Does the egg capsule protect against chronic UV-B radiation? A study based on encapsulated and decapsulated embryos of cuttlefish Sepia officinalis.

Although the egg capsule plays a crucial role in the embryonic development of cephalopods, its ability to protect embryos from Ultraviolet (UV) radiation is unknown. Our study evaluated the photoprotection mechanisms of S. officinalis to UV-B radiation and estimated the ability of the black capsule to act as a physical shield against it. Embryos with and without capsule and juveniles were exposed to four experimental UVB conditions for 55 days. The effects of different UVB doses were evaluated in terms of morphological abnormalities and differences in gene expression between each group. We observed that the development might be severely impaired in embryos exposed to UVB without capsule protection, and these effects were time- and UVB-dose-dependent. In addition, we found variations in gene expression levels (light-sensitive, stress response and DNA repair) in different tissues as a function of UVB doses. We suggest a relationship between morphological abnormalities and the limit of molecular regulation. These results suggest that the quantitative differences in expression are essential for defining the survivability of the embryo face to UVB. Thus, we demonstrated that the egg capsule could ensure successful embryonic development of the cuttlefish S. officinalis even at high doses of UVB.

Cephalopod palaeobiology: evolution and life history of the most intelligent invertebrates: Honoring Sigurd von Boletzky and his contributions to cephalopod research.

Sigurd von Boletzky was a cephalopod researcher who was world-renowned for his enthusiasm for his field of research, for his friendly and calm personality, and, of course, his publications. He dedicated most of his life as active researcher on the development, biology and evolution of coleoids. Nevertheless, he was always curious to learn about other cephalopods as well. Sigurd passed away in Switzerland on September 28th 2020. We dedicate this text and volume to his memory.

Diversity of Light Sensing Molecules and Their Expression During the Embryogenesis of the Cuttlefish (Sepia officinalis).

Eyes morphologies may differ but those differences are not reflected at the molecular level. Indeed, the ability to perceive light is thought to come from the same conserved gene families: opsins and cryptochromes. Even though cuttlefish (Cephalopoda) are known for their visually guided behaviors, there is a lack of data about the different opsins and cryptochromes orthologs represented in the genome and their expressions. Here we studied the evolutionary history of opsins, cryptochromes but also visual arrestins in molluscs with an emphasis on cephalopods. We identified 6 opsins, 2 cryptochromes and 1 visual arrestin in Sepia officinalis and we showed these families undergo several duplication events in Mollusca: one duplication in the arrestin family and two in the opsin family. In cuttlefish, we studied the temporal expression of these genes in the eyes of embryos from stage 23 to hatching and their expression in two extraocular tissues, skin and central nervous system (CNS = brain + optic lobes). We showed in embryos that some of these genes (Sof_CRY6, Sof_reti-1, Sof_reti-2, Sof_r-opsin1 and Sof_v-arr) are expressed in the eyes and not in the skin or CNS. By looking at a juvenile and an adult S. officinalis, it seems that some of these genes (Sof_r-opsin1 and Sof_reti1) are used for light detection in these extraocular tissues but that they set-up later in development than in the eyes. We also showed that their expression (except for Sof_CRY6) undergoes an increase in the eyes from stage 25 to 28 thus confirming their role in the ability of the cuttlefish embryos to perceive light through the egg capsule. This study raises the question of the role of Sof_CRY6 in the developing eyes in cuttlefish embryos and the role and localization of xenopsins and r-opsin2. Consequently, the diversity of molecular actors involved in light detection both in the eyes and extraocular tissues is higher than previously known. These results open the way for studying new molecules such as those of the signal transduction cascade.

Three-dimensional structural evolution of the cuttlefish Sepia officinalis shell from embryo to adult stages.

The cuttlefish shell is an internal structure with a composition and general organization unique among molluscs. Its formation and the structure-function relation are explored during Sepia officinalis development, using computerized axial tomography scanning (CAT-scan) three-dimensional analyses coupled to physical measurements and modelling. In addition to the evolution of the overall form, modifications of the internal structure were identified from the last third embryonic stages to adult. Most of these changes can be correlated to life cycle stages and environmental constraints. Protected by the capsule during embryonic life, the first internal chambers are sustained by isolated pillars formed from the dorsal to the ventral septum. After hatching, the formation of pillars appears to be a progressive process from isolated points to interconnected pillars forming a wall-delineated labyrinthine structure. We analysed the interpillar space, the connectivity and the tortuosity of the labyrinth. The labyrinthine pillar network is complete just prior to the wintering migration, probably to sustain the need to adapt to high pressure and to allow buoyancy regulation. At that time, the connectivity in the pillar network is compensated by an increase in tortuosity, most probably to reduce liquid diffusion in the shell. Altogether these results suggest adjustment of internal calcified structure development to both external forces and physiological needs.

Eye Development in Sepia officinalis Embryo: What the Uncommon Gene Expression Profiles Tell Us about Eye Evolution.

In metazoans, there is a remarkable diversity of photosensitive structures; their shapes, physiology, optical properties, and development are different. To approach the evolution of photosensitive structures and visual function, cephalopods are particularly interesting organisms due to their most highly centralized nervous system and their camerular eyes which constitute a convergence with those of vertebrates. The eye morphogenesis in numerous metazoans is controlled mainly by a conserved Retinal Determination Gene Network (RDGN) including pax, six, eya, and dac playing also key developmental roles in non-retinal structures and tissues of vertebrates and Drosophila. Here we have identified and explored the role of Sof-dac, Sof-six1/2, Sof-eya in eye morphogenesis, and nervous structures controlling the visual function in Sepia officinalis. We compare that with the already shown expressions in eye development of Sof-otx and Sof-pax genes. Rhodopsin is the pigment responsible for light sensitivity in metazoan, which correlate to correlate visual function and eye development. We studied Sof-rhodopsin expression during retina differentiation. By in situ hybridization, we show that (1) all of the RDGN genes, including Sof-pax6, are expressed in the eye area during the early developmental stages but they are not expressed in the retina, unlike Sof-otx, which could have a role in retina differentiation; (2) Sof-rhodopsin is expressed in the retina just before vision gets functional, from stage 23 to hatching. Our results evidence a role of Sof-six1/2, Sof-eya, and Sof-dac in eye development. However, the gene network involved in the retinal photoreceptor differentiation remains to be determined. Moreover, for the first time, Sof-rhodopsin expression is shown in the embryonic retina of cuttlefish suggesting the evolutionary conservation of the role of rhodopsin in visual phototransduction within metazoans. These findings are correlated with the physiological and behavioral observations suggesting that S. officinalis is able to react to light stimuli from stage 25 of organogenesis on, as soon as the first retinal pigments appear.

The Pax gene family: Highlights from cephalopods.

Pax genes play important roles in Metazoan development. Their evolution has been extensively studied but Lophotrochozoa are usually omitted. We addressed the question of Pax paralog diversity in Lophotrochozoa by a thorough review of available databases. The existence of six Pax families (Pax1/9, Pax2/5/8, Pax3/7, Pax4/6, Paxß, PoxNeuro) was confirmed and the lophotrochozoan Paxß subfamily was further characterized. Contrary to the pattern reported in chordates, the Pax2/5/8 family is devoid of homeodomain in Lophotrochozoa. Expression patterns of the three main pax classes (pax2/5/8, pax3/7, pax4/6) during Sepia officinalis development showed that Pax roles taken as ancestral and common in metazoans are modified in S. officinalis, most likely due to either the morphological specificities of cephalopods or to their direct development. Some expected expression patterns were missing (e.g. pax6 in the developing retina), and some expressions in unexpected tissues have been found (e.g. pax2/5/8 in dermal tissue and in gills). This study underlines the diversity and functional plasticity of Pax genes and illustrates the difficulty of using probable gene homology as strict indicator of homology between biological structures.

First proteomic analyses of the dorsal and ventral parts of the Sepia officinalis cuttlebone.

Protein compounds constituting mollusk shells are known for their major roles in the biomineralization processes. These last years, a great diversity of shell proteins have been described in bivalves and gastropods allowing a better understanding of the calcification control by organic compounds and given promising applications in biotechnology. Here, we analyzed for the first time the organic matrix of the aragonitic Sepia officinalis shell, with an emphasis on protein composition of two different structures: the dorsal shield and the chambered part. Our results highlight an organic matrix mainly composed of polysaccharide, glycoprotein and protein compounds as previously described in other mollusk shells, with quantitative and qualitative differences between the dorsal shield and the chamber part. Proteomic analysis resulted in identification of only a few protein compounds underlining the lack of reference databases for Sepiidae. However, most of them contain domains previously characterized in matrix proteins of aragonitic shell-builder mollusks, suggesting ancient and conserved mechanisms of the aragonite biomineralization processes within mollusks. BIOLOGICAL SIGNIFICANCE: The cuttlefish's inner shell, better known under the name "cuttlebone", is a complex mineral structure unique in mollusks and involved in tissue support and buoyancy regulation. Although it combines useful properties as high compressive strength, high porosity and high permeability, knowledge about organic compounds involved in its building remains limited. Moreover, several cuttlebone organic matrix studies reported data very different from each other or from other mollusk shells. Thus, this study provides 1) an overview of the organization of the main mineral structures found in the S. officinalis shell, 2) a reliable baseline about its organic composition, and 3) a first descriptive proteomic approach of organic matrices found in the two main parts of this shell. These data will contribute to the general knowledge about mollusk biomineralization as well as in the identification of protein compounds involved in the Sepiidae shell calcification.

Could FaRP-Like Peptides Participate in Regulation of Hyperosmotic Stress Responses in Plants?

The ability to respond to hyperosmotic stress is one of the numerous conserved cellular processes that most of the organisms have to face during their life. In metazoans, some peptides belonging to the FMRFamide-like peptide (FLP) family were shown to participate in osmoregulation via regulation of ion channels; this is, a well-known response to hyperosmotic stress in plants. Thus, we explored whether FLPs exist and regulate osmotic stress in plants. First, we demonstrated the response of Arabidopsis thaliana cultured cells to a metazoan FLP (FLRF). We found that A. thaliana express genes that display typical FLP repeated sequences, which end in RF and are surrounded by K or R, which is typical of cleavage sites and suggests bioactivity; however, the terminal G, allowing an amidation process in metazoan, seems to be replaced by W. Using synthetic peptides, we showed that amidation appears unnecessary to bioactivity in A. thaliana, and we provide evidence that these putative FLPs could be involved in physiological processes related to hyperosmotic stress responses in plants, urging further studies on this topic.