Ultrastructural evidence of a mechanosensory function of scale organs (sensilla) in sea snakes (Hydrophiinae)

The evolution of epidermal scales was a major innovation in lepidosaurs, providing a barrier to dehydration and physical stress, while functioning as a sensitive interface for detecting mechanical stimuli in the environment. In snakes, mechanoreception involves tiny scale organs (sensilla) that are concentrated on the surface of the head. The fully marine sea snakes (Hydrophiinae) are closely related to terrestrial hydrophiine snakes but have substantially more protruding (dome-shaped) scale organs that often cover a larger portion of the scale surface. Various divergent selection pressures in the marine environment could account for this morphological variation relating to detection of mechanical stimuli from direct contact with stimuli and/or indirect contact via water motion (i.e. ‘hydrodynamic reception’), or co-option for alternate sensory or non-sensory functions. We addressed these hypotheses using immunohistochemistry, and light and electron microscopy, to describe the cells and nerve connections underlying scale organs in two sea snakes, Aipysurus laevis and Hydrophis stokesii. Our results show ultrastructural features in the cephalic scale organs of both marine species that closely resemble the mechanosensitive Meissner-like corpuscles that underlie terrestrial snake scale organs. We conclude that the scale organs of marine hydrophiines have retained a mechanosensory function, but future studies are needed to examine whether they are sensitive to hydrodynamic stimuli.


Recommendation? Accept with minor revision (please list in comments)
Comments to the Author(s) The paper provides compelling anatomical evidence regarding the specialized mechanosensory function of cephalic and tail scale organs from two species of sea snake. I appreciate that the authors have used a variety of histological and EM techniques to describe the unique morphology of these organs, including careful measure of adjacent skin layers, suggesting specialized tactile function, similar to the mechanosensory organs noted in terrestrial snakes.
I have a few suggestions: 1) A summary schematic figure would serve this paper well. I suggest two (or more panels) representing the specialized cephalic organs, as well as the distinct tail organs. This could be prepared from observations from all the microscopic techniques in total (e.g., relative location of putative neuronal bundles from the PGP immunohistochemistry, numbers of center cells as based on the counts from TEM, etc.) and would serve the authors well in visually showing the differences in these organs, such as locations/numbers of discoid organs, lamellated corpuscles, differences in thickness of the skin strata.
2) The paper could benefit with a little more discussion regarding the potential innervation of these organs. The work of comparative neuroanatomists like Glenn Northcutt have showed elaborate branching patterns that suggest particular sensory function/importance in fish, reptile, and amphibian species, and these patterns have often been used to make evolutionary inferences. The authors touch upon this in the "Tail scale organs" section in the discussion but this can be taken a bit further. Innervation to the hydrodynamic sensors of the lateral line system-the neuromasts -arise through their own specialized cranial nerve system rather than trigeminal.
3) The authors could communicate with a little more confidence about the unlikely role of these sense organs in electroreception (as in the last sentence of the Dermal photoreception... section). Their histological sections do not seem to show any kind of canal or pore typical of passive electroreceptors, like ampullary-type organs. Also, based on the sections shown in the paper, it doesn't appear that their are more specialized active electroreceptive organs, like tuberous organs or mormyromasts seen in freshwater weakly-electric fish.
Other minor correction/comments: 1) The background white balancing looks unusual among panels in Figs. 5 and 6.
2) The labels are difficult to read in Fig. 7, with the black labeling on top of the black and white TEM. Perhaps the brightness or contrast could be adjusted, or the labels themselves colored.
3) The putative myelinated axon looks unusual with the sheathing appear very thin. The authors acknowledge that this is the best resolution available, however. 4) In paragraph 2 of the discussion, the authors state that they can see free nerve axons terminating in the alpha layers in Fig. 3. Free nerve endings are a specific kind of specialized sensory end organ, often described as related to pain sensation. The nerve bundles adjacent to the putative sense organs are visible, but free nerve endings terminating in the the distal epidermis don't seem visible here (maybe just at this resolution).
(p. 4, lines 94-95). Finally, it is suggested (p. 4, lines 80-82) that Acrochordus uses its scale organs to sense "water motion." I do not believe this is true. My understanding is that the snakes respond to physical touch of the fish. This should be checked.
(2) Throughout the paper the scale organs are referred to as "sensilla". I understand that this term is often used in the literature to refer to similar structures in squamate reptiles. However, it is inaccurate and misleading as a sensillum is a hair-like structure (usually in insects or other invertebrate taxa). It is also true that many lizard scale organs have a central hair-like protrusion that could more accurately be called a sensillum (exclusive of the rest of the receptor). Other terms are used in the literature for the sensory organs described in this paper, including 'scale organ' and 'integumentary sensory organ' or 'ISO'. These would seem to me to be far preferable than "sensilla/sensillum", but obviously this is the choice of the authors.
(3) Throughout the description of the sensory organs its inner, dermal component is referred to as a "dermal capsule". In my opinion, this is inaccurate and inconsistent with most anatomical usage. A capsule represents a discrete covering or sheath that surrounds something ["a membranous structure…that envelopes an organ"-Stedman's Medical Dictionary, 27th ed.], which is not the case here. The dermal protrusion into the epidermis is more accurately described as a 'dermal papilla', as parenthetically noted on p. 5, line 134. This should be the term used throughout the paper.
(4) Note that a finding of tactile/mechanoreception does not exclude all other possible functions, e.g., they could still function in modifying flow over the snake's surface (though unlikely).

EDITORIAL COMMENTS
Line 40: I do not believe that there is any evidence to support the assertion that snakes use mechanoreceptors to discriminate prey types. This is pure speculation based on mixed receptor types within the mouth. Evidence suggests that virtually all prey discrimination is chemosensory (gustatory and vomeronasal).
Line 45: change "stimulus" to 'stimuli' Line 95: delete hyphen in "Merkel-cell" Line 104: "collected 1 10 km offshore"; seems to be a typo, not clear what it should be Line 127: and elsewhere; insert 'trichrome after "Gomori's one-step"; also, do not capitalize "One-Step" Line 130: delete "the height (thickness)" and change to 'thickness' Line 184: It is unclear what is meant by "horizontally arranged" in reference to the 'central cells'; in the images they are either clustered, vertical or circular-never horizontal Line 187: re: the apparently basal taper in H. stokesii-are you sure that this is not simply a plane of section issue? Did you have serial sections across the width of the receptors to confirm this? From other images it appears that the dermal papilla extends outward/laterally toward the scale surface, i.e., the distal part of the papilla is wider than its base. As such, a section that just passes through the point that a lateral extension joins the central core would look tapered toward the base.

15-Feb-2019
Dear Ms Crowe-Riddell On behalf of the Editors, I am pleased to inform you that your Manuscript RSOS-182022 entitled "Ultrastructural evidence of a mechanosensory function of scale 'sensilla' in sea snakes (Hydrophiinae)" has been accepted for publication in Royal Society Open Science subject to minor revision in accordance with the referee suggestions. Please find the referees' comments at the end of this email.
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. Comments to the Author(s) The paper provides compelling anatomical evidence regarding the specialized mechanosensory function of cephalic and tail scale organs from two species of sea snake. I appreciate that the authors have used a variety of histological and EM techniques to describe the unique morphology of these organs, including careful measure of adjacent skin layers, suggesting specialized tactile function, similar to the mechanosensory organs noted in terrestrial snakes.
I have a few suggestions: 1) A summary schematic figure would serve this paper well. I suggest two (or more panels) representing the specialized cephalic organs, as well as the distinct tail organs. This could be prepared from observations from all the microscopic techniques in total (e.g., relative location of putative neuronal bundles from the PGP immunohistochemistry, numbers of center cells as based on the counts from TEM, etc.) and would serve the authors well in visually showing the differences in these organs, such as locations/numbers of discoid organs, lamellated corpuscles, differences in thickness of the skin strata.
2) The paper could benefit with a little more discussion regarding the potential innervation of these organs. The work of comparative neuroanatomists like Glenn Northcutt have showed elaborate branching patterns that suggest particular sensory function/importance in fish, reptile, and amphibian species, and these patterns have often been used to make evolutionary inferences. The authors touch upon this in the "Tail scale organs" section in the discussion but this can be taken a bit further. Innervation to the hydrodynamic sensors of the lateral line system-the neuromasts -arise through their own specialized cranial nerve system rather than trigeminal.
3) The authors could communicate with a little more confidence about the unlikely role of these sense organs in electroreception (as in the last sentence of the Dermal photoreception... section). Their histological sections do not seem to show any kind of canal or pore typical of passive electroreceptors, like ampullary-type organs. Also, based on the sections shown in the paper, it doesn't appear that their are more specialized active electroreceptive organs, like tuberous organs or mormyromasts seen in freshwater weakly-electric fish.
Other minor correction/comments: 1) The background white balancing looks unusual among panels in Figs. 5 and 6.
2) The labels are difficult to read in Fig. 7, with the black labeling on top of the black and white TEM. Perhaps the brightness or contrast could be adjusted, or the labels themselves colored.
3) The putative myelinated axon looks unusual with the sheathing appear very thin. The authors acknowledge that this is the best resolution available, however. 4) In paragraph 2 of the discussion, the authors state that they can see free nerve axons terminating in the alpha layers in Fig. 3. Free nerve endings are a specific kind of specialized sensory end organ, often described as related to pain sensation. The nerve bundles adjacent to the putative sense organs are visible, but free nerve endings terminating in the the distal epidermis don't seem visible here (maybe just at this resolution).

Reviewer: 2
Comments to the Author(s) In a previous study the authors found that fully marine, hydrophiine snakes had, on average, a higher density of scale organs on cranial scales than their terrestrial relatives. Scale organs also tended to be larger and cover a larger area of the scale, although there was broad overlap in these measures. In the present study the authors consider whether the derived scale organs of marine species have retained the ancestral mechanoreceptive (terrestrial) function, or if they have also changed in function. They use light and transmission electron microscopy, and immunohistochemistry to identify neuronal tissue to address the question. They show convincingly that the scale receptors retain a mechanoreceptive (touch) function.
The study is sound, the conclusions reasonable and supported by the data. I have only a few general comments and several small, editorial comments.

GENERAL COMMENTS
(1) [p. 4, 1st paragraph; p. 11, lines 335-336; p. 12, lines 339-340] There is some ambiguity/mischaracterization about the role of mechanoreceptors in sensing 'touch' vs. "hydrodynamic stimuli." I believe that this is a false distinction. The evidence is that the snake scale organs are mechanoreceptors sensitive to pressure. Pressure can take the form of physical touch or a compression wave traveling through water (a hydrodynamic stimulus). Thus there is not a dichotomy between 'touch receptors' and 'hydrodynamic receptors'. The evidence suggests, circumstantially, that the scale organs of snakes are adapted to be more sensitive to pressure/mechanical stimuli then their terrestrial counterparts, presumably because water pressure waves have less energy than a physical touch. To be clear, I believe that this is what the authors mean to say, but as written, it is either unclear or the distinction is overstated. The distinction relates to the nature or source of mechanical, pressure stimuli, not (necessarily) to the nature of the receptors.
Related to the above, note that the Merkel cell neurite complexes characteristic of crocodilian ISOs are also mechanosensory and hence would not actually play an "alternative sensory role" (p. 4, lines 94-95). Finally, it is suggested (p. 4, lines 80-82) that Acrochordus uses its scale organs to sense "water motion." I do not believe this is true. My understanding is that the snakes respond to physical touch of the fish. This should be checked.
(2) Throughout the paper the scale organs are referred to as "sensilla". I understand that this term is often used in the literature to refer to similar structures in squamate reptiles. However, it is inaccurate and misleading as a sensillum is a hair-like structure (usually in insects or other invertebrate taxa). It is also true that many lizard scale organs have a central hair-like protrusion that could more accurately be called a sensillum (exclusive of the rest of the receptor). Other terms are used in the literature for the sensory organs described in this paper, including 'scale organ' and 'integumentary sensory organ' or 'ISO'. These would seem to me to be far preferable than "sensilla/sensillum", but obviously this is the choice of the authors.
(3) Throughout the description of the sensory organs its inner, dermal component is referred to as a "dermal capsule". In my opinion, this is inaccurate and inconsistent with most anatomical usage. A capsule represents a discrete covering or sheath that surrounds something ["a membranous structure…that envelopes an organ"-Stedman's Medical Dictionary, 27th ed.], which is not the case here. The dermal protrusion into the epidermis is more accurately described as a 'dermal papilla', as parenthetically noted on p. 5, line 134. This should be the term used throughout the paper.
(4) Note that a finding of tactile/mechanoreception does not exclude all other possible functions, e.g., they could still function in modifying flow over the snake's surface (though unlikely).

EDITORIAL COMMENTS
Line 40: I do not believe that there is any evidence to support the assertion that snakes use mechanoreceptors to discriminate prey types. This is pure speculation based on mixed receptor types within the mouth. Evidence suggests that virtually all prey discrimination is chemosensory (gustatory and vomeronasal).
Line 45: change "stimulus" to 'stimuli' Line 95: delete hyphen in "Merkel-cell" Line 104: "collected 1 10 km offshore"; seems to be a typo, not clear what it should be Line 127: and elsewhere; insert 'trichrome after "Gomori's one-step"; also, do not capitalize "One-Step" Line 130: delete "the height (thickness)" and change to 'thickness' Line 184: It is unclear what is meant by "horizontally arranged" in reference to the 'central cells'; in the images they are either clustered, vertical or circular-never horizontal Line 187: re: the apparently basal taper in H. stokesii-are you sure that this is not simply a plane of section issue? Did you have serial sections across the width of the receptors to confirm this? From other images it appears that the dermal papilla extends outward/laterally toward the scale surface, i.e., the distal part of the papilla is wider than its base. As such, a section that just passes through the point that a lateral extension joins the central core would look tapered toward the base.
Line 204: change "skin" to 'scale' for clarity Line 238: change "present at base of dermal" to 'present at the base of the dermal' Line 247: replace the comma with a period; start new sentence with "The outer bumps…" Line 279: The comment about "cap cells" based on Jackson (1977) seems a bit pointless. There is no histological difference between the keratinocytes covering the dermal papilla and others. Obviously any cells in this position would provide abrasion resistance, but no moreso than anywhere else.
Lines 286-287: The figures do not show any direct evidence of discoid receptor innervation that I can see. Obviously they must be innervated and the nerves get pretty close within the scale organ, but no nerves leading directly to the discoid receptors, particularly the more distant, epidermal receptors, are evident.
Lines 296-299: It seems implausible to me that the central cells have no sensory function. What's the point of the whole organ structure then, particularly given that discoid receptors are distributed all over? How confident are you about this, i.e, what is the probability that you would have seen synaptic complexes? There are neurons all over within the dermal papilla and it seems unlikely that they are merely supplying discoid receptors. What else would they be doing? Just free nerve endings?
Lines 359-362: Catania (1995), ref. 59, does not provide any evidence for sensitivity to hydrodynamic stimuli in star-nosed moles, nor can I find any other reference to such a thing. They definitely use the star organ for direct touch of food objects while foraging within water, but again (see General Comment 1), this is no different from terrestrial touch. I did not check ref. 60 for the platypus, but I would confirm that they do, indeed, have receptors that are sensitive to water movement AND that they have been "co-opted" from terrestrial cutaneous touch receptors. In fact, I would confirm this for all of them. Direct touch underwater is not the same thing as being used to detect water movement (hydrodynamic stimuli). Pinniped whiskers are a good example for mammals (they use them to detect vortices indicating fish trails) [see review by Dehnhardt  Line 401: insert 'compared to a' after "differential sensitivity" Line 402: I don't see why the trigeminal or other cranial nerves that innervate cephalic cutaneous receptors are "specialized"… The sensory organs they innervate might be specialized receptors, but the cranial nerves, themselves, are not specialized.
Line 404: the "dorsal root ganglion" is not a "peripheral nerve of the spinal cord"-it is a part of the spinal nerve within which the sensory nerve bodies lie. For lines 402-404, it is sufficient to note that the cephalic receptors are innervated by cranial nerves while the postcranial receptors are innervated by spinal nerves, which is exactly what one would expect.
Line 406: the receptors are not used "to actively seek"-they are used while he snake actively seeks… Line 409: do you mean 'electrophysiological' rather than 'electrophysical'?

15-Mar-2019
Dear Ms Crowe-Riddell, I am pleased to inform you that your manuscript entitled "Ultrastructural evidence of a mechanosensory function of scale organs ('sensilla') in sea snakes (Hydrophiinae)" is now accepted for publication in Royal Society Open Science.
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Kind regards,

Jenna Crowe-Riddell and co-authors
Reviewer comments to Author: Reviewer: 1 Comments to the Author(s) The paper provides compelling anatomical evidence regarding the specialized mechanosensory function of cephalic and tail scale organs from two species of sea snake. I appreciate that the authors have used a variety of histological and EM techniques to describe the unique morphology of these organs, including careful measure of adjacent skin layers, suggesting specialized tactile function, similar to the mechanosensory organs noted in terrestrial snakes.

Reviewer 1 Reviewer 1 comments
Author's response 1) A summary schematic figure would serve this paper well. I suggest two (or more panels) representing the specialized cephalic organs, as well as the distinct tail organs. This could be prepared from observations from all the microscopic techniques in total (e.g., relative location of putative neuronal bundles from the PGP immunohistochemistry, numbers of center cells as based on the counts from TEM, etc.) and would serve the authors well in visually showing the differences in these organs, such as locations/numbers of discoid organs, lamellated corpuscles, differences in thickness of the skin strata. This is a great suggestion and would improve the paper. Unfortunately, we have not had time to make this change.
2) The paper could benefit with a little more discussion regarding the potential innervation of these organs. The work of comparative neuroanatomists like Glenn Northcutt have showed elaborate branching patterns that suggest particular sensory function/importance in fish, reptile, and amphibian species, and these patterns have often been used to make evolutionary inferences. The authors touch upon this in the "Tail scale organs" section in the discussion but this can be taken a bit further. Innervation to the hydrodynamic sensors of the We have incorporated relevant information from the Glenn Northcutt review paper on the comparative nerve patterns of lateral line systems in the 'Tail scale organs' section in the discussion and added the following sentence (lines 413-418): "Future studies should investigate the neural pathways and compare electrophysiological responses underlying scale mechanoreceptors distributed on the head and body of snakes. Such efforts may discover that sea snakes possess specialised nerve pathways lateral line system-the neuromasts -arise through their own specialized cranial nerve system rather than trigeminal. and/or responsive fields that are analogous to the cranial nerve canals of neuromasts in fish and amphibians, or the vibrissae of secondarilyaquatic systems in mammals (55,61), which would support a hydrodynamic function for cephalic scale organs." 3) The authors could communicate with a little more confidence about the unlikely role of these sense organs in electroreception (as in the last sentence of the Dermal photoreception... section). Their histological sections do not seem to show any kind of canal or pore typical of passive electroreceptors, like ampullary-type organs. Also, based on the sections shown in the paper, it doesn't appear that their are more specialized active electroreceptive organs, like tuberous organs or mormyromasts seen in freshwater weakly-electric fish.
We have made this change to paragraph 'Tail scale organs' in the discussion, such that (lines 432-435): "An electro-magneto-sense is plausible (5), but our histological sections do not show canals or pores that are indicative of passive electroreceptors (e.g. ampullary-type organs) or specialised active electroreceptive organs (e.g. tuberous organs or mormyromasts of weaklyelectric fish) (66,67)." Other minor correction/comments: 1) The background white balancing looks unusual among panels in Figs. 5 and 6.
The white balance has been corrected for Figs. 5 and 6.
2) The labels are difficult to read in Fig. 7, with the black labeling on top of the black and white TEM. Perhaps the brightness or contrast could be adjusted, or the labels themselves colored. Font size has been increased and changed to bold. Scale bars and font have also been increased.
3) The putative myelinated axon looks unusual with the sheathing appear very thin. The authors acknowledge that this is the best resolution available, however.
Unfortunately, we were unable to achieve a higher resolution image of the putative myelinated axon. 4) In paragraph 2 of the discussion, the authors state that they can see free nerve axons terminating in the alpha layers in Fig. 3. Free nerve endings are a specific kind of specialized sensory end organ, often described as related to pain sensation. The nerve bundles adjacent to the putative sense organs are visible, but free nerve endings terminating in the the distal epidermis don't seem visible here (maybe just at this resolution).
This was a typo-meant to reference Figure 5 (immunohistochemical results), this change has made as well as removing "free" before "nerve axons"

Reviewer: 2
Comments to the Author(s) In a previous study the authors found that fully marine, hydrophiine snakes had, on average, a higher density of scale organs on cranial scales than their terrestrial relatives. Scale organs also tended to be larger and cover a larger area of the scale, although there was broad overlap in these measures. In the present study the authors consider whether the derived scale organs of marine species have retained the ancestral mechanoreceptive (terrestrial) function, or if they have also changed in function. They use light and transmission electron microscopy, and immunohistochemistry to identify neuronal tissue to address the question. They show convincingly that the scale receptors retain a mechanoreceptive (touch) function.
The study is sound, the conclusions reasonable and supported by the data. I have only a few general comments and several small, editorial comments.

Reviwer 2 comments
Author's response (1) [p. 4, 1st paragraph; p. 11, lines 335-336; p. 12, lines 339-340] There is some ambiguity/mischaracterization about the role of mechanoreceptors in sensing 'touch' vs. "hydrodynamic stimuli." I believe that this is a false distinction. The evidence is that the snake scale organs are mechanoreceptors sensitive to pressure. Pressure can take the form of physical touch or a compression wave traveling through water (a hydrodynamic stimulus). Thus there is not a dichotomy between 'touch receptors' and 'hydrodynamic receptors'. The evidence suggests, circumstantially, that the scale organs of snakes are adapted to be more sensitive to pressure/mechanical stimuli then their terrestrial counterparts, presumably because water pressure waves have less energy than a physical touch. To be clear, I believe that this is what the authors mean to say, but as written, it is either unclear or the distinction is overstated. The distinction relates to the nature or source of mechanical, pressure stimuli, not (necessarily) to the nature of the receptors.
We understand the reviewer's concern and have clarified the relationship between mechanosensitivity to touch versus water motion in the Abstract (lines 19-22), Introduction (lines 76-80; 91-93) and Discussion (lines 340-346; 360-364) However, we are reluctant to use of the term 'pressure' for the following reasons. Water movement can consist of mid water 'pressure waves' or water surface waves. But the term 'pressure wave' has a complex and often confused interpretation. A vibrating object in the water, for example, can produce change in pressure (this is what we call 'sound', and we can measure it in Pascals but will also produce displacement of the particles in the medium (which is NOT pressure). To detect the pressure wave (in Pascal), you need a pressuresensitive device (pressure -displacement transducer). Pressure detection is mediated by the inner ears of land vertebrates (which can be considered mechanoreceptors), and by the swim bladders of some fishes. It is possible that the inner ears of sea snakes also detect pressure, but this remains to be proven. Based on this, we consider it likely that the mechanoreceptors in the skin of sea snakes cannot detect pressure, but can detect motion displacement, velocity or acceleration, similar to the mechanosensitive neuromasts in the lateral line of fishes and other animals that detect motion, but not pressure. We briefly mention the possibility that scale organs are sensitive to pressure (i.e. baroreception) in the Discussion (lines 436-442).
Related to the above, note that the Merkel cell neurite complexes characteristic of crocodilian ISOs are also mechanosensory and hence would not actually play an "alternative sensory role" (p. 4, lines 94-95).
Indeed, Merkel cell neurite complexes are associated with mechanoreception. We have modified text to reflect this (lines 89-95): "We aimed to better understand the evolution of scale organs in sea snakes by describing their ultrastructure in two fully-aquatic species, Aipysurus laevis and Hydrophis stokesii, using immunohistochemistry, and light and electron microscopy. If sea snake scale organs are retained for a mechanosensory role, either closecontact touch or detection of water motion (deflected off objects or prey/predators), we would expect them to have retained the ultrastructure described in terrestrial snakes, and possibly contain other sensory cells such as the Merkel-cell neurite complexes of crocodilian ISOs." Finally, it is suggested (p. 4, lines 80-82) that Acrochordus uses its scale organs to sense "water motion." I do not believe this is true. My understanding is that the snakes respond to physical touch of the fish. This should be checked.
We have checked the original reference (Povel and van der Kooij, 1997) and subsequent refs (Lillywhite 2014), in which the authors confirm mechanosensory function to scale 'sensillae' in Acrochordus and speculate that they are sensitive to water motion generated by fish prey given its analogous structure to hair-cells within fish neuromasts. We have modified the text to reflect the uncertainty in function in the literature. In the introduction (lines 80-83): "Indeed, two independently aquatic snakes, Erpeton and Acrochordus, are distantly related to hydrophiine sea snakes but have protruding organs that are likely sensitive to water motion generated by the movement of fish prey (5,35)." In the Discussion (lines 373-374): "Scale 'sensillae' in file snakes (Acrochordus) are thought to be sensitive to the hydrodynamic motion generated by the movement of fish prey." (2) Throughout the paper the scale organs are referred to as "sensilla". I understand that this term is often used in the literature to refer to similar structures in squamate reptiles. However, it is inaccurate and misleading as a sensillum is a hair-like structure (usually in insects or other invertebrate taxa). It is also true that many lizard scale organs have a central hair-like protrusion that could more accurately be called a sensillum (exclusive of the rest of the receptor). Other terms are used in the literature for the sensory organs described in this paper, including 'scale organ' and 'integumentary sensory organ' or 'ISO'. These would seem to me to be far preferable than "sensilla/sensillum", but obviously this is the choice of the authors.
We agree with Review 2 and welcome the opportunity to use a more accurate term. After defining the term in the introduction (lines 15-16), we have replaced 'sensilla/sensillum' with 'scale organ/s' throughout the ms.
(3) Throughout the description of the sensory organs its inner, dermal component is referred to as a "dermal capsule". In my opinion, this is inaccurate and inconsistent with most anatomical usage. A capsule represents a discrete covering or sheath that surrounds something ["a membranous structure…that envelopes an organ"-Stedman's Medical Dictionary, 27th ed.], which is not the case here. The dermal protrusion into the epidermis is more accurately described as a 'dermal Concurring with Reviewers 2 point above (2), 'dermal capsule' is anatomically inaccurate term for the underlying organ structure that we describe in the ms. Accordingly, we have replaced all instances of 'dermal capsule' with 'dermal papilla' throughout the ms.
papilla', as parenthetically noted on p. 5, line 134. This should be the term used throughout the paper.
(4) Note that a finding of tactile/mechanoreception does not exclude all other possible functions, e.g., they could still function in modifying flow over the snake's surface (though unlikely).
We have added the following sentence to paragraph 'Dermal photoreception and other cutaneous sensory modalities' in the discussion to address this point (lines 448-450): "Finally, these sensory hypotheses do not exclude other non-sensory functions for scale organs, e.g. modifying boundary layer of skin, so these roles should be considered in future studies in the scale organs of sea snakes." EDITORIAL COMMENTS Line 40: I do not believe that there is any evidence to support the assertion that snakes use mechanoreceptors to discriminate prey types. This is pure speculation based on mixed receptor types within the mouth. Evidence suggests that virtually all prey discrimination is chemosensory (gustatory and vomeronasal).
The paper referenced in the text (Nishida et al. 2000) describes ultrastructure of papillae in the mouth of Elaphe snakes, which provides compelling evidence for both chemo and mechanoreceptive functions for these oral organs. Nevertheless, we agree that the evidenced that these mechanoreceptors are used to discriminate prey types is indeed speculative. We have changed the text to clarify that these organs may be used in feeding (as appose to discriminating prey types) (lines 39-41): "Snakes are likely to use these mechanosensory organs to explore and navigate substrate (7,8), during courtship (11) and feeding behaviours (9,10), but the anatomy and neurophysiology of scale organs are conspicuously understudied in comparison to other sensory organs" Line 45: change "stimulus" to 'stimuli' This change has been made.
Line 95: delete hyphen in "Merkelcell" This change has been made throughout the ms.
Line 104: "collected 1 10 km offshore"; seems to be a typo, not clear what it should be A hypen was deleted during the conversion to pdf, I have changes to '1 to 10 km offshore' Line 127: and elsewhere; insert 'trichrome after "Gomori's one-step"; also, do not capitalize "One-Step" This change has been made.
Line 130: delete "the height (thickness)" and change to 'thickness' This change has been made.
Line 184: It is unclear what is meant by "horizontally arranged" in reference to the 'central cells'; in the images they are either clustered, vertical or circular-never horizontal We have removed this term from the text.
Line 187: re: the apparently basal taper in H. stokesii-are you sure that this is not simply a plane of section issue? Did you have serial sections I have re-examined my images of serial sections and have come to the same conclusion as the reviewer: the basal taper is likely the result of the plane of the section. We across the width of the receptors to confirm this? From other images it appears that the dermal papilla extends outward/laterally toward the scale surface, i.e., the distal part of the papilla is wider than its base. As such, a section that just passes through the point that a lateral extension joins the central core would look tapered toward the base.
have made the following change to the ms (lines 189-190): "The dermal papilla was occasionally tapered at its basal end in H. stokesii ( Figure 3A), but this likely to be an artefact of tissue sectioning." Line 204: change "skin" to 'scale' for clarity This change has been made.
Line 238: change "present at base of dermal" to 'present at the base of the dermal' This change has been made.
Line 247: replace the comma with a period; start new sentence with "The outer bumps…" This change has been made.
Line 279: The comment about "cap cells" based on Jackson (1977) seems a bit pointless. There is no histological difference between the keratinocytes covering the dermal papilla and others. Obviously any cells in this position would provide abrasion resistance, but no moreso than anywhere else We agree with this comment and have removed the term 'cap cells' throughout the ms and referred to them simply as 'the keratinocytes above the dermal papilla' as necessary. However, we have kept the term 'cap cells' as  is one of the only available previous studies that have described scale organs in snakes and so we believe the terminology should be noted in the ms.
Lines 286-287: The figures do not show any direct evidence of discoid receptor innervation that I can see. Obviously they must be innervated and the nerves get pretty close within the scale organ, but no nerves leading directly to the discoid receptors, particularly the more distant, epidermal receptors, are evident.
Indeed. Previous studies on snake skin have found that nerves originating in the dermis terminate in epidermal 'discoid' receptors ). However, we did not observe direct evidence in our serial sections of this, therefore the text has been changed accordingly (lines 214-216): "Dermal axons travelled to the scale organs ( Figure 5C), then meandered through the central dermal papilla before innervating the outer epidermis and presumably terminate as distinct discoid endings in the alpha layer ( Figure 5A, B)." Lines 296-299: It seems implausible to me that the central cells have no sensory function. What's the point of the whole organ structure then, particularly given that discoid receptors are distributed all over? How confident are you about this, i.e, what is the probability that you would have seen synaptic complexes? There are neurons all over within the dermal papilla and it seems unlikely that they are merely supplying discoid receptors. What else would they be doing? Just free nerve endings?
We concur with Reviewer 2, this sentence now reads as such (lines 301-304): "We did not find synaptic contacts between axons and central cells, which is consistent with light microscopy studies of other colubroid snakes (e.g. Elaphe) (23). Nevertheless, the presence of discoid receptors superior to the dermal papilla suggest that the central cells have a functional role in transducing mechanical stimuli." Lines 359-362: Catania (1995), ref. 59, does not provide any evidence for sensitivity to hydrodynamic stimuli in star-nosed moles, nor can I find any other reference to such a thing. They definitely use the star organ for direct touch of food objects while foraging within water, but again (see General Comment 1), this is no different from terrestrial touch. I did not check ref. 60 for the platypus, but I would confirm that they do, indeed, have receptors that are sensitive to water movement AND that they have been "co-opted" from terrestrial cutaneous touch receptors. In fact, I would confirm this for all of them. Direct touch underwater is not the same thing as being used to detect water movement (hydrodynamic stimuli). Pinniped whiskers are a good example for mammals (they use them to detect vortices indicating fish trails) [see review by Dehnhardt  We have deleted these references from the text and included pinniped vibrissae as a prime example of cooption of cutaneous mechanoreceptors.
Line 401: insert 'compared to a' after "differential sensitivity" This change has been made.
Line 402: I don't see why the trigeminal or other cranial nerves that innervate cephalic cutaneous receptors are "specialized"… The sensory organs they innervate might be specialized receptors, but the cranial nerves, themselves, are not specialized. This line has been deleted, and new sentence has been added to add clarity (lines 414-419): "Future studies should investigate the neural pathways and compare electrophysiological responses underlying scale mechanoreceptors distributed on the head and body of snakes. Such efforts may discover that sea snakes possess specialised nerve pathways and/or responsive fields that are analogous to the cranial nerve canals of neuromasts in fish and amphibians, or the vibrissae of secondarily-aquatic systems in mammals (55,61), which would support a hydrodynamic function for cephalic scale organs." Line 404: the "dorsal root ganglion" is not a "peripheral nerve of the spinal cord"-it is a part of the spinal nerve within which the sensory nerve bodies lie. For lines 402-404, it is sufficient to note that the cephalic receptors are innervated by cranial nerves while the This line has been deleted (see change in text in previous point). postcranial receptors are innervated by spinal nerves, which is exactly what one would expect. Line 406: the receptors are not used "to actively seek"-they are used while he snake actively seeks… This line has been deleted (see change in text in previous point).
Indeed, this change has been made.
Lines 429-432: there is also no histological support for a magnetic sense We have changes this line to make a stronger assertion in line with Reviewer 1 and Reviewer 2 comments on magnetic sense (lines 432-436): "Several other sensory functions have been tentatively attributed to the scale organs of sea snakes, but these currently lack supporting evidence. An electro-magnetosense is plausible (5), but our histological sections do not show canals or pores that are indicative of passive electroreceptors (e.g. ampullary-type organs) or specialised active electroreceptive organs (e.g. tuberous organs or mormyromasts of weakly-electric fish) (66,67)." FIGURE LEGENDS: 2A: no plane of section is give for the image; 2B: delete "cross-" (redundant); 'hematoxylin' is misspelled "hemotoxylin"; 2C: insert 'trichrome' after "Gomori one-step"; 3A: no plane of section given; 3B,C: delete "cross-"; 'transverse' is misspelled "traverse"; 4: check for above changes; also, Latin binomial name not italicized; "deeper cross-section" doesn't make sense to me, please clarify; 7: "keratin filaments tonofilaments (t)"? must be a typo-missing parentheses?
These changes have been made. (1-3). The epidermal scales also provide the primary surface for mechanoreception, which is 36 the ability to sense mechanical stimuli that result from pressure or physical displacement 37 (vibration) (4). Scale organs ('sensilla' or ('tubercles' sensu (5-7) are small 38 mechanoreceptors that protrude from the surface of epidermal scales of the head and body of 39 snakes. Snakes are likely to use these mechanosensory organs to explore and navigate 40 substrate (8,9), discriminate prey types (9,10) and and duringengage in courtship behaviours 41 3 (10), and feeding (11,12) behaviorus, but the anatomy and neurophysiology of scale 42 sensillaorgans are conspicuously understudied in comparison to other sensory organs (e.g. , 43 for example, eyes (13), , auditory structures (14), vomeronasal organ (15) and, heat-sensing 44 pits (16). Hydrophiine snakes (Elapidae) provide a useful comparative framework to investigate 68 the evolution of squamate scale sensillaorgans in response to major ecological transitions (7). 69 The fully marine, viviparous sea snakes comprise a clade of more than 60 species that 70 evolved within the terrestrial Australian hydrophiine radiation (tiger snakes, death adders, 71 taipans) approximately 9 to 18 million years ago (30). Previous work has found that the 72 cephalic scale sensillaorgans of sea snakes are substantially more protruding (dome-shaped) 73 compared to their terrestrial counterparts, and in some lineages cover a much larger 74 proportion of the scale surface (> 6% versus < 2.5% in sampled taxa) (7), . This divergence in 75  Table 1 and Figure  119 1. A single specimen of Oxyuranus scutellatus (the Australian taipan) was sourced from a 120 captive breeding population (Venom Supplies Pty Ltd, South Australia) to sample brain 121 tissue for antibody controls (see below) because this species is closely related to viviparous 122 the sea snakes (30). All samples were fixed by immersion in either 4% paraformaldehyde for 123 immunohistochemistry, or 1.5% glutaraldehyde and 4% paraformaldehyde for electron-124 microscopy. After immersion in fixative for 24 hours, samples were washed and stored in 125 phosphate buffered saline (PBS; pH 7.4) with sucrose, before being transferred into 126 phosphate buffer with 0.05% sodium azide. 127

Stereo and light microscopy 128
The outer skin morphology of museum specimens was examined using a stereomicroscope 129 with a mounted camera (SMZ25, Nikon Inc., Japan). Specimens were submerged in water 130 and illuminated by a ring of light-emitting diodes (P2-FIRL LED Ring Illumination Unit, 131 Nikon Inc., Japan) to reduce specular reflections from the scales. A high-depth-of-field 132 photographic image was composed using imaging software (NIS-Elements Advanced 133 Research v5.10, Nikon Inc., Japan). 134 The general cellular morphology of the skin samples was examined using light 135 microscopy. Samples were dehydrated by successive immersion in alcohol, then paraffin-136 embedded for serial sectioning (10 µm). Slides were stained with hemotoxylin-eosin or 137 Gomori's One-Step (40), scanned using a digital slide scanner (Nanozoomer, Hamamatsu 138 Photonics, Japan) and measurements taken using imaging software (Nanozoomer Digital 139 6 Pathology v2.6, Hamamatsu Photonics, Japan). We measured the height (thickness) of the 140 epidermis located above scale sensillaorgans, and at adjacent areas of skin that did not 141 contain sensillaorgans. Because the outer layer of hardened skin (beta layer) sometimes 142 became artificially separated from surrounding layers during tissue processing, we measured 143 only the living (nucleated) epidermal layer (stratum germinativum). The diameter and height 144 of dermal capsulespapillae (papilla) and other dermal structures were measured and the ratio 145 of diameter:height calculated. 146

Statistics 147
We used the two-sample t-test (unpaired) to examine differences in epidermal thickness 148 between scale organs and adjacent skin that did not contain scale organs. Before statistical 149 analyses, we checked that data were normally distributed using Bartlett's test. Statistical 150 analyses were performed using base packages in R v3.5.1 (R Core Team, 2017). 151

Immunohistochemistry 152
Immunohistochemistry was performed on paraffin-embedded serial sections (10 µm) for a 153 neuronal marker, protein gene product 9.5 (PgP9.5). Briefly, slides were blocked for 154 endogenous peroxidase with 0.5% hydrogen peroxide in methanol at room temperature for 30 155 minutes (min). Slides were rinsed in PBS and processed in 10 mM sodium citrate (pH 6.0) for 156 heat-induced epitope retrieval. Slides were washed twice in PBS, before blocking in 3% 157 normal horse serum (NHS) in PBS for 30 min. Sections were incubated with mouse 158 monoclonal anti-PgP9.5 antibody (dilution 1:2000 with 3% NHS) at room temperature 159 overnight. Sections were then washed twice in PBS and incubated with a peroxidase-160 conjugated secondary antibody (IgG anti-mouse, 1:500 diluted in PBS with 3% NHS) for 30 161 min, then incubated with streptavin peroxide (dilution 1:1000 with 3% NHS) for 1 hour. 162 Binding sites were revealed using a red chromogen (NovaRed Peroxidase Substrate Kit, 163 Vector, USA) according to manufacturer instructions and incubated for 2 to 3 min. Slides 164 were washed in distilled water for 5 min before counterstaining in Harris hematoxylin for 30 165 to 60 seconds and allowed to air dry. A primary antibody control was performed using the 166 above protocol on snake (taipan) brain tissue; a secondary antibody control was performed 167 using the above protocol, with the primary antibody incubation step omitted, on snake brain 168 and cephalic skin tissue. Slides were imaged using an optical microscope (BX51, Olympus, 169 Australia) and the saturation and hue of images was adjusted using imaging software (Adobe 170 Photoshop v2017.1.1, Adobe Systems Inc., USA). Unfortunately, due to preservation issues 7 we were unable to perform immunohistochemistry on these cephalic skin sections in A.  (Table 1)  188 were in the resting phase of epidermal shedding cycle; skin samples viewed under the 189 electron microscope (Table 3.1) appeared to be in pre-renewal phase. 190

Cephalic scale organs 191
Observed under a stereomicroscope, the cephalic scale sensillaorgans appeared as 192 unpigmented external elevations ('bumps') of outer skin (Figure 1). Observed under light 193 microscopy, the cephalic scale sensillaorgans of A. laevis ( Figure 2) and H. stokesii (Figure  194 3) shared a similar structure that consisted of a cluster of 9 to 11 cells ('central cells'), which 195 were horizontally arranged, originatinged in the dermis and evaginated evaginating the 196 epidermis to create a dermal capsulepapilla ('papilla'). The ratio of length to diameter of the 197 dermal capsulepapilla was approximately 1:1 for both A. laevis and H. stokesii (Table S1). 198 The dermal capsulepapilla was occasionally tapered at its basal end in H. stokesii (Figure  199 3A), but but this likely to be an artefact of tissue sectioning. remained expanded in A. laevis 200 ( Figure 2B, C). In some dermal capsulespapillae we were able to identify a blood vessel 201 leading to (and thus presumably vascularising) the central cells ( Figure 2B). In H. stokesii, 202  Figure 5A, B). These discoid endings were primarily located above the dermal 228 capsulepapilla, but were also present in flat epidermis that did not contain sensillaorgans 229 ( Figure 6A). Unfortunately, the second type of dermal capsulespapillae in H. stokesii 230 (described above; Figure 4) were not present in the sections stained for 231 immunohistochemistry. 232 Immunoreactions were also localised to ovoid structures within the cephalic dermis of 233 H. stokesii (Figure 3.6). These structures corresponded to lamellar cells that were ovoid in 234 shape and resembled small Pacinian-like corpuscles (mean length 29 ± 15 µm and mean 235 diameter of 22 ± 12 µm; Table S1). The location of these 'lamellar corpuscles' in H. stokesii 236 9 ranged from 61 to 124 µm (mean 93 µm) depth from the basal layer of the epidermis. 237 Lamellar corpuscles were also identified in the cephalic dermis of A. laevis that were a 238 similar ovoid shape (mean length 37 ± 26 µm and mean diameter 25 ± 5 µm; Figure 2C) to 239 those found on the cephalic dermis of H. stokesii. The location of the lamellar corpuscles in 240 A. laevis ranged from 53 to 168 µm (mean 118 µm; Table S1) depth from the basal layer of 241 the epidermis. Although the lamellar corpuscles were dispersed throughout the dermis 242 (stratum laxum), they were often subjacent to scale sensillaorgans ( Figure 2C; Figure 3B,C). 243 Unfortunately, due to preservation issues we were unable to perform immunohistochemistry 244 on these cephalic skin sections in A. laevis. 245 The dermal capsulepapilla of a scale sensillumorgan was observed in A. laevis using 246 electron microscopy (Figure 7). High magnification images showed a cluster of central cells 247 within the dermal capsulepapilla ( Figure 7B). These central cells were distinguished from 248 surrounding keratinocytes by their round shape and lack of tonofilaments ( Figure 7B  10 with a ratio of length and diameter of 1:1 (Table S1; Figure 8B). Although the dermal 270 capsulepapilla displaced the surrounding epidermal layer (including the columnar cells of the 271 stratum germinativum) this did not result in elevations of the outer epidermis ( Figure 6B). 272 The epidermis above the dermal capsulepapilla was 42% thinner than adjacent epidermis that 273 did not contain dermal capsulespapillae (11 µm; t = −4.65, 86 d.f., P < 0.001) and slightly 274 thinner (5.5 µm) than the cap cellsepidermis above of cephalic scale sensillaorgans (t = 2.59, 275 18 d.f., P = 0.02). Subjacent to these tail dermal capsulespapillae, collagen fibres in the 276 dermis (stratum laxum) were dispersed and melanosomes could not be seen ( Figure 8B). 277 Unfortunately, due to preservation issues we were unable to perform immunohistochemistry 278 on these tail sections. 279

Cephalic scale organs 281
Scale sensillaorgans and dermal capsulepapillae 282 Previous work found that the cephalic sensillascale organs of sea snakes are substantially 283 more protruding and often cover a larger proportion of the scale surface than the sensillascale 284 organs of terrestrial hydrophiine snakes (7). The present study shows that, despite these 285 differences, sensillaorgans in sea snakes have retained a similar underlying ultrastructure to 286 their terrestrial counterparts. The sensillascale organs examined in Aipysurus laevis and 287 Hydrophis stokesii are characterised by a dermal capsulepapilla that consists of an 288 aggregation of central cells with collagen fibres, blood vessels and nerve axons in the 289 intercellular domain that together displace the surrounding epidermis (Figure 2-7). A similar 290 underlying structure has been reported for the cephalic scale sensillaorgans of ten terrestrial 291 species representing the several major phylogenetic groups of snakes squamates including 292 (agamids, henophidians, iguanids scolecophidians, and colubroids, ) and lizards (agamids, 293 iguanids and varanids) (10,21-23,42-45). In terrestrial snakes and sea snakes, the epidermis 294 above the dermal capsulepapilla is comprised of columnar keratinocytes (i.e. stratum 295 germinavatum) that form a layer that is 15% to 50% thinner than the epidermis of adjacent 296 flat skin. The columnar keratinocytes above the dermal capsulepapilla have been described as 297 'cap cells' in snakes and suggested to provide protection against abrasion or aid in 298 transducing mechanosensory stimuli (22). 299 We discovered that sea snake skin contained free nerve axons that extend from the dermis 300 and terminate within the alpha layer (epidermis) as distinct discoid structures (Figure 35). In 301 Commented [JC3]: 'Squamates' as snakes and lizards are not monophyletic groups terrestrial colubroid snakes, these structures have variously been described as 'discoid 302 receptors' (21), 'end bulbs' (20) and 'button-like' (10) nerve endings. In the sea snake skin, 303 we found discoid receptors distributed throughout the epidermis, but aggregated above the 304 dermal capsulespapillae (Figure 5A The dome-shape and often high scale-coverage of scale mechanoreceptors in sea snakes 359 suggests divergent selection on these organs in marine environments, either for retained 360 (ancestral) enhanced sensitivity to tactile stimuli or a derived sensitivity to hydrodynamic 361 stimuli. Sea snakes forage in benthic habitats, frequently probing burrows and crevices as do 362 terrestrial snakes on land (49)., but there There is no obvious reason thatwhy sea snakes 363 should require a heightened tactile sense compared to terrestrial species. Sea snakes forage in 364 benthic habitats, frequently probing burrows and crevices as do terrestrial snakes on land 365 (51). It seems more likely that sea snakes have experienced selection pressures for sensitivity 366 to hydrodynamic stimuli (7). Observations of the sea snake Hydrophis (Pelamis) platurus 367 approaching and biting a vibrating object (50) provides some behavioural evidence that sea but our understanding of the spatial ecology, and thus long-range navigation abilities, of sea 503 snakes is limited. 504

Conclusions 505
Our study shows that the ultrastructure of cephalic sensillascale organs of in sea snakes 506 closely resembles the mechanosensitive Meissner-like corpuscles that underlie the scale 507 sensillaorgans inof terrestrial snakes. This provides evidence that the sensillascale organs of 508 marine hydrophiines lineages have retained an ancestral mechanosensory function. Our