Figure - available from: Frontiers in Marine Science
This content is subject to copyright.
Advantageous Culture Traits of Several Cephalopod Models. Comparison of cephalopod species previously used in laboratory experiments. “Lifecycle closed” refers to a species being cultured across at least one generation. An animal is considered capable of group rearing if minimal aggression and cannibalism is observed, and the stress of group rearing prevents successful culturing efforts. “Multiple spawner” indicates normal multiple spawning events completed by one female. “Precocious offspring” refers to hatchling behaviors similar to adults (including predation). “Small at maturity” refers to an animal with a dorsal mantle length less than 6 cm. Some cephalopod species have evolved a light organ that is bioluminescent. The tree is based on results published by Anderson and Lindgren (2020).
Source publication
Cephalopod research remains limited by the inability to culture species under laboratory conditions for multiple generations to provide continuous access to animals at all stages of the life cycle. Here, we describe a multi-generational laboratory culture system for two emerging cephalopod models: the hummingbird or Berry’s bobtail squid, Euprymna...
Citations
... The Euprymna berryi (E. berryi) bobtail hummingbird squid is an emerging new model organism amenable to genetic manipulation 3,4 . Bobtail squids have primarily been studied for their unique symbiotic relationships with bioluminescent bacteria Vibrio fischeri 5 and likely use extensive A-to-I RNA editing to sense and adapt to their environment, similar to other cephalopod species 6 . ...
Squid primary cells from various tissues and ages are isolated, maintained in culture, and express exogenous genes. This protocol opens up numerous opportunities in molecular biology, neuroscience, and marine biology, enabling molecular and cellular-level investigations into processes specific to squids, including their complex behaviors, rapid color change mechanisms, RNA editing capabilities, with broader implications in basic cell physiology. We also describe procedures that harness life-stage tractability in Euprymna berryi squids towards future studies of aging in this model marine organism.
Highlights
- Squid primary cells are isolated from optic lobes, gills, eyes, and the skin
- Cells from these tissues can be dissociated from any life stage of the squid
- Isolated cells can express exogenous genes through mRNA transfections and are subject to live and/or fixed cell imaging
- Specific details for optimized media conditions, trypsinization, plating, and passaging are included
... Moreover, E. scolopes pioneered bobtail squids in general as emerging fruitful model systems for molecular biology thanks to their small body size, relatively easy maintenance protocols 17,18 and emerging transgenic approaches 19 . ...
Coleoid cephalopods possess numerous complex, species-specific morphological and behavioural adaptations, e.g., a uniquely structured nervous system that is the largest among the invertebrates. The Hawaiian bobtail squid (Euprymna scolopes) is one of the most established cephalopod species. With its recent publication of the chromosomal-scale genome assembly and regulatory genomic data, it also emerges as a key model for cephalopod gene regulation and evolution. However, the latest genome assembly has been lacking a native gene model set. Our manuscript describes the generation of new long-read transcriptomic data and, made using this combined with a plethora of publicly available transcriptomic and protein sequence data, a new reference annotation for E. scolopes.
... This combination of factors only occurs in some coastal species of the loliginids and octopods, and increasingly model organisms like Euprymna spp. (Hanlon et al., 1997;Nyholm and McFall-Ngai, 2021;Jolly et al., 2022). Laboratory-based studies likely represents the shortest path for studying ecology and behavior of many life stages of coastal and oceanic cephalopods. ...
Life cycle definitions provide the background for conceptualizing meaningful questions to address the mechanisms that generate different life cycle patterns. This review provides explicit definitions and explanations of the steps in a cephalopod life cycle, from fertilization to death. Each large step, or phase, is characterized by a particular developmental process and morphology. Each phase is composed of smaller developmentally distinct steps, or stages. The cephalopod life cycle is comprised of all or some of the following phases: Embryonic, Paralarval, Juvenile, Subadult, Adult and Senescent, and each life cycle is taxon-specific. All cephalopods have direct development and maintain a consistent body plan throughout ontogeny (i.e., no true larval phase and no metamorphosis). Most cephalopods have a life cycle marked by a long early life and a short adult life followed by senescence. Cephalopods have two developmental modes: they produce either small planktonic hatchlings as paralarvae, or large hatchlings as juveniles. All cephalopods go through a Hatchling stage soon after eclosion during which they rely on two modes of nutrition: endogenous (yolk) and exogenous (prey). Many cephalopods with planktonic paralarvae will become benthic early in their life cycle during their Settlement stage or remain pelagic during their Metapelagic stage. Juvenile growth is fast and ontogenetic changes (outside of gonadal maturation) generally cease at the end of the Juvenile phase. The Subadult phase begins when the definitive adult morphology (except for size and body proportions) is acquired (e.g., full complement of photophores). Sexual organs undergo most of their development during the Subadult phase. The Adult phase starts with spawning competency and concludes when gonads are spent. The Senescent phase begins with spent gonads and ends with death. Using this new terminology, we examine the patterns of cephalopod life cycles and find that there are four main patterns based on the presence of a Paralarval phase and the habitat occupied by each phase: Holopelagic (all phases are pelagic), Holobenthic (all phases are benthic), Merobenthic and Meropelagic (phases alternate between benthic and pelagic environments). In these two last patterns, the main difference is the presence of a Paralarval phase in Merobenthic species. The definitions and terminology proposed here provide a unifying framework for future ecological, evolutionary and life cycles research on cephalopods.
... In this study, we have turned to Euprymna berryi, a bobtail squid from the Indo-Pacific that possesses many important traits for its use as a genetically tractable model system. 25,26 E. berryi (1) can be raised in the lab through its entire life cycle, (2) produces frequent clutches of embryos over a 3-4 month span that can be microinjected and then cultured to hatching in an incubator, (3) reaches sexual maturity in about 3 months, (4) has an assembled genome, 27 and (5) is only 3-5 cm in mantle length as an adult. 26,[28][29][30] What is lacking in this emerging model organism is the development of genetic tools and defined genetic strains to facilitate biological discovery. ...
... 25,26 E. berryi (1) can be raised in the lab through its entire life cycle, (2) produces frequent clutches of embryos over a 3-4 month span that can be microinjected and then cultured to hatching in an incubator, (3) reaches sexual maturity in about 3 months, (4) has an assembled genome, 27 and (5) is only 3-5 cm in mantle length as an adult. 26,[28][29][30] What is lacking in this emerging model organism is the development of genetic tools and defined genetic strains to facilitate biological discovery. For example, the ability to image neural activity non-invasively is a cornerstone of modern neuroscience and would be an indispensable tool for cephalopod neurobiology. ...
... First, we introduce E. berryi as a model for molecular genetics. Previous work has demonstrated the potential utility of this species 26 : it is small, reaches sexual maturity relatively rapidly (3-4 months, depending on the culture temperature), and is robust in culture. E. berryi displays similar characteristics to Euprymna scolopes, a morphologically comparable congener that has been an important and well-studied model for bacterial-animal symbioses. ...
Cephalopods are remarkable among invertebrates for their cognitive abilities, adaptive camouflage, novel structures, and propensity for recoding proteins through RNA editing. Due to the lack of genetically tractable cephalopod models, however, the mechanisms underlying these innovations are poorly understood. Genome editing tools such as CRISPR-Cas9 allow targeted mutations in diverse species to better link genes and function. One emerging cephalopod model, Euprymna berryi, produces large numbers of embryos that can be easily cultured throughout their life cycle and has a sequenced genome. As proof of principle, we used CRISPR-Cas9 in E. berryi to target the gene for tryptophan 2,3 dioxygenase (TDO), an enzyme required for the formation of ommochromes, the pigments present in the eyes and chromatophores of cephalopods. CRISPR-Cas9 ribonucleoproteins targeting tdo were injected into early embryos and then cultured to adulthood. Unexpectedly, the injected specimens were pigmented, despite verification of indels at the targeted sites by sequencing in injected animals (G0s). A homozygote knockout line for TDO, bred through multiple generations, was also pigmented. Surprisingly, a gene encoding indoleamine 2,3, dioxygenase (IDO), an enzyme that catalyzes the same reaction as TDO in vertebrates, was also present in E. berryi. Double knockouts of both tdo and ido with CRISPR-Cas9 produced an albino phenotype. We demonstrate the utility of these albinos for in vivo imaging of Ca2+ signaling in the brain using two-photon microscopy. These data show the feasibility of making gene knockout cephalopod lines that can be used for live imaging of neural activity in these behaviorally sophisticated organisms.
... In the past ten years the Euprymna genus has emerged as a genetic model candidate, and there is intensive study currently of its genome [15] , nervous system [10,11,16], and behavior [17][18][19] Of the two primary species of Euprymna common in research labs, Euprymna scolopes has a long history of use in studies of symbiosis and host-microbe interactions, as a result of its unique co-evolution with a bioluminescent microbe which is cultured by the squid in a specialised light organ [20][21][22][23][24] . E. scolopes is challenging to rear in captivity, with rearing success rates varying from 50% down to almost zero [25] In contrast, the slightly larger species Euprymna berryi is considerably easier to rear, permitting rearing successes up to 90% of hatchlings raised until adulthood [26] and R. Crook, unpubl.). Sepiolid squid are distinct in physiology and behavior from other squid genera and from cuttlefish, suggesting that their responses to anesthesia and analgesia may differ in important ways too. ...
Cephalopods’ remarkable behavior and complex neurobiology make them valuable comparative model organisms, but studies aimed at enhancing welfare of captive cephalopods remain uncommon. Increasing regulation of cephalopods in research laboratories has resulted in growing interest in welfare-oriented refinements, including analgesia and anesthesia. Although general and local anesthesia in cephalopods have received limited prior study, there have been no studies of systemic analgesics in cephalopods to date. Here we show that analgesics from several different drug classes may be effective in E. berryi. Buprenorphine, ketorolac and dexmedetomidine, at doses similar to those used in fish, showed promising effects on baseline nociceptive thresholds, excitability of peripheral sensory nerves, and on behavioral responses to transient noxious stimulation. We found no evidence of positive effects of acetaminophen or ketamine administered at doses that are effective in vertebrates. Bioinformatic analyses suggested conserved candidate receptors for dexmedetomidine and ketorolac, but not buprenorphine. We also show that rapid general immersion anesthesia using a mix of MgCl2 and ethanol was successful in E. berryi at multiple age classes, similar to findings in other cephalopods. These data indicate that systemic analgesia and general anesthesia in Euprymna berryi are achievable welfare enhancing interventions, but further study and refinement is warranted.
Functional studies of host-microbe interactions benefit from natural model systems that enable exploration of molecular mechanisms at the host-microbe interface. Bioluminescent Vibrio fischeri colonize the light organ of the Hawaiian bobtail squid, Euprymna scolopes , and this binary model has enabled advances in understanding host-microbe communication, colonization specificity, in vivo biofilms, intraspecific competition, and quorum sensing. The hummingbird bobtail squid, Euprymna berryi, can be generationally bred and maintained in lab settings and has had multiple genes deleted by CRISPR approaches. The prospect of expanding the utility of the light organ model system by producing multigenerational host lines led us to determine the extent to which the E. berryi light organ symbiosis parallels known processes in E. scolopes . However, the nature of the E. berryi light organ, including its microbial constituency and specificity for microbial partners, have not been examined. In this report, we isolate bacteria from E. berryi animals and tank water. Assays of bacterial behaviors required in the host, as well as host responses to bacterial colonization, illustrate largely parallel phenotypes in E. berryi and E. scolopes hatchlings. This study reveals E. berryi to be a valuable comparative model to complement studies in E. scolopes .
IMPORTANCE
Microbiome studies have been substantially advanced by model systems that enable functional interrogation of the roles of the partners and the molecular communication between those partners. The Euprymna scolopes-Vibrio fischeri system has contributed foundational knowledge, revealing key roles for bacterial quorum sensing broadly and in animal hosts, for bacteria in stimulating animal development, for bacterial motility in accessing host sites, and for in vivo biofilm formation in development and specificity of an animal’s microbiome. Euprymna berryi is a second bobtail squid host, and one that has recently been shown to be robust to laboratory husbandry and amenable to gene knockout. This study identifies E. berryi as a strong symbiosis model host due to features that are conserved with those is E. scolopes , which will enable extension of functional studies in bobtail squid symbioses.
Coleoid cephalopods (octopus, squid and cuttlefish) have unusually complex nervous systems. The coleoid nervous system is also the only one currently known to recode the majority of expressed proteins through A-to-I RNA editing. The deamination of adenosine by adenosine deaminase acting on RNA (ADAR) enzymes produces inosine, which is interpreted as guanosine during translation. If this occurs in an open reading frame, which is the case for tens of thousands of editing sites in coleoids, it can recode the encoded protein. Here, we describe recent findings aimed at deciphering the mechanisms underlying high-level recoding and its adaptive potential. We describe the complement of ADAR enzymes in cephalopods, including a recently discovered novel domain in sqADAR1. We further summarize current evidence supporting an adaptive role of high-level RNA recoding in coleoids, and review recent studies showing that a large proportion of recoding sites is temperature-sensitive. Despite these new findings, the mechanisms governing the high level of RNA recoding in coleoid cephalopods remain poorly understood. Recent advances using genome editing in squid may provide useful tools to further study A-to-I RNA editing in these animals.
Two new pygmy squid from the Ryukyu archipelago, Japan, are described: Kodama jujutsu, n. gen., n. sp. and Idiosepius kijimuna, n. sp. They differ from all other nominal species in a combination of traits, including the number of tentacular club suckers, shape of the funnel-mantle locking-cartilage, modification of the male hectocotylus and the structure of the gladius and nuchal-locking cartilage, in addition to mitochondrial DNA markers (12S, 16S and COI). They are both known from Okinawa Island and there is some overlap in their distributions. In a molecular phylogeny that includes all nominal Idiosepiidae, Kodama jujutsu, n. gen., n. sp. is sister taxon to a clade containing Xipholeptos Reid & Strugnell, 2018 and Idiosepius Steenstrup, 1881. Xipholeptos and Idiosepius are sister taxa. Idiosepius spp. now includes seven nominal species. In addition, aspects of the behaviour of the new species are described.
Interest in cephalopods as comparative models in neuroscience, cognition, behavior and ecology is surging due to recent advances in culture and experimental techniques. Although cephalopods have a long history in research, their use had remained limited due to the challenges of funding work on comparative models, the lack of modern techniques applicable to them and the small number of labs with the facilities to keep and house large numbers of healthy animals for long periods. Breakthroughs in each of these areas are now creating new interest in cephalopods from researchers who trained and worked in other models, as well as allowing established cephalopod labs to grow and collaborate more widely. This broadening of the field is essential to its long-term health, but also brings with it new and heightened scrutiny from animal rights organizations, federal regulatory agencies, and members of the public. As a community, it is critical that scientists working with cephalopods engage in discussions, studies and communication that promote high standards for cephalopod welfare. The concept of “social license to operate,” more commonly encountered in industry, recreation and agriculture, provides a useful lens through which to view proactive steps the cephalopod research community may take to ensure a strong future for our field.
In this Perspective, I discuss recent progress in cephalopod ethics and welfare studies, and use the conceptual framework of Social License to Operate to propose a forward-looking, public-facing strategy for parallel development of welfare-focused best-practices and scientific breakthroughs.