Transcriptome sequencing of seven deep marine invertebrates.

We present 4k video and whole transcriptome data for seven deep-sea invertebrate animals collected in the Eastern Pacific Ocean during a research expedition onboard the Schmidt Ocean Institute's R/V Falkor in August of 2021. The animals include one jellyfish (Atolla sp.), three siphonophores (Apolemia sp., Praya sp., and Halistemma sp.), one larvacean (Bathochordaeus mcnutti), one tunicate (Pyrosomatidae sp.), and one ctenophore (Lampocteis sp.). Four of the animals were sequenced with long-read RNA sequencing technology, such that the reads themselves define a reference assembly for those animals. The larvacean tissues were successfully preserved in situ and has paired long-read reference data and short read quantitative transcriptomic data for within-specimen analyses of gene expression. Additionally, for three animals we provide quantitative image data, and a 3D model for one siphonophore. The paired image and transcriptomic data can be used for species identification, species description, and reference genetic data for these deep-sea animals.

Contextual and combinatorial structure in sperm whale vocalisations.

Sperm whales (Physeter macrocephalus) are highly social mammals that communicate using sequences of clicks called codas. While a subset of codas have been shown to encode information about caller identity, almost everything else about the sperm whale communication system, including its structure and information-carrying capacity, remains unknown. We show that codas exhibit contextual and combinatorial structure. First, we report previously undescribed features of codas that are sensitive to the conversational context in which they occur, and systematically controlled and imitated across whales. We call these rubato and ornamentation. Second, we show that codas form a combinatorial coding system in which rubato and ornamentation combine with two context-independent features we call rhythm and tempo to produce a large inventory of distinguishable codas. Sperm whale vocalisations are more expressive and structured than previously believed, and built from a repertoire comprising nearly an order of magnitude more distinguishable codas. These results show context-sensitive and combinatorial vocalisation can appear in organisms with divergent evolutionary lineage and vocal apparatus.

An in situ digital synthesis strategy for the discovery and description of ocean life.

Revolutionary advancements in underwater imaging, robotics, and genomic sequencing have reshaped marine exploration. We present and demonstrate an interdisciplinary approach that uses emerging quantitative imaging technologies, an innovative robotic encapsulation system with in situ RNA preservation and next-generation genomic sequencing to gain comprehensive biological, biophysical, and genomic data from deep-sea organisms. The synthesis of these data provides rich morphological and genetic information for species description, surpassing traditional passive observation methods and preserved specimens, particularly for gelatinous zooplankton. Our approach enhances our ability to study delicate mid-water animals, improving research in the world's oceans.

Transcriptomics of a Greenlandic Snailfish Reveals Exceptionally High Expression of Antifreeze Protein Transcripts.

Polar fishes have evolved antifreeze proteins (AFPs) that allow them to survive in subzero temperatures. We performed deep transcriptomic sequencing on a postlarval/juvenile variegated snailfish, Liparis gibbus (Actinopterygii: Scorpaeniformes: Cottoidei: Liparidae), living in an iceberg habitat (-2°C) in Eastern Greenland and report detection of highly expressed transcripts that code for putative AFPs from 2 gene families, Type I and LS-12-like proteins (putative Type IV AFPs). The transcripts encoding both proteins have expression levels among the top <1% of expressed genes in the fish. The Type I AFP sequence is different from a reported Type I AFP from the same species, possibly expressed from a different genetic locus. While prior findings from related adult sculpins suggest that LS-12-like/Type IV AFPs may not have a role in antifreeze protection, our finding of very high relative gene expression of the LS-12-like gene suggests that highly active transcription of the gene is important to the fish in the iceberg habitat and raises the possibility that weak or combinatorial antifreeze activity could be beneficial. These findings highlight the physiological importance of antifreeze proteins to the survival of fishes living in polar habitats.

Toward understanding the communication in sperm whales.

Machine learning has been advancing dramatically over the past decade. Most strides are human-based applications due to the availability of large-scale datasets; however, opportunities are ripe to apply this technology to more deeply understand non-human communication. We detail a scientific roadmap for advancing the understanding of communication of whales that can be built further upon as a template to decipher other forms of animal and non-human communication. Sperm whales, with their highly developed neuroanatomical features, cognitive abilities, social structures, and discrete click-based encoding make for an excellent model for advanced tools that can be applied to other animals in the future. We outline the key elements required for the collection and processing of massive datasets, detecting basic communication units and language-like higher-level structures, and validating models through interactive playback experiments. The technological capabilities developed by such an undertaking hold potential for cross-applications in broader communities investigating non-human communication and behavioral research.

Advances and future outlooks in soft robotics for minimally invasive marine biology.

This Viewpoint describes interdisciplinary research that aims to maximize understanding of deep marine life, while concurrently being minimally invasive. We describe the synthesis of multiple modern approaches (spanning robotics, biology, biomechanics, engineering, imaging, and genomic sequencing) and present future directions that hold the potential for a paradigm shift in marine biology.

Selection, drift, and constraint in cypridinid luciferases and the diversification of bioluminescent signals in sea fireflies.

Understanding the genetic causes of evolutionary diversification is challenging because differences across species are complex, often involving many genes. However, cases where single or few genetic loci affect a trait that varies dramatically across a radiation of species provide tractable opportunities to understand the genetics of diversification. Here, we begin to explore how diversification of bioluminescent signals across species of cypridinid ostracods ("sea fireflies") was influenced by evolution of a single gene, cypridinid-luciferase. In addition to emission spectra ("colour") of bioluminescence from 21 cypridinid species, we report 13 new c-luciferase genes from de novo transcriptomes, including in vitro assays to confirm function of four of those genes. Our comparative analyses suggest some amino acid sites in c-luciferase evolved under episodic diversifying selection and may be associated with changes in both enzyme kinetics and colour, two enzymatic functions that directly impact the phenotype of bioluminescent signals. The analyses also suggest multiple other amino acid positions in c-luciferase evolved neutrally or under purifying selection, and may have impacted the variation of colour of bioluminescent signals across genera. Previous mutagenesis studies at candidate sites show epistatic interactions, which could constrain the evolution of c-luciferase function. This work provides important steps toward understanding the genetic basis of diversification of behavioural signals across multiple species, suggesting different evolutionary processes act at different times during a radiation of species. These results set the stage for additional mutagenesis studies that could explicitly link selection, drift, and constraint to the evolution of phenotypic diversification.

A putative chordate luciferase from a cosmopolitan tunicate indicates convergent bioluminescence evolution across phyla.

Pyrosomes are tunicates in the phylum Chordata, which also contains vertebrates. Their gigantic blooms play important ecological and biogeochemical roles in oceans. Pyrosoma, meaning "fire-body", derives from their brilliant bioluminescence. The biochemistry of this light production is unknown, but has been hypothesized to be bacterial in origin. We found that mixing coelenterazine-a eukaryote-specific luciferin-with Pyrosoma atlanticum homogenate produced light. To identify the bioluminescent machinery, we sequenced P. atlanticum transcriptomes and found a sequence match to a cnidarian luciferase (RLuc). We expressed this novel luciferase (PyroLuc) and, combined with coelenterazine, it produced light. A similar gene was recently predicted from a bioluminescent brittle star, indicating that RLuc-like luciferases may have evolved convergently from homologous dehalogenases across phyla (Cnidaria, Echinodermata, and Chordata). This report indicates that a widespread gene may be able to functionally converge, resulting in bioluminescence across animal phyla, and describes and characterizes the first putative chordate luciferase.

Evolutionary Traits that Enable Scleractinian Corals to Survive Mass Extinction Events.

Scleractinian "stony" corals are major habitat engineers, whose skeletons form the framework for the highly diverse, yet increasingly threatened, coral reef ecosystem. Fossil coral skeletons also present a rich record that enables paleontological analysis of coral origins, tracing them back to the Triassic (~241 Myr). While numerous invertebrate lineages were eradicated at the last major mass extinction boundary, the Cretaceous-Tertiary/K-T (66 Myr), a number of Scleractinian corals survived. We review this history and assess traits correlated with K-T mass extinction survival. Disaster-related "survival" traits that emerged from our analysis are: (1) deep water residing (>100 m); (2) cosmopolitan distributions, (3) non-symbiotic, (4) solitary or small colonies and (5) bleaching-resistant. We then compared these traits to the traits of modern Scleractinian corals, using to IUCN Red List data, and report that corals with these same survival traits have relatively stable populations, while those lacking them are presently decreasing in abundance and diversity. This shows corals exhibiting a similar dynamic survival response as seen at the last major extinction, the K-T. While these results could be seen as promising, that some corals may survive the Anthropocene extinction, they also highlight how our relatively-fragile Primate order does not possess analogous "survival" characteristics, nor have a record of mass extinction survival as some corals are capable.

Ultra-gentle soft robotic fingers induce minimal transcriptomic response in a fragile marine animal.

Tessler et al. demonstrate that a 'soft' robot causes less stress to a jellyfish while handling compared to a traditional 'hard' robot.

Disrupting Fluorescence by Mutagenesis in a Green Fluorescent Fatty Acid Binding Protein from a Marine Eel.

Biofluorescence has been found to be an increasingly widespread phenomenon in the ocean. The reclusive Caribbean chlopsid eel, Kaupichthys hyoproroides displays bright green fluorescence in its native marine environment. We have previously shown the fluorescence to be attributed to a fluorescent fatty acid-binding protein, Chlopsid FP, part of a larger family of fluorescent fatty acid-binding proteins, including the homologous UnaG. All require the addition of exogenous bilirubin for fluorescence. Here, we report the generation of a series of point mutants, and deletions that result in the quenching of fluorescence in Chlopsid FP. In addition, we report the binding constants of bilirubin to Chlopsid FP and mutants, measured by fluorescence titration. This study provides key insights into the potential mechanism of fluorescence in this class of fluorescent fatty acid-binding proteins.

Publisher Correction: Deep Machine Learning Techniques for the Detection and Classification of Sperm Whale Bioacoustics.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Deep Machine Learning Techniques for the Detection and Classification of Sperm Whale Bioacoustics.

We implemented Machine Learning (ML) techniques to advance the study of sperm whale (Physeter macrocephalus) bioacoustics. This entailed employing Convolutional Neural Networks (CNNs) to construct an echolocation click detector designed to classify spectrograms generated from sperm whale acoustic data according to the presence or absence of a click. The click detector achieved 99.5% accuracy in classifying 650 spectrograms. The successful application of CNNs to clicks reveals the potential of future studies to train CNN-based architectures to extract finer-scale details from cetacean spectrograms. Long short-term memory and gated recurrent unit recurrent neural networks were trained to perform classification tasks, including (1) "coda type classification" where we obtained 97.5% accuracy in categorizing 23 coda types from a Dominica dataset containing 8,719 codas and 93.6% accuracy in categorizing 43 coda types from an Eastern Tropical Pacific (ETP) dataset with 16,995 codas; (2) "vocal clan classification" where we obtained 95.3% accuracy for two clan classes from Dominica and 93.1% for four ETP clan types; and (3) "individual whale identification" where we obtained 99.4% accuracy using two Dominica sperm whales. These results demonstrate the feasibility of applying ML to sperm whale bioacoustics and establish the validity of constructing neural networks to learn meaningful representations of whale vocalizations.

Bioluminescent flashes drive nighttime schooling behavior and synchronized swimming dynamics in flashlight fish.

Schooling fishes, like flocking birds and swarming insects, display remarkable behavioral coordination. While over 25% of fish species exhibit schooling behavior, nighttime schooling has rarely been observed or reported. This is due to vision being the primary modality for schooling, which is corroborated by the fact that most fish schools disperse at critically low light levels. Here we report on a large aggregation of the bioluminescent flashlight fish Anomalops katoptron that exhibited nighttime schooling behavior during multiple moon phases, including the new moon. Data were recorded with a suite of low-light imaging devices, including a high-speed, high-resolution scientific complementary metal-oxide-semiconductor (sCMOS) camera. Image analysis revealed nighttime schooling using synchronized bioluminescent flashing displays, and demonstrated that school motion synchrony exhibits correlation with relative swim speed. A computer model of flashlight fish schooling behavior shows that only a small percentage of individuals need to exhibit bioluminescence in order for school cohesion to be maintained. Flashlight fish schooling is unique among fishes, in that bioluminescence enables schooling in conditions of no ambient light. In addition, some members can still partake in the school while not actively exhibiting their bioluminescence. Image analysis of our field data and model demonstrate that if a small percentage of fish become motivated to change direction, the rest of the school follows. The use of bioluminescence by flashlight fish to enable schooling in shallow water adds an additional ecological application to bioluminescence and suggests that schooling behavior in mesopelagic bioluminescent fishes may be also mediated by luminescent displays.

Bright Green Biofluorescence in Sharks Derives from Bromo-Kynurenine Metabolism.

Although in recent years there has been an increased awareness of the widespread nature of biofluorescence in the marine environment, the diversity of the molecules responsible for this luminescent phenotype has been mostly limited to green fluorescent proteins (GFPs), GFP-like proteins, and fluorescent fatty acid-binding proteins (FABPs). In the present study, we describe a previously undescribed group of brominated tryptophan-kynurenine small molecule metabolites responsible for the green biofluorescence in two species of sharks and provide their structural, antimicrobial, and spectral characterization. Multi-scale fluorescence microscopy studies guided the discovery of metabolites that were differentially produced in fluorescent versus non-fluorescent skin, as well as the species-specific structural details of their unusual light-guiding denticles. Overall, this study provides the detailed description of a family of small molecules responsible for marine biofluorescence and opens new questions related to their roles in central nervous system signaling, resilience to microbial infections, and photoprotection.

Ultragentle manipulation of delicate structures using a soft robotic gripper.

Here, we present ultragentle soft robotic actuators capable of grasping delicate specimens of gelatinous marine life. Although state-of-the-art soft robotic manipulators have demonstrated gentle gripping of brittle animals (e.g., corals) and echinoderms (e.g., sea cucumbers) in the deep sea, they are unable to nondestructively grasp more fragile soft-bodied organisms, such as jellyfish. Through an exploration of design parameters and laboratory testing of individual actuators, we confirmed that our nanofiber-reinforced soft actuators apply sufficiently low contact pressure to ensure minimal harm to typical jellyfish species. We then built a gripping device using several actuators and evaluated its underwater grasping performance in the laboratory. By assessing the gripper's region of acquisition and robustness to external forces, we gained insight into the necessary precision and speed with which grasping maneuvers must be performed to achieve successful collection of samples. Last, we demonstrated successful manipulation of three live jellyfish species in an aquarium setting using a hand-held prototype gripper. Overall, our ultragentle gripper demonstrates an improvement in gentle sample collection compared with existing deep-sea sampling devices. Extensions of this technology may improve a variety of in situ characterization techniques used to study the ecological and genetic features of deep-sea organisms.

A Dexterous, Glove-Based Teleoperable Low-Power Soft Robotic Arm for Delicate Deep-Sea Biological Exploration.

Modern marine biologists seeking to study or interact with deep-sea organisms are confronted with few options beyond industrial robotic arms, claws, and suction samplers. This limits biological interactions to a subset of "rugged" and mostly immotile fauna. As the deep sea is one of the most biologically diverse and least studied ecosystems on the planet, there is much room for innovation in facilitating delicate interactions with a multitude of organisms. The biodiversity and physiology of shallow marine systems, such as coral reefs, are common study targets due to the easier nature of access; SCUBA diving allows for in situ delicate human interactions. Beyond the range of technical SCUBA (~150 m), the ability to achieve the same level of human dexterity using robotic systems becomes critically important. The deep ocean is navigated primarily by manned submersibles or remotely operated vehicles, which currently offer few options for delicate manipulation. Here we present results in developing a soft robotic manipulator for deep-sea biological sampling. This low-power glove-controlled soft robot was designed with the future marine biologist in mind, where science can be conducted at a comparable or better means than via a human diver and at depths well beyond the limits of SCUBA. The technology relies on compliant materials that are matched with the soft and fragile nature of marine organisms, and uses seawater as the working fluid. Actuators are driven by a custom proportional-control hydraulic engine that requires less than 50 W of electrical power, making it suitable for battery-powered operation. A wearable glove master allows for intuitive control of the arm. The manipulator system has been successfully operated in depths exceeding 2300 m (3500 psi) and has been field-tested onboard a manned submersible and unmanned remotely operated vehicles. The design, development, testing, and field trials of the soft manipulator is placed in context with existing systems and we offer suggestions for future work based on these findings.

Luciferin production and luciferase transcription in the bioluminescent copepod Metridia lucens.

Bioluminescent copepods are often the most abundant marine zooplankton and play critical roles in oceanic food webs. Metridia copepods exhibit particularly bright bioluminescence, and the molecular basis of their light production has just recently begun to be explored. Here we add to this body of work by transcriptomically profiling Metridia lucens, a common species found in temperate, northern, and southern latitudes. In this previously molecularly-uncharacterized species, we find the typical luciferase paralog gene set found in Metridia. More surprisingly, we recover noteworthy putative luciferase sequences that had not been described from Metridia species, indicating that bioluminescence produced by these copepods may be more complex than previously known. This includes another copepod luciferase, as well as one from a shrimp. Furthermore, feeding experiments using mass spectrometry and 13C labelled L-tyrosine and L-phenylalanine firmly establish that M. lucens produces its own coelenterazine luciferin rather than acquiring it through diet. This coelenterazine synthesis has only been directly confirmed in one other copepod species.

Shipboard design and fabrication of custom 3D-printed soft robotic manipulators for the investigation of delicate deep-sea organisms.

Soft robotics is an emerging technology that has shown considerable promise in deep-sea marine biological applications. It is particularly useful in facilitating delicate interactions with fragile marine organisms. This study describes the shipboard design, 3D printing and integration of custom soft robotic manipulators for investigating and interacting with deep-sea organisms. Soft robotics manipulators were tested down to 2224m via a Remotely-Operated Vehicle (ROV) in the Phoenix Islands Protected Area (PIPA) and facilitated the study of a diverse suite of soft-bodied and fragile marine life. Instantaneous feedback from the ROV pilots and biologists allowed for rapid re-design, such as adding "fingernails", and re-fabrication of soft manipulators at sea. These were then used to successfully grasp fragile deep-sea animals, such as goniasterids and holothurians, which have historically been difficult to collect undamaged via rigid mechanical arms and suction samplers. As scientific expeditions to remote parts of the world are costly and lengthy to plan, on-the-fly soft robot actuator printing offers a real-time solution to better understand and interact with delicate deep-sea environments, soft-bodied, brittle, and otherwise fragile organisms. This also offers a less invasive means of interacting with slow-growing deep marine organisms, some of which can be up to 18,000 years old.

A Modular Soft Robotic Wrist for Underwater Manipulation.

This article presents the development of modular soft robotic wrist joint mechanisms for delicate and precise manipulation in the harsh deep-sea environment. The wrist consists of a rotary module and bending module, which can be combined with other actuators as part of a complete manipulator system. These mechanisms are part of a suite of soft robotic actuators being developed for deep-sea manipulation via submersibles and remotely operated vehicles, and are designed to be powered hydraulically with seawater. The wrist joint mechanisms can also be activated with pneumatic pressure for terrestrial-based applications, such as automated assembly and robotic locomotion. Here we report the development and characterization of a suite of rotary and bending modules by varying fiber number and silicone hardness. Performance of the complete soft robotic wrist is demonstrated in normal atmospheric conditions using both pneumatic and hydraulic pressures for actuation and under high ambient hydrostatic pressures equivalent to those found at least 2300 m deep in the ocean. This rugged modular wrist holds the potential to be utilized at full ocean depths (>10,000 m) and is a step forward in the development of jointed underwater soft robotic arms.