Bobtail Squid Research Articles

The complete mitochondrial genome of the pygmy squid, Idiosepius (Cephalopoda: Decapodiformes): the first representative from the family Idiosepiidae

We report the first complete mitochondrial genome of the pygmy squid, Idiosepius, (Idiosepiidae). The mtDNA genome is 16,183 bp long with an AT content of 75.4%. All conserved metazoan mitochondrial genes are identified with the addition of a 1018 bp non-coding region. Idiosepius gene order most closely resembles that of the bobtail squid Semirossia (Sepiolidae).

The northernmost record of Sepietta oweniana (Cephalopoda: Sepiolidae) and comments on boreo-subtropical cephalopod species occurrence in the Arctic

The first observation of the common bobtail squid Sepietta oweniana has been made in the Barents Sea (Tromso Bank; 70°54′N 19°46.8′E). It is the northernmost area of this species distribution. No changes were found in the reproductive system structure. But the food spectrum was changed, particularly, juvenile fish of the Lotidae family was found. The bobtail squid of the Sepiolinae subfamily have never been recorded as fish eaters previously. The expansion of boreo-subtropical species of cephalopods into the Arctic during the last decade can be divided into two groups: (1) a foraging migration happening relatively regularly; and (2) a range expansion due to the ongoing Arctic warming. The case of S. oweniana obviously belongs to the second group.

Eye-specification genes in the bacterial light organ of the bobtail squid Euprymna scolopes, and their expression in response to symbiont cues

The squid Euprymna scolopes has evolved independent sets of tissues capable of light detection, including a complex eye and a photophore or ‘light organ’, which houses the luminous bacterial symbiont Vibrio fischeri. As the eye and light organ originate from different embryonic tissues, we examined whether the eye-specification genes, pax6, eya, six, and dac, are shared by these two organs, and if so, whether they are regulated in the light organ by symbiosis. We obtained sequences of the four genes with PCR, confirmed orthology with phylogenetic analysis, and determined that each was expressed in the eye and light organ. With in situ hybridization (ISH), we localized the gene transcripts in developing embryos, comparing the patterns of expression in the two organs. The four transcripts localized to similar tissues, including those associated with the visual system ∼1/4 into embryogenesis (Naef stage 18) and the light organ ∼3/4 into embryogenesis (Naef stage 26). We used ISH and quantitative real-time PCR to examine transcript expression and differential regulation in postembryonic light organs in response to the following colonization conditions: wild-type, luminescent V. fischeri; a mutant strain defective in light production; and as a control, no symbiont. In ISH experiments light organs showed down regulation of the pax6, eya, and six transcripts in response to wild-type V. fischeri. Mutant strains also induced down regulation of the pax6 and eya transcripts, but not of the six transcript. Thus, luminescence was required for down regulation of the six transcript. We discuss these results in the context of symbiont-induced light-organ development. Our study indicates that the eye-specification genes are expressed in light-interacting tissues independent of their embryonic origin and are capable of responding to bacterial cues. These results offer evidence for evolutionary tinkering or the recruitment of eye development genes for use in a light-sensing photophore.

Distribution and reproductive biology of the lentil bobtail squid, Rondeletiola minor (Cephalopoda: Sepiolidae) from the Portuguese Atlantic Coast

The present study investigated the distribution and general and reproductive biology (size, maturation, fecundity) of the bobtail squid Rondeletiola minor from the Portuguese Atlantic Coast, based on 387 specimens collected between 1997 and 2009, of which 240 were fully analysed. The species was mainly encountered on the southern shelf of the Algarve, in depths of 175–375 m. Females had mantle lengths (ML) of 10.9–21.2 mm and were mature from 13.4 mm, while ML in males ranged from 10.4 to 18.9 mm with the smallest mature specimen being 10.4 mm long. The potential fecundity varied from 5 to 460 (mean 228). Mature ova ranged in numbers from 2 to 115 (mean 26), having a diameter of 1.5–3.0 mm (mean 2.0). Oocytes were present in the ovary at different stages of maturity. We also encountered mature specimens with elevated reproductive indices in all sampled months, which is a sign of a multiple spawning strategy with asynchronous ovulation. A peak in reproductive activity is indicated in autumn. R. minor seems to exhibit a comparatively long spawning season, adapted to a life in variable environmental conditions.

Micro-CT in cephalopod research: Investigating the internal anatomy of a sepiolid squid using a non-destructive technique with special focus on the ganglionic system

Most investigations on the internal organization of soft-bodied animals such as cephalopods are based on classical serial sectioning (i.e. histological). Here, we demonstrate a state-of-the-art-technique of X-ray microtomography (micro-CT) for 3D imaging of soft-bodied organisms without mineralized structures. As a model, we chose the Hawaiian bobtail squid Euprymna scolopes that is considered a key-organism for study of decabrachiate development. While neurodevelopmental as well as gene expression data are available for this species, basic information on its organogenesis is still lacking. We therefore focused our study on major neural ganglia during development, to assess the suitability of micro-CT as non-destructive method for ontogenetic studies. We show that micro-CT in combination with contrast-enhancing substances, such as iodine or phosphotungstic acid, can provide detailed 3D information on the anatomy of cephalopod embryonic structures including the nervous system. The technique eases computer-assisted 3D-reconstructions and modeling due to the perfectly aligned, distortion-free image stacks produced by the micro-CT scans. Non-destructive micro-CT applications have great mapping potential when combined with other classic techniques such as histology, immunocytochemistry, and gene expression studies.

Quorum Sensing in the Squid-Vibrio Symbiosis

Quorum sensing is an intercellular form of communication that bacteria use to coordinate group behaviors such as biofilm formation and the production of antibiotics and virulence factors. The term quorum sensing was originally coined to describe the mechanism underlying the onset of luminescence production in cultures of the marine bacterium Vibrio fischeri. Luminescence and, more generally, quorum sensing are important for V. fischeri to form a mutualistic symbiosis with the Hawaiian bobtail squid, Euprymna scolopes. The symbiosis is established when V. fischeri cells migrate via flagella-based motility from the surrounding seawater into a specialized structure injuvenile squid called the light organ. The cells grow to high cell densities within the light organ where the infection persists over the lifetime of the animal. A hallmark of a successful symbiosis is the luminescence produced by V. fischeri that camouflages the squid at night by eliminating its shadow within the water column. While the regulatory networks governing quorum sensing are critical for properly regulating V. fischeri luminescence within the squid light organ, they also regulate luminescence-independent processes during symbiosis. In this review, we discuss the quorum-sensing network of V. fischeri and highlight its impact at various stages during host colonization.

The complete mitochondrial genomes of deep-sea squid (Bathyteuthis abyssicola), bob-tail squid (Semirossia patagonica) and four giant cuttlefish (Sepia apama, S. latimanus, S. lycidas and S. pharaonis), and their application to the phylogenetic analysis of Decapodiformes

We determined the complete mitochondrial (mt) genomes of the deep-sea squid (Bathyteuthis abyssicola; supperfamily Bathyteuthoidea), the bob-tail squid (Semirossia patagonica; order Sepiolida) and four giant cuttlefish (Sepia apama, S. latimanus, S. lycidas and S. pharaonis; order Sepiida). The unique structures of the mt genomes of Bathyteuthis and Semirossia provide new information about the evolution of decapodiform mt genomes. We show that the mt genome of B. abyssicola, like those of other oegopsids studied so far, has two long duplicated regions that include seven genes (COX1–3, ATP6 and ATP8, tRNAAsn, and either ND2 or ND3) and that one of the duplicated COX3 genes has lost its function. The mt genome of S. patagonica is unlike any other decapodiforms and, like Nautilus, its ATP6 and ATP8 genes are not adjacent to each other. The four giant cuttlefish have identical mt gene order to other cuttlefish determined to date. Molecular phylogenetic analyses using maximum likelihood and Bayesian methods suggest that traditional order Sepioidea (Sepiolida+Sepiida) is paraphyletic and Sepia (cuttlefish) has the sister-relationship with all other decapodiforms. Taking both the phylogenetic analyses and the mt gene order analyses into account, it is likely that the octopus-type mt genome is an ancestral state and that it had maintained from at least the Cephalopoda ancestor to the common ancestor of Oegopsida, Myopsida and Sepiolida.

Genetic determinants of swimming motility in the squid light‐organ symbiont Vibrio fischeri

Bacterial flagellar motility is a complex cellular behavior required for the colonization of the light-emitting organ of the Hawaiian bobtail squid, Euprymna scolopes, by the beneficial bioluminescent symbiont Vibrio fischeri. We characterized the basis of this behavior by performing (i) a forward genetic screen to identify mutants defective in soft-agar motility, as well as (ii) a transcriptional analysis to determine the genes that are expressed downstream of the flagellar master regulator FlrA. Mutants with severe defects in soft-agar motility were identified due to insertions in genes with putative roles in flagellar motility and in genes that were unexpected, including those predicted to encode hypothetical proteins and cell division–related proteins. Analysis of mutants for their ability to enter into a productive symbiosis indicated that flagellar motility mutants are deficient, while chemotaxis mutants are able to colonize a subset of juvenile squid to light-producing levels. Thirty-three genes required for normal motility in soft agar were also downregulated in the absence of FlrA, suggesting they belong to the flagellar regulon of V. fischeri. Mutagenesis of putative paralogs of the flagellar motility genes motA motB, and fliL revealed that motA1 motB1, and both fliL1 and fliL2, but not motA2 and motB2, likely contribute to soft-agar motility. Using these complementary approaches, we have characterized the genetic basis of flagellar motility in V. fischeri and furthered our understanding of the roles of flagellar motility and chemotaxis in colonization of the juvenile squid, including identifying 11 novel mutants unable to enter into a productive light-organ symbiosis.

Exploring host-microbiota interactions in animal models and humans.

The animal and bacterial kingdoms have coevolved and coadapted in response to environmental selective pressures over hundreds of millions of years. The meta'omics revolution in both sequencing and its analytic pipelines is fostering an explosion of interest in how the gut microbiome impacts physiology and propensity to disease. Gut microbiome studies are inherently interdisciplinary, drawing on approaches and technical skill sets from the biomedical sciences, ecology, and computational biology. Central to unraveling the complex biology of environment, genetics, and microbiome interaction in human health and disease is a deeper understanding of the symbiosis between animals and bacteria. Experimental model systems, including mice, fish, insects, and the Hawaiian bobtail squid, continue to provide critical insight into how host-microbiota homeostasis is constructed and maintained. Here we consider how model systems are influencing current understanding of host-microbiota interactions and explore recent human microbiome studies.

Radiance of the kraken

Radiance of the kraken

Quorum sensing

When the Hawaiian bobtail squid rises from the sand each evening to hunt, it swims cloaked from detection by predators and prey because it casts no moonlight shadow, a trick it owes to compartments full of glowing bacteria on its underside. It tunes these mini-floodlights to the right intensity using light sensors on its back, adjusting a shutter on its body to match the moonlight perfectly (1).

Chemoreceptor VfcA Mediates Amino Acid Chemotaxis in Vibrio fischeri

Flagellar motility and chemotaxis by Vibrio fischeri are important behaviors mediating the colonization of its mutualistic host, the Hawaiian bobtail squid. However, none of the 43 putative methyl-accepting chemotaxis proteins (MCPs) encoded in the V. fischeri genome has been previously characterized. Using both an available transposon mutant collection and directed mutagenesis, we isolated mutants for 19 of these genes, and screened them for altered chemotaxis to six previously identified chemoattractants. Only one mutant was defective in responding to any of the tested compounds; the disrupted gene was thus named vfcA (Vibrio fischeri chemoreceptor A; locus tag VF_0777). In soft-agar plates, mutants disrupted in vfcA did not exhibit the serine-sensing chemotactic ring, and the pattern of migration in the mutant was not affected by the addition of exogenous serine. Using a capillary chemotaxis assay, we showed that, unlike wild-type V. fischeri, the vfcA mutant did not undergo chemotaxis toward serine and that expression of vfcA on a plasmid in the mutant was sufficient to restore the behavior. In addition to serine, we demonstrated that alanine, cysteine, and threonine are strong attractants for wild-type V. fischeri and that the attraction is also mediated by VfcA. This study thus provides the first insights into how V. fischeri integrates information from one of its 43 MCPs to respond to environmental stimuli.

Changing Views of the Evolution of Immunity

Changing Views of the Evolution of Immunity

Vibrio fischeriexhibit the growth advantage in stationary-phase phenotype

Vibrio fischeri are bioluminescent marine bacteria that can be isolated from their symbiotic animal partners or from ocean water. A V. fischeri population increases exponentially inside the light organ of the Hawaiian bobtail squid (Euprymna scolopes) while the host is quiescent during the day. This bacterial light organ population reaches stationary phase and then remains high during the night, when the squid use bacterial bioluminescence as a counter-predation strategy. At dawn, host squid release 90%-95% of the light organ contents into the ocean water prior to burying in the sand for the day. As the squid sleeps, the cycle of bacterial population growth in the light organ begins again. These V. fischeri cells that are vented into the ocean must persist under typical marine low nutrient conditions until they encounter another opportunity to colonize a host. We hypothesized that because V. fischeri regularly encounter cycles of feast and famine in nature, they would exhibit the growth advantage in stationary phase (GASP) phenotype. We found that older V. fischeri cells exhibit a Class 2 GASP response in which old cells increase dramatically in frequency while the population of young V. fischeri cells remains almost constant during co-incubation.

Recombinant reflectin‐based optical materials

AbstractReflectins are a unique group of structural proteins involved in dynamic optical systems in cephalopods that modulate incident light or bioluminescence. We describe cloning, structural characterization, and optical properties of a reflectin‐based domain, refCBA, from reflectin 1a of Hawaiian bobtail squid, Euprymna scolopes. Thin films created from the recombinant protein refCBA display interesting optical features when the recombinant protein is appropriately organized. RefCBA was cloned and expressed as a soluble protein enabling purification, with little structural organization found using Fourier transform infrared spectroscopy and circular dichroism. Single‐layer and multi‐layer thin films of refCBA were then produced by flow coating and spin coating, and displayed colors due to thin film interference. Diffraction experiments showed the assemblies were ordered enough to work as diffraction gratings to generate diffraction patterns. Nano‐spheres and lamellar microstructures of refCBA samples were observed by scanning electron microscopy and atomic force microscopy. Despite the reduced complexity of the refCBA protein compared to natural reflectins, unique biomaterials with similar properties to reflectins were generated by self‐assembled reflectin‐based refCBA molecules. © 2012 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys, 2013

Host/Microbe Interactions Revealed Through “Omics” in the Symbiosis Between the Hawaiian Bobtail SquidEuprymna scolopesand the Bioluminescent BacteriumVibrio fischeri

The association between Euprymna scolopes, the Hawaiian bobtail squid, and Vibrio fischeri, a bioluminescent bacterium, has served as a model for beneficial symbioses for over 25 years. The experimental tractability of this association has helped researchers characterize many of the colonization events necessary for symbiosis. Recent technological advances, such as the sequenced genome of V. fischeri, DNA microarrays, and high-throughput transcriptomics and proteomics, have allowed for the identification of host and symbiont factors that are important in establishing and maintaining specificity in the association. We highlight some of these findings pertaining to quorum sensing, luminescence, responses to reactive oxygen and nitrogen species, recognition of microbe-associated molecular patterns by the innate immune system of the host, and a diel rhythm that helps regulate the symbiont population. We also discuss how comparative genomics has allowed the identification of symbiont factors important for specificity and why sequencing the host's genome should be a priority for the research community.

A Semi-quantitative Approach to Assess Biofilm Formation Using Wrinkled Colony Development

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a biofilm, bacterial cells often exhibit altered physiology, including enhanced resistance to antibiotics and other environmental stresses. Additionally, biofilms can play important roles in host-microbe interactions. Biofilms develop when bacteria transition from individual, planktonic cells to form complex, multi-cellular communities. In the laboratory, biofilms are studied by assessing the development of specific biofilm phenotypes. A common biofilm phenotype involves the formation of wrinkled or rugose bacterial colonies on solid agar media. Wrinkled colony formation provides a particularly simple and useful means to identify and characterize bacterial strains exhibiting altered biofilm phenotypes, and to investigate environmental conditions that impact biofilm formation. Wrinkled colony formation serves as an indicator of biofilm formation in a variety of bacteria, including both Gram-positive bacteria, such as Bacillus subtilis, and Gram-negative bacteria, such as Vibrio cholerae, Vibrio parahaemolyticus, Pseudomonas aeruginosa, and Vibrio fischeri. The marine bacterium V. fischeri has become a model for biofilm formation due to the critical role of biofilms during host colonization: biofilms produced by V. fischeri promote its colonization of the Hawaiian bobtail squid Euprymna scolopes. Importantly, biofilm phenotypes observed in vitro correlate with the ability of V. fischeri cells to effectively colonize host animals: strains impaired for biofilm formation in vitro possess a colonization defect, while strains exhibiting increased biofilm phenotypes are enhanced for colonization. V. fischeri therefore provides a simple model system to assess the mechanisms by which bacteria regulate biofilm formation and how biofilms impact host colonization. In this report, we describe a semi-quantitative method to assess biofilm formation using V. fischeri as a model system. This method involves the careful spotting of bacterial cultures at defined concentrations and volumes onto solid agar media; a spotted culture is synonymous to a single bacterial colony. This 'spotted culture' technique can be utilized to compare gross biofilm phenotypes at single, specified time-points (end-point assays), or to identify and characterize subtle biofilm phenotypes through time-course assays of biofilm development and measurements of the colony diameter, which is influenced by biofilm formation. Thus, this technique provides a semi-quantitative analysis of biofilm formation, permitting evaluation of the timing and patterning of wrinkled colony development and the relative size of the developing structure, characteristics that extend beyond the simple overall morphology.

A Semi-quantitative Approach to Assess Biofilm Formation Using Wrinkled Colony Development

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a biofilm, bacterial cells often exhibit altered physiology, including enhanced resistance to antibiotics and other environmental stresses 1. Additionally, biofilms can play important roles in host-microbe interactions. Biofilms develop when bacteria transition from individual, planktonic cells to form complex, multi-cellular communities 2. In the laboratory, biofilms are studied by assessing the development of specific biofilm phenotypes. A common biofilm phenotype involves the formation of wrinkled or rugose bacterial colonies on solid agar media 3. Wrinkled colony formation provides a particularly simple and useful means to identify and characterize bacterial strains exhibiting altered biofilm phenotypes, and to investigate environmental conditions that impact biofilm formation. Wrinkled colony formation serves as an indicator of biofilm formation in a variety of bacteria, including both Gram-positive bacteria, such as Bacillus subtilis 4, and Gram-negative bacteria, such as Vibrio cholerae 5, Vibrio parahaemolyticus 6, Pseudomonas aeruginosa 7, and Vibrio fischeri 8. The marine bacterium V. fischeri has become a model for biofilm formation due to the critical role of biofilms during host colonization: biofilms produced by V. fischeri promote its colonization of the Hawaiian bobtail squid Euprymna scolopes 8-10. Importantly, biofilm phenotypes observed in vitro correlate with the ability of V. fischeri cells to effectively colonize host animals: strains impaired for biofilm formation in vitro possess a colonization defect 9,11, while strains exhibiting increased biofilm phenotypes are enhanced for colonization 8,12. V. fischeri therefore provides a simple model system to assess the mechanisms by which bacteria regulate biofilm formation and how biofilms impact host colonization. In this report, we describe a semi-quantitative method to assess biofilm formation using V. fischeri as a model system. This method involves the careful spotting of bacterial cultures at defined concentrations and volumes onto solid agar media; a spotted culture is synonymous to a single bacterial colony. This 'spotted culture' technique can be utilized to compare gross biofilm phenotypes at single, specified time-points (end-point assays), or to identify and characterize subtle biofilm phenotypes through time-course assays of biofilm development and measurements of the colony diameter, which is influenced by biofilm formation. Thus, this technique provides a semi-quantitative analysis of biofilm formation, permitting evaluation of the timing and patterning of wrinkled colony development and the relative size of the developing structure, characteristics that extend beyond the simple overall morphology.

Multiple Fitness Benefits of Polyandry in a Cephalopod

BackgroundSex differences in reproductive investment play a crucial role in sexual conflict. One intriguing aspect of sexual conflict is the evolution of female multiple mating (polyandry), particularly in systems where females receive no obvious direct benefits from males, and where mating is highly costly. Here, theory predicts that polyandrous females can increase their reproductive success by taking advantage of the genetic benefits of mating with multiple males. Cephalopods provide a model system for addressing this question, as all species mate multiply. Here we examine differences in reproductive success between monandrous, multiply mated (to the same male) and polyandrous female dumpling squid (Euprymna tasmanica).Methodology/Principal FindingsWe mated females in the laboratory with two different males (polyandrous; controlling for mating order), or with a single male (monandrous). To control for mating frequency, we mated monandrous females either once (monandrous 1), or with the same male twice (monandrous 2), and measured reproductive success for each of the three treatments (polyandrous, monandrous 1, monandrous 2). Females mated to two different males produced eggs faster and had larger hatchlings relative to egg mass than females mated once with a single male.Conclusions/SignificanceThe benefits of polyandry demonstrated here are the first, to our knowledge, in any cephalopod. These benefits may outweigh the significant costs associated with mating and help to explain how multiple mating has evolved (or is maintained) in this group.

Squid-Derived Chitin Oligosaccharides Are a Chemotactic Signal during Colonization by Vibrio fischeri

Chitin, a polymer of N-acetylglucosamine (GlcNAc), is noted as the second most abundant biopolymer in nature. Chitin serves many functions for marine bacteria in the family Vibrionaceae ("vibrios"), in some instances providing a physical attachment site, inducing natural genetic competence, and serving as an attractant for chemotaxis. The marine luminous bacterium Vibrio fischeri is the specific symbiont in the light-emitting organ of the Hawaiian bobtail squid, Euprymna scolopes. The bacterium provides the squid with luminescence that the animal uses in an antipredatory defense, while the squid supports the symbiont's nutritional requirements. V. fischeri cells are harvested from seawater during each host generation, and V. fischeri is the only species that can complete this process in nature. Furthermore, chitin is located in squid hemocytes and plays a nutritional role in the symbiosis. We demonstrate here that chitin oligosaccharides produced by the squid host serve as a chemotactic signal for colonizing bacteria. V. fischeri uses the gradient of host chitin to enter the squid light organ duct and colonize the animal. We provide evidence that chitin serves a novel function in an animal-bacterial mutualism, as an animal-produced bacterium-attracting synomone.