurotransmitters.
The quality of Figure 6 is low and the indication of points of interest in it looks
arbitrary. Why was the same section used to demonstrate the presence of
glutamate (6d) and glycine (6f)? This can be seen from the contours of the cut
and the presence identical cell structures on both parts of the figure.
Is it possible that a mixture of antibodies was used for identification?
If so, this was not indicated in METHODS. DISCUSSION Lines 267-271. What general conclusion can be drawn by comparing the results
of the two groups? Lines 323-324. In this work, of the transmitters mentioned here, only glutamate
was studied, so the comparison of the presented results with the data of
D'Aniello et al. is incorrect. Line 325. Why Dopamine? Thus, despite the discovery of some new facts and a number of interesting
questions raised by the authors in the discussion, the manuscript under review
contains very preliminary observations. In the light of the above comments,
the MS can not be considered for acceptance even in abridged form.
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Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors have done their best to give adequate answers to all the raised queries.
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Reviewer 2 Report
Comments and Suggestions for Authorsplease see attached.
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Reviewer 3 Report
Comments and Suggestions for AuthorsThe previously raised issue of Figure 6 remains unclear. As explained by the authors, figures 6d and 6f present two adjacent consecutive slices. In the upper part of both figures, two large cells are clearly visible, which are indicated by an arrow. In panel 6d it is listed as containing glycine and in figure 6f as containing GABA. Judging by the location, there is no doubt that this is the same cell. If the authors are confident in the adequacy of the immunolabeling methods used, then the discussion should pay attention to the presence of two mediators in one cell, if at all possible.
One more remark. Sentence (lines 99-100) “//////in Loligo pealeii, ommin granules , with a spectral sensitivity equivalent to 100% rhodopsin, could migrate to the distal end of the of the outer retinal layer,///// ” needs to be edited. As presented, this means that the spectral sensitivity of ommin granules corresponds to 100% rhodopsin. In fact, we are talking about the spectrum of sensitivity of the motile reaction of the pigment.
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