Cephalopod Sex Determination and its Ancient Evolutionary Origin Revealed by Chromosome-level Assembly of the California Two-Spot Octopus.

Sex chromosomes are critical elements of sexual reproduction in many animal and plant taxa, however they show incredible diversity and rapid turnover even within clades. Here, using a chromosome-level assembly generated with long read sequencing, we report the first evidence for genetic sex determination in cephalopods. We have uncovered a sex chromosome in California two-spot octopus (Octopus bimaculoides) in which males/females show ZZ/ZO karyotypes respectively. We show that the octopus Z chromosome is an evolutionary outlier with respect to divergence and repetitive element content as compared to other chromosomes and that it is present in all coleoid cephalopods that we have examined. Our results suggest that the cephalopod Z chromosome originated between 455 and 248 million years ago and has been conserved to the present, making it the among the oldest conserved animal sex chromosomes known.

Functional organization of visual responses in the octopus optic lobe.

Cephalopods are highly visual animals with camera-type eyes, large brains, and a rich repertoire of visually guided behaviors. However, the cephalopod brain evolved independently from those of other highly visual species, such as vertebrates; therefore, the neural circuits that process sensory information are profoundly different. It is largely unknown how their powerful but unique visual system functions, as there have been no direct neural measurements of visual responses in the cephalopod brain. In this study, we used two-photon calcium imaging to record visually evoked responses in the primary visual processing center of the octopus central brain, the optic lobe, to determine how basic features of the visual scene are represented and organized. We found spatially localized receptive fields for light (ON) and dark (OFF) stimuli, which were retinotopically organized across the optic lobe, demonstrating a hallmark of visual system organization shared across many species. An examination of these responses revealed transformations of the visual representation across the layers of the optic lobe, including the emergence of the OFF pathway and increased size selectivity. We also identified asymmetries in the spatial processing of ON and OFF stimuli, which suggest unique circuit mechanisms for form processing that may have evolved to suit the specific demands of processing an underwater visual scene. This study provides insight into the neural processing and functional organization of the octopus visual system, highlighting both shared and unique aspects, and lays a foundation for future studies of the neural circuits that mediate visual processing and behavior in cephalopods.

Cephalopod-omics: Emerging Fields and Technologies in Cephalopod Biology.

Few animal groups can claim the level of wonder that cephalopods instill in the minds of researchers and the general public. Much of cephalopod biology, however, remains unexplored: the largest invertebrate brain, difficult husbandry conditions, and complex (meta-)genomes, among many other things, have hindered progress in addressing key questions. However, recent technological advancements in sequencing, imaging, and genetic manipulation have opened new avenues for exploring the biology of these extraordinary animals. The cephalopod molecular biology community is thus experiencing a large influx of researchers, emerging from different fields, accelerating the pace of research in this clade. In the first post-pandemic event at the Cephalopod International Advisory Council (CIAC) conference in April 2022, over 40 participants from all over the world met and discussed key challenges and perspectives for current cephalopod molecular biology and evolution. Our particular focus was on the fields of comparative and regulatory genomics, gene manipulation, single-cell transcriptomics, metagenomics, and microbial interactions. This article is a result of this joint effort, summarizing the latest insights from these emerging fields, their bottlenecks, and potential solutions. The article highlights the interdisciplinary nature of the cephalopod-omics community and provides an emphasis on continuous consolidation of efforts and collaboration in this rapidly evolving field.

Functional organization of visual responses in the octopus optic lobe.

Cephalopods are highly visual animals with camera-type eyes, large brains, and a rich repertoire of visually guided behaviors. However, the cephalopod brain evolved independently from that of other highly visual species, such as vertebrates, and therefore the neural circuits that process sensory information are profoundly different. It is largely unknown how their powerful but unique visual system functions, since there have been no direct neural measurements of visual responses in the cephalopod brain. In this study, we used two-photon calcium imaging to record visually evoked responses in the primary visual processing center of the octopus central brain, the optic lobe, to determine how basic features of the visual scene are represented and organized. We found spatially localized receptive fields for light (ON) and dark (OFF) stimuli, which were retinotopically organized across the optic lobe, demonstrating a hallmark of visual system organization shared across many species. Examination of these responses revealed transformations of the visual representation across the layers of the optic lobe, including the emergence of the OFF pathway and increased size selectivity. We also identified asymmetries in the spatial processing of ON and OFF stimuli, which suggest unique circuit mechanisms for form processing that may have evolved to suit the specific demands of processing an underwater visual scene. This study provides insight into the neural processing and functional organization of the octopus visual system, highlighting both shared and unique aspects, and lays a foundation for future studies of the neural circuits that mediate visual processing and behavior in cephalopods. Highlights: The functional organization and visual response properties of the cephalopod visual system are largely unknownUsing calcium imaging, we performed mapping of visual responses in the octopus optic lobeVisual responses demonstrate localized ON and OFF receptive fields with retinotopic organizationON/OFF pathways and size selectivity emerge across layers of the optic lobe and have distinct properties relative to other species.

Cell types and molecular architecture of the Octopus bimaculoides visual system.

Cephalopods have a remarkable visual system, with a camera-type eye and high acuity vision that they use for a wide range of sophisticated visually driven behaviors. However, the cephalopod brain is organized dramatically differently from that of vertebrates and invertebrates, and beyond neuroanatomical descriptions, little is known regarding the cell types and molecular determinants of their visual system organization. Here, we present a comprehensive single-cell molecular atlas of the octopus optic lobe, which is the primary visual processing structure in the cephalopod brain. We combined single-cell RNA sequencing with RNA fluorescence in situ hybridization to both identify putative molecular cell types and determine their anatomical and spatial organization within the optic lobe. Our results reveal six major neuronal cell classes identified by neurotransmitter/neuropeptide usage, in addition to non-neuronal and immature neuronal populations. We find that additional markers divide these neuronal classes into subtypes with distinct anatomical localizations, revealing further diversity and a detailed laminar organization within the optic lobe. We also delineate the immature neurons within this continuously growing tissue into subtypes defined by evolutionarily conserved developmental genes as well as novel cephalopod- and octopus-specific genes. Together, these findings outline the organizational logic of the octopus visual system, based on functional determinants, laminar identity, and developmental markers/pathways. The resulting atlas presented here details the "parts list" for neural circuits used for vision in octopus, providing a platform for investigations into the development and function of the octopus visual system as well as the evolution of visual processing.