Shuichi Shigeno

Cephalopod Genomics: A Plan of Strategies and Organization

The Cephalopod Sequencing Consortium (CephSeq Consortium) was established at a NESCent Catalysis Group Meeting, “Paths to Cephalopod Genomics-Strategies, Choices, Organization,” held in Durham, North Carolina, USA on May 24–27, 2012. Twenty-eight participants representing nine countries (Austria, Australia, China, Denmark, France, Italy, Japan, Spain and the USA) met to address the pressing need for genome sequencing of cephalopod mollusks. This group, drawn from cephalopod biologists, neuroscientists, developmental and evolutionary biologists, materials scientists, bioinformaticians and researchers active in sequencing, assembling and annotating genomes, agreed on a set of cephalopod species of particular importance for initial sequencing and developed strategies and an organization (CephSeq Consortium) to promote this sequencing. The conclusions and recommendations of this meeting are described in this white paper.

Molecular Evidence for Convergence and Parallelism in Evolution of Complex Brains of Cephalopod Molluscs: Insights from Visual Systems

Coleoid cephalopods show remarkable evolutionary convergence with vertebrates in their neural organization, including (1) eyes and visual system with optic lobes, (2) specialized parts of the brain controlling learning and memory, such as vertical lobes, and (3) unique vasculature supporting such complexity of the central nervous system. We performed deep sequencing of eye transcriptomes of pygmy squids (Idiosepius paradoxus) and chambered nautiluses (Nautilus pompilius) to decipher the molecular basis of convergent evolution in cephalopods. RNA-seq was complemented by in situ hybridization to localize the expression of selected genes. We found three types of genomic innovations in the evolution of complex brains: (1) recruitment of novel genes into morphogenetic pathways, (2) recombination of various coding and regulatory regions of different genes, often called evolutionary tinkering or co-option, and (3) duplication and divergence of genes. Massive recruitment of novel genes occurred in the evolution of the camera eye from nautilus pinhole eye. We also showed that the type-2 co-option of transcription factors played important roles in the evolution of the lens and visual neurons. In summary, the cephalopod convergent morphological evolution of the camera eyes was driven by a mosaic of all types of gene recruitments. In addition, our analysis revealed unexpected variations of squids opsins, retinochromes, and arrestins, providing more detailed information, valuable for further research on intra-ocular and extra-ocular photoreception of the cephalopods.

Effects of mass sedimentation events after the 2011 off the Pacific coast of Tohoku Earthquake on benthic prokaryotes and meiofauna inhabiting the upper bathyal sediments

We examined the effects of mass sedimentation events caused by the 2011xa0off the Pacific coast of Tohoku Earthquake on abundances and vertical distributions of prokaryotes and metazoan meiofauna in sediments, using sediment cores collected from eight bathyal stations off Tohoku 1xa0year after the M9.0 earthquake. Event deposits 1–7xa0cm thick were observed at the topmost part of the sediment cores at all sampling stations. At some stations, prokaryotic cell abundances were lower in the surface event-deposit layers compared to those in deeper sediments. These variations were explained by environmental parameters such as a dimensionless sorting factor and mean grain size, suggesting that turbidite sedimentation affected prokaryotic cell abundances. Nematodes had anomalously higher subsurface abundances at the stations where subsurface peak prokaryotic cell numbers were observed, whereas copepods always showed peak densities in the sediment surface layer. Although there are no available data for prokaryotic cell abundances and meiofaunal densities before the earthquake from the same sites, it is likely that the subsurface peaks in prokaryotic cell numbers and nematode densities resulted from the sedimentation events. The effects of sedimentation events on the organisms were observed 1xa0year after the earthquake, indicating that episodic sedimentation events on scales of several centimeters have a large effect on small organisms inhabiting sediments.

Evidence for a cordal, not ganglionic, pattern of cephalopod brain neurogenesis.

IntroductionFrom the large-brained cephalopods to the acephalic bivalves, molluscs show a vast range of nervous system centralization patterns. Despite this diversity, molluscan nervous systems, broadly considered, are organized either as medullary cords, as seen in chitons, or as ganglia, which are typical of gastropods and bivalves. The cephalopod brain is exceptional not just in terms of its size; its relationship to a molluscan cordal or ganglionic plan has not been resolved from the study of its compacted adult structure. One approach to clarifying this puzzle is to investigate the patterns of early cephalopod brain neurogenesis, where molecular markers for cephalopod neural development may be informative.ResultsWe report here on early brain pattern formation in the California two-spot octopus, Octopus bimaculoides. Employing gene expression analysis with the pan-bilaterian neuronal marker ELAV and the atonal-related neuronal differentiation genes NEUROGENIN and NEUROD, as well as immunostaining using a Distalless-like homeoprotein antibody, we found that the octopus central brain forms from concentric cords rather than bilaterally distributed pairs of ganglia.ConclusionWe conclude that the cephalopod brain, despite its great size and elaborate specializations, retains in its development the hypothesized ancestral molluscan nervous system plan of medullary cords, as described for chitons and other aculiferan molluscs.

Sensing deep extreme environments: the receptor cell types, brain centers, and multi-layer neural packaging of hydrothermal vent endemic worms.

IntroductionDeep-sea alvinellid worm species endemic to hydrothermal vents, such as Alvinella and Paralvinella, are considered to be among the most thermotolerant animals known with their adaptability to toxic heavy metals, and tolerance of highly reductive and oxidative stressful environments. Despite the number of recent studies focused on their overall transcriptomic, proteomic, and metabolic stabilities, little is known regarding their sensory receptor cells and electrically active neuro-processing centers, and how these can tolerate and function in such harsh conditions.ResultsWe examined the extra- and intracellular organizations of the epidermal ciliated sensory cells and their higher centers in the central nervous system through immunocytochemical, ultrastructural, and neurotracing analyses. We observed that these cells were rich in mitochondria and possessed many electron-dense granules, and identified specialized glial cells and serial myelin-like repeats in the head sensory systems of Paralvinella hessleri. Additionally, we identified the major epidermal sensory pathways, in which a pair of distinct mushroom bodies-like or small interneuron clusters was observed. These sensory learning and memory systems are commonly found in insects and annelids, but the alvinellid inputs are unlikely derived from the sensory ciliary cells of the dorsal head regions.ConclusionsOur evidence provides insight into the cellular and system-wide adaptive structure used to sense, process, and combat the deep-sea hydrothermal vent environment. The alvinellid sensory cells exhibit characteristics of annelid ciliary types, and among the most unique features were the head sensory inputs and structure of the neural cell bodies of the brain, which were surrounded by multiple membranes. We speculated that such enhanced protection is required for the production of normal electrical signals, and to avoid the breakdown of the membrane surrounding metabolically fragile neurons from oxidative stress. Such pivotal acquisition is not broadly found in the all body parts, suggesting the head sensory inputs are specific, and these heterogenetic protection mechanisms may be present in alvinellid worms.

Beyond Brain Size

Despite prolonged interest in comparing brain size and behavioral proxies of ‘intelligence’ across taxa, the adaptive and cognitive significance of brain size variation remains elusive. Central to this problem is the continued focus on hominid cognition as a benchmark, and the assumption that behavioral complexity has a simple relationship with brain size. Although comparative studies of brain size have been criticized for not reflecting how evolution actually operates, and for producing spurious, inconsistent results, the causes of these limitations have received little discussion. We show how these issues arise from implicit assumptions about what brain size measures and how it correlates with behavioral and cognitive traits. We explore how inconsistencies can arise through heterogeneity in evolutionary trajectories and selection pressures on neuroanatomy or neurophysiology across taxa. We examine how interference from ecological and life history variables complicates interpretations of brain-behavior correlations, and point out how this problem is exacerbated by the limitations of brain and cognitive measures. These considerations, and the diversity of brain morphologies and behavioral capacities, suggest that comparative brain-behavior research can make greater progress by focusing on specific neuroanatomical and behavioral traits within relevant ecological and evolutionary contexts. We suggest that a synergistic combination of the ‘bottom up’ approach of classical neuroethology and the ‘top down’ approach of comparative biology/psychology within closely related but behaviorally diverse clades can limit the effects of heterogeneity, interference, and noise. We argue this shift away from broad-scale analyses of superficial phenotypes will provide deeper, more robust insights into brain evolution.

Brain Evolution as an Information Flow Designer: The Ground Architecture for Biological and Artificial General Intelligence

For centuries, neuroscientists have identified a number of neural systems involved in sensory, motor, state control, and cognitive functions. Modern comparative studies have proposed their diversity, origins, and basic functionality across animal phyla. Despite a number of attempts, however, a common functional plan of the complex brain remains controversial. For example, there is currently no prominent theory of how neural networks are structurally comparable between phylogenetically distant animals such as vertebrates, octopuses, worms, and insects, in which there are distinguishably different brain architectures. This chapter attempts to identify the types of information flow patterns that were specialized during brain evolution, when these patterns appeared as a prototype, and how the flow systems have been shaped based on the common morphological architecture. In a notable case, a number of sensory associative centers show comparable patterns in mammalian, insect, and octopus brains, representing a common input and output flow of information. One can speculate that a common underlying structure is shared between various animals because of common functionalities that produce highly effective learning, memory, and autonomous cognitive tasks. Such an underlying structure could help establish a large-scale framework for comparison between phylogenetically distant animal brains and perhaps even form the groundwork for artificial general intelligence.

Dual Cellular Supporters: Multi-Layer Glial Wrapping and the Penetrative Matrix Specialized in Deep-Sea Hydrothermal Vent Endemic Scale-Worms

Hydrothermal vent organisms undergo extreme environments that may require unique innovations. The present study reports a distinct case of cellular supportive systems in the nervous systems of a scale-worm, Branchinotogluma japonica, endemic to deep-sea hydrothermal vents. We found two organizations in the tissues of these animals. First, multi-layers of glia ensheath the ventral cell bodies of the brain and ventral nerve cord, in a manner similar to that of myelin or lamellar ensheathments. Second, matrices of numerous penetrative fibers, or tonofilaments, composed of bundles of ca. 20-nm fibers, are directly connected with the basal parts of epidermal cuticles and run into the diffuse intercellular spaces of the brain neuropils and peripheral nerves. Both types of tissue might be mechanical supportive structures for the neuronal cell bodies. In addition, as a glial function, the multi-layer membranes and the epithelial support cells may be required for physicochemical homeostatic regulation to filter toxic heavy metals and for inhibiting breakdown of glial membrane integrity under strong oxidative stress imposed by hypoxia in the hydrothermal vent environment. Similar functions are known in the well-studied cases of the blood-brain barrier in mammalian brains, including in human stroke.

Highly sensitive avoidance plays a key role in sensory adaptation to deep-sea hydrothermal vent environments

The environments around deep-sea hydrothermal vents are very harsh conditions for organisms due to the possibility of exposure to highly toxic compounds and extremely hot venting there. Despite such extreme environments, some indigenous species have thrived there. Alvinellid worms (Annelida) are among the organisms best adapted to high-temperature and oxidatively stressful venting regions. Although intensive studies of the adaptation of these worms to the environments of hydrothermal vents have been made, little is known about the worms’ sensory adaptation to the severe chemical conditions there. To examine the sensitivity of the vent-endemic worm Paralvinella hessleri to low pH and oxidative stress, we determined the concentration of acetic acid and hydrogen peroxide that induced avoidance behavior of this worm, and compared these concentrations to those obtained for related species inhabiting intertidal zones, Thelepus sp. The concentrations of the chemicals that induced avoidance behavior of P. hessleri were 10–100 times lower than those for Thelepus sp. To identify the receptors for these chemicals, chemical avoidance tests were performed with the addition of ruthenium red, a blocker of transient receptor potential (TRP) channels. This treatment suppressed the chemical avoidance behavior of P. hessleri, which suggests that TRP channels are involved in the chemical avoidance behavior of this species. Our results revealed for the first time hypersensitive detection systems for acid and for oxidative stress in the vent-endemic worm P. hessleri, possibly mediated by TRP channels, suggesting that such sensory systems may have facilitated the adaptation of this organism to harsh vent environments.

Calcium imaging method to visualize the spatial patterns of neural responses in the pygmy squid, Idiosepius paradoxus, central nervous system

BACKGROUNDnCephalopods exhibit unique behaviors such as camouflage and tactile learning. The brain functions correlated to these behaviors have long been analyzed through behavioral observations of animals subject to surgical manipulation or electrical stimulation of brain lobes. However, physiological methods have rarely been introduced to investigate the functions of each individual lobe, though physiological work on giant axons and slices of the vertical lobe system of the cephalopods have provided deep insights into ion conductance of nerves and long-term synaptic plasticity. The lack of in vivo physiological work is partly due to difficulties in immobilizing the brain which is contained within the soft body and applying calcium indicators to the cephalopod central nervous system.nnnNEW METHODnWe here present a calcium imaging method to visualize neural responses in the central nervous system of the smallest squid, Idiosepius paradoxus.nnnRESULTSnWe injected calcium indicator Cal-520 into the brachial lobes and revealed a spatiotemporal pattern of neural responses to the electrical stimulations of the axial nerve cord in the first arm.nnnCOMPARISON WITH EXISTING METHODSnWe established a method to immobilize the central nervous system which is contained within the soft body and record the calcium responses from the intact central nervous system.nnnCONCLUSIONSnOur method provides a novel approach to investigate the mechanisms of how the characteristic organization of the cephalopod brain functions to induce their unique behaviors.