The neuronal clock network in the polar key species Antarctic krill ( Euphausia superba )
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eLife Assessment
This foundational and valuable study expands our understanding of circadian clock work in non-model taxa in wider environmental niches, using solid methods for protein and RNA detection to describe the expression pattern of PDH, cry2, and per in the central nervous system of Euphausia superba. While the anatomical annotation is extensive, support for the identification of the clock network is incomplete.
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Abstract
Organisms are exposed to predictable daily and seasonal environmental oscillations. Biological clocks enable organisms to anticipate these changes and coordinate physiology and behaviour accordingly. While circadian mechanisms are well studied in terrestrial model organisms, little is known about the neuronal organisation of biological clocks in ecologically important species, especially in the marine environment. Antarctic krill ( Euphausia superba ) is central to the functioning of the Southern Ocean ecosystem and relies on precise timing to cope with the extreme, high-latitude fluctuations in photoperiod, food availability, and sea-ice cover in its habitat. Despite evidence for circadian and seasonal rhythms in krill behaviour and physiology, the neuronal architecture underlying these timing processes has remained unresolved. In this study, we use in situ hybridisation and antibody staining to characterise the circadian clock in the krill brain. Immunostaining with an antibody against crustacean β-Pigment-dispersing hormone (β-PDH) reveals distinct clusters of PDH-positive neurons in the optic lobes and dorsal central brain, along with an extensive PDH-positive fibre network. We further localise transcripts of the core clock genes cryptochrome-2 ( cry2 ) and period ( per ) in cell clusters in the optic lobes, which also include the PDH-positive neurons. More specifically, PDH-positive neurons are a subgroup of the cry2 and per -positive cells. Together, these findings provide the first description of the neuronal architecture of the circadian clock in Antarctic krill and establish essential groundwork for future studies on biological timing, environmental adaptation, and the resilience of this key species in a rapidly changing Southern Ocean.
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-
eLife Assessment
This foundational and valuable study expands our understanding of circadian clock work in non-model taxa in wider environmental niches, using solid methods for protein and RNA detection to describe the expression pattern of PDH, cry2, and per in the central nervous system of Euphausia superba. While the anatomical annotation is extensive, support for the identification of the clock network is incomplete.
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Reviewer #1 (Public review):
Summary:
Hüppe and colleagues characterized the network of neurons in the central nervous system of Antarctic krill that contained pigment-dispersing hormone (PDH), an important output factor in the circadian clock of insects. These neurons in the brain are putative clock neurons since a subset also expressed the clock genes period and cryptochrome 2. As one of the ocean's major contributors to biomass, krill is an ecologically important marine species that experiences challenging daily and seasonal environmental fluctuations in its high-latitude habitat. A comprehensive study of krill's internal clock may help to understand the extent of its resilience to the rapidly changing climate.
The authors used antibody staining against PDH across the whole central nervous system and additional in situ hybridization …
Reviewer #1 (Public review):
Summary:
Hüppe and colleagues characterized the network of neurons in the central nervous system of Antarctic krill that contained pigment-dispersing hormone (PDH), an important output factor in the circadian clock of insects. These neurons in the brain are putative clock neurons since a subset also expressed the clock genes period and cryptochrome 2. As one of the ocean's major contributors to biomass, krill is an ecologically important marine species that experiences challenging daily and seasonal environmental fluctuations in its high-latitude habitat. A comprehensive study of krill's internal clock may help to understand the extent of its resilience to the rapidly changing climate.
The authors used antibody staining against PDH across the whole central nervous system and additional in situ hybridization for cry2 and per mRNA, with a focus on the supraesophageal ganglion. There, they identified the major neuropils in the eye stalks and central brain of Antarctic krill. The resulting staining pattern aligns with the identified circadian clock network in insects and PDH-expressing networks in other crustaceans, making these neurons highly likely candidates for krill clock neurons.
Strengths:
(1) This study provides the first clues about the circadian clock architecture in a non-model organism in chronobiology, Antarctic krill, with a clear 3D reconstruction of the putative clock network.
(2) The authors effectively place their results within the extensive body of literature on arthropod circadian clock networks to argue that the neurons they describe are likely the circadian clock in krill.
Weaknesses:
(1) The data presented here are not sufficient to support the claim that the described network is the circadian clock because functional evidence is missing.
(2) Additionally, the study falls short of identifying any elements of the positive limb of the canonical circadian clock transcriptional-translational feedback loop, e.g., clk or cyc, in the PDH-expressing neurons.
(3) No sample sizes are reported, making it difficult for readers to assess the generalizability of the presented data.
-
Reviewer #2 (Public review):
Summary:
This study advances our understanding of the neuronal basis of the circadian clock in pancrustaceans. It extends our knowledge on the pigment-dispersing hormone system and provides links to information on the expression of core clock components, cryptochrome 2, and period. The data are sound and well-documented.
Comments:
The neuronal components of the arthropod circadian clock system have been analysed extensively in insects. Much less information on this system is available on malacostraca crustacea crustaceans. However, considering that malacostracan crustaceans and insects go back to a common pancrustacean ancestor and considering that we know that the brain architecture in these two groups shares many commonalities (see, e. g., extensive reviews by N. J. Strausfeld), we have to expect that …
Reviewer #2 (Public review):
Summary:
This study advances our understanding of the neuronal basis of the circadian clock in pancrustaceans. It extends our knowledge on the pigment-dispersing hormone system and provides links to information on the expression of core clock components, cryptochrome 2, and period. The data are sound and well-documented.
Comments:
The neuronal components of the arthropod circadian clock system have been analysed extensively in insects. Much less information on this system is available on malacostraca crustacea crustaceans. However, considering that malacostracan crustaceans and insects go back to a common pancrustacean ancestor and considering that we know that the brain architecture in these two groups shares many commonalities (see, e. g., extensive reviews by N. J. Strausfeld), we have to expect that crustaceans and insects share many of the characteristics of the circadian system. This is the case, e. g., for the network of pigment-dispersing hormone-positive neurons. The authors cite these studies, although late in the paper (discussion, line 339ff), and I suggest to move this info into the introduction: "339 ff: The arborization pattern of the PDH-network has been described in various malacostracan crustaceans, including Carcinus maenas (Alexander et al., 2020; Mangerich & Keller, 1988; Mangerich et al., 1987), Cancer productus (Hsu et al., 2008), Orconectes limosus (de Kleijn et al., 1993; Mangerich & Keller, 1988; Mangerich et al., 1987), Homarus americanus (Harzsch etal., 2009), Cherax destructor, Procambarus clarkii (Sullivan et al., 2009), and Procambarus virginalis (Luna et al., 2010)."
The strength of this paper is that it extends our knowledge on the PDH system and brings together neuroanatomical information on PDH-positive neurons with information on the expression of core clock components, cryptochrome 2, and period. That way, it advances our understanding of the neuronal basis of the circadian clock in pancrustaceans. The data are sound and well documented, and the authors are to be applauded for the superb dissection presented in Figure 1.
Below, please find some essential suggestions on how to further improve the paper.
(1) Framing of the study:
I know that krill is a key element of the Southern Ocean's food webs, but my sense is that discussing the current findings in a context of resilience of this species to global ocean change means largely overselling this study:
- Lines 47, 48: "and the resilience of this key species in a rapidly changing Southern Ocean."
- Lines 70 ff: "Hence, understanding the mechanisms of adaptation, including biological clocks, is crucial for predicting how species, populations, and whole ecosystems will respond to climate change."
- 154 ff: "The Southern Ocean environment experiences rapid change (Abram et al., 2025; Meredith et al., 2019; Thomalla et al., 2023). To assess krill's resilience to environmental changes, understanding the mechanisms that govern daily and seasonal timing in krill is essential."
- 325 ff: "The rhythmic adaptation of krill to its high-latitude environment is key to its success in the Southern Ocean, which in turn represents a cornerstone for the well-being of the whole krill centred ecosystem. To predict krill's resilience to rapid environmental changes, it is essential to understand the mechanisms that govern daily and seasonal timing in krill."
- 597 ff: "A detailed mechanistic understanding of the flexibility of clock-based processes is therefore essential to predict krill resilience in a changing Southern Ocean."
My understanding is that duration of day length is one of the most predictable environmental drivers, and - despite the seasonal changes of day length - nevertheless a very stable one compared to fluctuations of environmental drivers such as temperature or salinity (see, e.g. this recent review on environmental driver fluctuations on nervous system functioning in crustaceans: Stein W, Harzsch S (2021) The Neurobiology of Ocean Change - insights from decapod crustaceans. Zoology: 125887. https://www.sciencedirect.com/science/article/pii/S094420062030146X).
I do not see how global ocean change may significantly change day length, and what this study has to do with understanding this species' resilience against ocean change. I suggest that you explain in more detail why the light day length will change in the future or strongly tone this aspect. Statements such as Line 76 ff: "Due to their disproportionate importance for ecosystem function, understanding the resilience of ecological key species is essential in assessing the fate of ecosystems in the future." are completely out of focus here and, again, trying to oversell the current study.
(2) Uncited essential studies of crustacean neuroanatomy, missing connection to contemporary crustacean neurobiology:
- Line 157: "despite the ecological importance of E. superba, only very little is known about its neurobiology".
- Line 329: "However, so far, little was known about the neurobiology of krill in general."
I agree that this species' brain is understudied, but this makes it even more important to cite the little information that IS available. Please consider this essential reading for any crustacean neurobiologist: "Sandeman, D.C., Scholtz, G., Sandeman, R.E., 1993. Brain evolution in decapod crustacea. J. Exp. Zool. 265, 112-133." to find information on the basic brain anatomy in E. superba.
The manuscript in many places seems to reinvent the wheel and raises the impression that our knowledge of crustacean brain morphology is close to zero. The authors in places seem to operate in a vacuum, and I find it disturbing that in a study on the crustacean brain, very few references are provided to studies on crustacean brain anatomy, such as the following essential book chapter: "Schmidt, M., 2016. Malacostraca. In: Schmidt-Rhaesa, A., Harzsch, S., Purschke, G. (Eds.), Structure & Evolution of Invertebrate Nervous Systems. Oxford University Press, Oxford, pp. 529-582. https://www.researchgate.net/publication/315366157"
In terms of brain anatomy, I would like to know if the authors have a hypothesis on whether and how their target species' brain structure may be similar or different to the brains of other "shrimps" as described, e. g., in the following studies. If so, please elaborate in the introduction:
Krieger J, Hörnig MK, Sandeman RE, Sandeman DC, Harzsch S (2020), Masters of communication: The brain of the banded cleaner shrimp Stenopus hispidus (Olivier, 1811) with an emphasis on sensory processing areas. Journal of Comparative Neurology 528(9): 1561-1587.
Meth R, Wittfoth C, Harzsch S (2017) Brain architecture of the Pacific White Shrimp Penaeus vannamei Boone, 1931 (Malacostraca, Dendrobranchiata): correspondence of brain structure and sensory input? Cell and Tissue Research 369(2): 255-271.
(3) Lacking rigor and command of crustacean brain nomenclature
I suggest that for their brain nomenclature, the authors should rigorously stick to that laid out by Sandeman et al. 1992 (not yet cited in the ms): Sandeman, D.C., Sandeman, R.E., Derby, C.D., Schmidt, M., 1992. Morphology of the brain of crayfish, crabs, and spiny lobsters: a common nomenclature for homologous structures. Biol. Bull. 183, 304-326.
More specifically, in lines 41, 163, 199, 204, 207, and throughout the paper, the authors use the terms "Optic lobes" or "optic lobe neuropils". To the best of my knowledge, "optic lobe" is not a term used in crustacean neuroanatomy at all (as opposed to insects). Lamina, medulla, and lobula are collectively referred to as "visual neuropils" (see Krieger, J., Hörnig, M. K., Sandeman, R. E., Sandeman, D. C., & Harzsch, S. (2020). Masters of communication: The brain of the banded cleaner shrimp Stenopus hispidus (Olivier, 1811) with an emphasis on sensory processing areas. Journal of Comparative Neurology, 528(9), 1561-1587. https://doi.org/10.1002/CNE.24831). The medulla terminalis and mushroom bodies are referred to as "lateral protocerebrum". All afore-mentioned neuropils are summarized as "eyestalk neuropils" (compare nomenclature in Schmidt 2016 as referenced above).
Line 170, 172, 175 ff, and Figure 1. "abdomen", "abdominal ganglia": Contra the book chapter by Siegel 2016 "Introducing Antarctic Krill Euphausia superba Dana, 1850", his Fig. 1.2, the "tail" of crustaceans in most books on crustacean anatomy is not called "abdomen" but instead "pleon"; hence the name "pleopods" for the appendages of the pleon (instead of "abdomipods"). What is more, I suggest using the terms "pleon ganglia" instead of "abdominal ganglia", following the terminology suggested in "Harzsch S, Sandeman D, Chaigneau J (2012) Morphology and development of the central nervous system. In: Forest J and von Vaupel Klein JC (Eds.). Treatise on Zoology - Anatomy, Taxonomy, Biology. The Crustacea Vol. 3. Brill, Leiden pp. 9-236."
Line 174: "thoracic ganglia". In Figure 1, there is a labelling mistake as these ganglia are named "thoracaic ganglia".
Line 176, and throughout the paper: "supraesophageal ganglion". Following the standard nomenclature for crustaceans (see, e. g., Schmidt, M., 2016. Malacostraca. In: Schmidt-Rhaesa, A., Harzsch, S., Purschke, G. (Eds.), Structure & Evolution of Invertebrate Nervous Systems. Oxford University Press, Oxford, pp. 529-582. https://www.researchgate.net/publication/315366157", this structure (as in insects) is typically called a "brain". For terminology, also consult the following nomenclature paper: "Richter, S., Loesel, R., Purschke, G., Schmidt-Rhaesa, A., Scholtz, G., Stach, T., Vogt, L., Wanninger, A., Brenneis, G., Döring, C., Faller, S., Fritsch, M., Grobe, P., Heuer, C. M., Kaul, S., Møller, O. S., Müller, C. H. G., Rieger, V., Rothe, B. H., Stegner, M., Harzsch, S. (2010). Invertebrate neurophylogeny: Suggested terms and definitions for a neuroanatomical glossary. Frontiers in Zoology, 7. https://doi.org/10.1186/1742-9994-7-29".
Line 212, and throughout the paper - hemielliposoid body: please refer to Harzsch Krieger 2011 and the numerous references to studies by Strausfeld cited therein in crustaceans. Strausfeld has provided compelling evidence that the crustacean hemiellipsoid body is equivalent to the insect mushroom body, so this term should be replaced. Harzsch, S., & Krieger, J. (2021). Genealogical relationships of mushroom bodies, hemiellipsoid bodies, and their afferent pathways in the brains of Pancrustacea: Recent progress and open questions. Arthropod Structure & Development, 65, 101100. HYPERLINK "https://doi.org/10.1016/J.ASD.2021.101100" https://doi.org/10.1016/J.ASD.2021.101100.
Legend, figure 2, and others, and throughout the paper: "The olfactory neuropiles comprise the lateral antennal neuropile (LAN, ochre), the olfactory lobes (OL, yellow), and the antennal neuropile (AnN, green)." This is a strange terminological mix that you should urgently revise according to the standard terminology by Sandeman et al. 1992 (as referenced above). The LAN is the lateral antenna 1 neuropil. The AnN is the antenna 2 neuropil. The AnN is NOT deutocerebral but tritocerebral.
-
Author response:
Reviewer #1 (Public review):
Summary:
Hüppe and colleagues characterized the network of neurons in the central nervous system of Antarctic krill that contained pigment-dispersing hormone (PDH), an important output factor in the circadian clock of insects. These neurons in the brain are putative clock neurons since a subset also expressed the clock genes period and cryptochrome 2. As one of the ocean's major contributors to biomass, krill is an ecologically important marine species that experiences challenging daily and seasonal environmental fluctuations in its high-latitude habitat. A comprehensive study of krill's internal clock may help to understand the extent of its resilience to the rapidly changing climate.
The authors used antibody staining against PDH across the whole central nervous system and additional in …
Author response:
Reviewer #1 (Public review):
Summary:
Hüppe and colleagues characterized the network of neurons in the central nervous system of Antarctic krill that contained pigment-dispersing hormone (PDH), an important output factor in the circadian clock of insects. These neurons in the brain are putative clock neurons since a subset also expressed the clock genes period and cryptochrome 2. As one of the ocean's major contributors to biomass, krill is an ecologically important marine species that experiences challenging daily and seasonal environmental fluctuations in its high-latitude habitat. A comprehensive study of krill's internal clock may help to understand the extent of its resilience to the rapidly changing climate.
The authors used antibody staining against PDH across the whole central nervous system and additional in situ hybridization for cry2 and per mRNA, with a focus on the supraesophageal ganglion. There, they identified the major neuropils in the eye stalks and central brain of Antarctic krill. The resulting staining pattern aligns with the identified circadian clock network in insects and PDH-expressing networks in other crustaceans, making these neurons highly likely candidates for krill clock neurons.
Strengths:
(1) This study provides the first clues about the circadian clock architecture in a non-model organism in chronobiology, Antarctic krill, with a clear 3D reconstruction of the putative clock network.
(2) The authors effectively place their results within the extensive body of literature on arthropod circadian clock networks to argue that the neurons they describe are likely the circadian clock in krill.
Weaknesses:
(1) The data presented here are not sufficient to support the claim that the described network is the circadian clock because functional evidence is missing.
(2) Additionally, the study falls short of identifying any elements of the positive limb of the canonical circadian clock transcriptional-translational feedback loop, e.g., clk or cyc, in the PDH-expressing neurons.
(3) No sample sizes are reported, making it difficult for readers to assess the generalizability of the presented data.
We thank the reviewer for recognizing the contribution of this study to advancing our understanding of clock systems in non-traditional model organisms. We acknowledge that definitive functional evidence would require the generation of null mutants of core clock components, which is currently not feasible in this species. In a revised version, we will adjust our claims to more precisely reflect the evidence presented and include sample sizes to allow the reader to better assess the representativeness of the results.
Reviewer #2 (Public review):
Summary:
This study advances our understanding of the neuronal basis of the circadian clock in pancrustaceans. It extends our knowledge on the pigment-dispersing hormone system and provides links to information on the expression of core clock components, cryptochrome 2, and period. The data are sound and well-documented.
Comments:
The neuronal components of the arthropod circadian clock system have been analysed extensively in insects. Much less information on this system is available on malacostraca crustacea crustaceans. However, considering that malacostracan crustaceans and insects go back to a common pancrustacean ancestor and considering that we know that the brain architecture in these two groups shares many commonalities (see, e. g., extensive reviews by N. J. Strausfeld), we have to expect that crustaceans and insects share many of the characteristics of the circadian system. This is the case, e. g., for the network of pigment-dispersing hormone-positive neurons. The authors cite these studies, although late in the paper (discussion, line 339ff), and I suggest to move this info into the introduction: "339 ff: The arborization pattern of the PDH-network has been described in various malacostracan crustaceans, including Carcinus maenas (Alexander et al., 2020; Mangerich & Keller, 1988; Mangerich et al., 1987), Cancer productus (Hsu et al., 2008), Orconectes limosus (de Kleijn et al., 1993; Mangerich & Keller, 1988; Mangerich et al., 1987), Homarus americanus (Harzsch etal., 2009), Cherax destructor, Procambarus clarkii (Sullivan et al., 2009), and Procambarus virginalis (Luna et al., 2010)."
The strength of this paper is that it extends our knowledge on the PDH system and brings together neuroanatomical information on PDH-positive neurons with information on the expression of core clock components, cryptochrome 2, and period. That way, it advances our understanding of the neuronal basis of the circadian clock in pancrustaceans. The data are sound and well documented, and the authors are to be applauded for the superb dissection presented in Figure 1.
Below, please find some essential suggestions on how to further improve the paper.
(1) Framing of the study:
I know that krill is a key element of the Southern Ocean's food webs, but my sense is that discussing the current findings in a context of resilience of this species to global ocean change means largely overselling this study:
Lines 47, 48: "and the resilience of this key species in a rapidly changing Southern Ocean."
Lines 70 ff: "Hence, understanding the mechanisms of adaptation, including biological clocks, is crucial for predicting how species, populations, and whole ecosystems will respond to climate change."
154 ff: "The Southern Ocean environment experiences rapid change (Abram et al., 2025; Meredith et al., 2019; Thomalla et al., 2023). To assess krill's resilience to environmental changes, understanding the mechanisms that govern daily and seasonal timing in krill is essential."
325 ff: "The rhythmic adaptation of krill to its high-latitude environment is key to its success in the Southern Ocean, which in turn represents a cornerstone for the well-being of the whole krill centred ecosystem. To predict krill's resilience to rapid environmental changes, it is essential to understand the mechanisms that govern daily and seasonal timing in krill."
597 ff: "A detailed mechanistic understanding of the flexibility of clock-based processes is therefore essential to predict krill resilience in a changing Southern Ocean."
My understanding is that duration of day length is one of the most predictable environmental drivers, and - despite the seasonal changes of day length - nevertheless a very stable one compared to fluctuations of environmental drivers such as temperature or salinity (see, e.g. this recent review on environmental driver fluctuations on nervous system functioning in crustaceans: Stein W, Harzsch S (2021) The Neurobiology of Ocean Change - insights from decapod crustaceans. Zoology: 125887. https://www.sciencedirect.com/science/article/pii/S094420062030146X).
I do not see how global ocean change may significantly change day length, and what this study has to do with understanding this species' resilience against ocean change. I suggest that you explain in more detail why the light day length will change in the future or strongly tone this aspect. Statements such as Line 76 ff: "Due to their disproportionate importance for ecosystem function, understanding the resilience of ecological key species is essential in assessing the fate of ecosystems in the future." are completely out of focus here and, again, trying to oversell the current study.
(2) Uncited essential studies of crustacean neuroanatomy, missing connection to contemporary crustacean neurobiology:
Line 157: "despite the ecological importance of E. superba, only very little is known about its neurobiology".
Line 329: "However, so far, little was known about the neurobiology of krill in general."
I agree that this species' brain is understudied, but this makes it even more important to cite the little information that IS available. Please consider this essential reading for any crustacean neurobiologist: "Sandeman, D.C., Scholtz, G., Sandeman, R.E., 1993. Brain evolution in decapod crustacea. J. Exp. Zool. 265, 112-133." to find information on the basic brain anatomy in E. superba.
The manuscript in many places seems to reinvent the wheel and raises the impression that our knowledge of crustacean brain morphology is close to zero. The authors in places seem to operate in a vacuum, and I find it disturbing that in a study on the crustacean brain, very few references are provided to studies on crustacean brain anatomy, such as the following essential book chapter: "Schmidt, M., 2016. Malacostraca. In: Schmidt-Rhaesa, A., Harzsch, S., Purschke, G. (Eds.), Structure & Evolution of Invertebrate Nervous Systems. Oxford University Press, Oxford, pp. 529-582. https://www.researchgate.net/publication/315366157"
In terms of brain anatomy, I would like to know if the authors have a hypothesis on whether and how their target species' brain structure may be similar or different to the brains of other "shrimps" as described, e. g., in the following studies. If so, please elaborate in the introduction:
Krieger J, Hörnig MK, Sandeman RE, Sandeman DC, Harzsch S (2020), Masters of communication: The brain of the banded cleaner shrimp Stenopus hispidus (Olivier, 1811) with an emphasis on sensory processing areas. Journal of Comparative Neurology 528(9): 1561-1587.
Meth R, Wittfoth C, Harzsch S (2017) Brain architecture of the Pacific White Shrimp Penaeus vannamei Boone, 1931 (Malacostraca, Dendrobranchiata): correspondence of brain structure and sensory input? Cell and Tissue Research 369(2): 255-271.
(3) Lacking rigor and command of crustacean brain nomenclature
I suggest that for their brain nomenclature, the authors should rigorously stick to that laid out by Sandeman et al. 1992 (not yet cited in the ms): Sandeman, D.C., Sandeman, R.E., Derby, C.D., Schmidt, M., 1992. Morphology of the brain of crayfish, crabs, and spiny lobsters: a common nomenclature for homologous structures. Biol. Bull. 183, 304-326.
More specifically, in lines 41, 163, 199, 204, 207, and throughout the paper, the authors use the terms "Optic lobes" or "optic lobe neuropils". To the best of my knowledge, "optic lobe" is not a term used in crustacean neuroanatomy at all (as opposed to insects). Lamina, medulla, and lobula are collectively referred to as "visual neuropils" (see Krieger, J., Hörnig, M. K., Sandeman, R. E., Sandeman, D. C., & Harzsch, S. (2020). Masters of communication: The brain of the banded cleaner shrimp Stenopus hispidus (Olivier, 1811) with an emphasis on sensory processing areas. Journal of Comparative Neurology, 528(9), 1561-1587. https://doi.org/10.1002/CNE.24831). The medulla terminalis and mushroom bodies are referred to as "lateral protocerebrum". All afore-mentioned neuropils are summarized as "eyestalk neuropils" (compare nomenclature in Schmidt 2016 as referenced above).
Line 170, 172, 175 ff, and Figure 1. "abdomen", "abdominal ganglia": Contra the book chapter by Siegel 2016 "Introducing Antarctic Krill Euphausia superba Dana, 1850", his Fig. 1.2, the "tail" of crustaceans in most books on crustacean anatomy is not called "abdomen" but instead "pleon"; hence the name "pleopods" for the appendages of the pleon (instead of "abdomipods"). What is more, I suggest using the terms "pleon ganglia" instead of "abdominal ganglia", following the terminology suggested in "Harzsch S, Sandeman D, Chaigneau J (2012) Morphology and development of the central nervous system. In: Forest J and von Vaupel Klein JC (Eds.). Treatise on Zoology - Anatomy, Taxonomy, Biology. The Crustacea Vol. 3. Brill, Leiden pp. 9-236."
Line 174: "thoracic ganglia". In Figure 1, there is a labelling mistake as these ganglia are named "thoracaic ganglia".
Line 176, and throughout the paper: "supraesophageal ganglion". Following the standard nomenclature for crustaceans (see, e. g., Schmidt, M., 2016. Malacostraca. In: Schmidt-Rhaesa, A., Harzsch, S., Purschke, G. (Eds.), Structure & Evolution of Invertebrate Nervous Systems. Oxford University Press, Oxford, pp. 529-582. https://www.researchgate.net/publication/315366157", this structure (as in insects) is typically called a "brain". For terminology, also consult the following nomenclature paper: "Richter, S., Loesel, R., Purschke, G., Schmidt-Rhaesa, A., Scholtz, G., Stach, T., Vogt, L., Wanninger, A., Brenneis, G., Döring, C., Faller, S., Fritsch, M., Grobe, P., Heuer, C. M., Kaul, S., Møller, O. S., Müller, C. H. G., Rieger, V., Rothe, B. H., Stegner, M., Harzsch, S. (2010). Invertebrate neurophylogeny: Suggested terms and definitions for a neuroanatomical glossary. Frontiers in Zoology, 7. https://doi.org/10.1186/1742-9994-7-29".
Line 212, and throughout the paper - hemielliposoid body: please refer to Harzsch Krieger 2011 and the numerous references to studies by Strausfeld cited therein in crustaceans. Strausfeld has provided compelling evidence that the crustacean hemiellipsoid body is equivalent to the insect mushroom body, so this term should be replaced. Harzsch, S., & Krieger, J. (2021). Genealogical relationships of mushroom bodies, hemiellipsoid bodies, and their afferent pathways in the brains of Pancrustacea: Recent progress and open questions. Arthropod Structure & Development, 65, 101100. HYPERLINK "https://doi.org/10.1016/J.ASD.2021.101100" https://doi.org/10.1016/J.ASD.2021.101100.
Legend, figure 2, and others, and throughout the paper: "The olfactory neuropiles comprise the lateral antennal neuropile (LAN, ochre), the olfactory lobes (OL, yellow), and the antennal neuropile (AnN, green)." This is a strange terminological mix that you should urgently revise according to the standard terminology by Sandeman et al. 1992 (as referenced above). The LAN is the lateral antenna 1 neuropil. The AnN is the antenna 2 neuropil. The AnN is NOT deutocerebral but tritocerebral.
We thank the reviewer for acknowledging this paper's contribution to our understanding of the neuronal basis of the circadian clock in Pancrustaceans, as well as for the positive evaluation of the data documentation and presentation.
We would like to clarify that we are aware of the existing body of literature on crustacean neuroanatomy and did not intend to present our data as a first in this field. This study intersects multiple communities (e.g., chronobiology, crustacean neurobiology, krill ecology), and the current focus of the manuscript arose from an attempt to make the paper as accessible to these communities as possible. We acknowledge, however, that the current version falls short in its engagement with the existing literature on crustacean brain anatomy. We therefore thank the reviewer for the input on crustacean neuroanatomy and its nomenclature, which will help us improve the manuscript in these respects. In a revised version, we plan to adjust the framing of the study to more precisely reflect the data presented. This will include better situating the present findings within the existing literature on crustacean neuroanatomy and its specific nomenclature, while toning down the emphasis on ecological importance and implications.
Reviewer #3 (Public review):
Summary:
A solid and very descriptive study of gene expression of three factors in krill, PDH, per, and cry2 that are important for circadian rhythms in insects. The results reveal optic areas in which PDH colocalises with each or per and cry2, and central brain areas where it does not. The authors speculate on the functional implications of their results for biological rhythms.
Comments:
This manuscript describes a detailed anatomical study of the brain of krill in a circadian gene expression context. The results are well described, and the work is well done considering the obvious technical/practical difficulties of working with this species. Having stated that, the authors in their Methods write that the animals, after being caught, were placed in constant darkness. Is there any idea at all of when in ZT these brains were processed? Are the representations of gene expression taken at random around the clock? Perhaps the authors might make this explicit somewhere in the ms as it is an important point.
The manuscript focuses mostly on PDH and its overlap or not with per or cry2. I found Figures 5 and 6 particularly confusing. The panels show PDH colocalising (or not-filled or unfilled arrows) with cry2 or with per. What they do not show (to me) is that per and cry2 colocalise. Now, of course, they probably do, but Figure 5 does not show this - or am I misinterpreting it? In Figure 6 again, I cannot see any panels with per and cry2 overlaid. Seems different sections were used for each probe? Is that what 'Areas with high per/ cry2-expression are marked by white arrowheads' means? I see that lines 493 and 494 confirm my suspicions that per/cry were not shown to be colocalised. Perhaps the authors could make this clearer up front than halfway through the Discussion, and clarify this in their legends, which are a little misleading in this respect?
We thank the reviewer for his positive evaluation of our work, acknowledging the difficulty when working with this organism, and for the constructive comments. In a revised version of the manuscript, we will clarify the sampling time in the Methods. We will also state upfront — and in the figure legends — that per and cry2 were assessed on separate sections and their direct co-localization was therefore not demonstrated. However, as both components were independently shown to co-localize with PDH, their spatial overlap is nevertheless suggested by the shared co-localization with PDH. We will make this reasoning explicit earlier in the manuscript to avoid any misleading implications.
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