Biomedical potential of ß-chitosan from cuttlebone of cephalopods.

Polymeric ß-chitosan allomorph characterized by parallel arrangement of linear polysaccharide comprised of ß-(1 â†’ 4)-linked-D-glucosamine and N-acetyl-D-glucosamine was isolated and characterized from the gladius of Indian Ocean Squid (Uroteuthis duvaucelii) and spineless cuttlefish (Sepiella inermis). The ß-chitosan from U. duvaucelii displayed considerably greater attenuation potential against hydroxymethylglutaryl coenzyme-A reductase, dipeptidyl peptidase-4, I converting enzyme, and 5-lipoxygenase (IC50 0.15-0.53 mg mL-1) than those exhibited by α-chitosan of comparable molecular weight. Comparatively lesser conformational rigidity of ß-chitin could result in its greater susceptibility to deacetylation (86-87%) contrasted to the α-allomorph (~83%), consequently delivering enhanced functionalities than those exhibited by α-chitosan. Porous ß-chitosan scaffolds displayed an average pore size of lesser than 50 µm, and its binding capacity was significantly higher than that exhibited by α-chitosan (p < 0.05). Potential pharmacological activities reinforced by lesser binding affinities and intermolecular energy of ß-chitosan with the target enzymes recognized its prospective biomedical applications.

A lasting symbiosis: how Vibrio fischeri finds a squid partner and persists within its natural host.

As our understanding of the human microbiome progresses, so does the need for natural experimental animal models that promote a mechanistic understanding of beneficial microorganism-host interactions. Years of research into the exclusive symbiosis between the Hawaiian bobtail squid, Euprymna scolopes, and the bioluminescent bacterium Vibrio fischeri have permitted a detailed understanding of those bacterial genes underlying signal exchange and rhythmic activities that result in a persistent, beneficial association, as well as glimpses into the evolution of symbiotic competence. Migrating from the ambient seawater to regions deep inside the light-emitting organ of the squid, V. fischeri experiences, recognizes and adjusts to the changing environmental conditions. Here, we review key advances over the past 15 years that are deepening our understanding of these events.

A lasting symbiosis: how the Hawaiian bobtail squid finds and keeps its bioluminescent bacterial partner.

For more than 30 years, the association between the Hawaiian bobtail squid, Euprymna scolopes, and the bioluminescent bacterium Vibrio fischeri has been studied as a model system for understanding the colonization of animal epithelia by symbiotic bacteria. The squid-vibrio light-organ system provides the exquisite resolution only possible with the study of a binary partnership. The impact of this relationship on the partners' biology has been broadly characterized, including their ecology and evolutionary biology as well as the underlying molecular mechanisms of symbiotic dynamics. Much has been learned about the factors that foster initial light-organ colonization, and more recently about the maturation and long-term maintenance of the association. This Review synthesizes the results of recent research on the light-organ association and also describes the development of new horizons for E. scolopes as a model organism that promises to inform biology and biomedicine about the basic nature of host-microorganism interactions.

A resonant squid-inspired robot unlocks biological propulsive efficiency.

Elasticity has been linked to the remarkable propulsive efficiency of pulse-jet animals such as the squid and jellyfish, but reports that quantify the underlying dynamics or demonstrate its application in robotic systems are rare. This work identifies the pulse-jet propulsion mode used by these animals as a coupled mass-spring-mass oscillator, enabling the design of a flexible self-propelled robot. We use this system to experimentally demonstrate that resonance greatly benefits pulse-jet swimming speed and efficiency, and the robot's optimal cost of transport is found to match that of the most efficient biological swimmers in nature, such as the jellyfish Aurelia aurita The robot also exhibits a preferred Strouhal number for efficient swimming, thereby bridging the gap between pulse-jet propulsion and established findings in efficient fish swimming. Extensions of the current robotic framework to larger amplitude oscillations could combine resonance effects with optimal vortex formation to further increase propulsive performance and potentially outperform biological swimmers altogether.

Multiple observations of Bigfin Squid (Magnapinna sp.) in the Great Australian Bight reveal distribution patterns, morphological characteristics, and rarely seen behaviour.

One of the most remarkable groups of deep-sea squids is the Magnapinnidae, known for their large fins and strikingly long arm and tentacle filaments. Little is known of their biology and ecology as most specimens are damaged and juvenile, and in-situ sightings are sparse, numbering around a dozen globally. As part of a recent large-scale research programme in the Great Australian Bight, Remotely Operated Vehicles and a towed camera system were deployed in depths of 946-3258 m resulting in five Magnapinna sp. sightings. These represent the first records of Bigfin Squid in Australian waters, and more than double the known records from the southern hemisphere, bolstering a hypothesis of cosmopolitan distribution. As most previous observations have been of single Magnapinna squid these multiple sightings have been quite revealing, being found in close spatial and temporal proximity of each other. Morphological differences indicate each sighting is of an individual rather than multiple sightings of the same squid. In terms of morphology, previous in-situ measurements have been roughly based on nearby objects of known size, but this study used paired lasers visible on the body of a Magnapinna squid, providing a more accurate scaling of size. Squid of a juvenile size were also recorded and are confirmed to possess the long distal filaments which have thus far been mostly missing from specimens due to damage. We have described fine-scale habitat, in-situ colouration, and behavioural components including a horizontal example of the 'elbow' pose, and coiling of distal filaments: a behaviour not previously seen in squid. These sightings add to our knowledge of this elusive and intriguing genus, and reinforce the value of imagery as a tool in deep-sea squid research.

Squids use multiple escape jet patterns throughout ontogeny.

Throughout their lives, squids are both predators and prey for a multitude of animals, many of which are at the top of ocean food webs, making them an integral component of the trophic structure of marine ecosystems. The escape jet, which is produced by the rapid expulsion of water from the mantle cavity through a funnel, is central to a cephalopod's ability to avoid predation throughout its life. Although squid undergo morphological and behavioral changes and experience remarkably different Reynolds number regimes throughout their development, little is known about the dynamics and propulsive efficiency of escape jets throughout ontogeny. We examine the hydrodynamics and kinematics of escape jets in squid throughout ontogeny using 2D/3D velocimetry and high-speed videography. All life stages of squid produced two escape jet patterns: (1) 'escape jet I' characterized by short rapid pulses resulting in vortex ring formation and (2) 'escape jet II' characterized by long high-volume jets, often with a leading-edge vortex ring. Paralarvae exhibited higher propulsive efficiency than adult squid during escape jet ejection, and propulsive efficiency was higher for escape jet I than escape jet II in juveniles and adults. These results indicate that although squid undergo major ecological transitions and morphology changes from paralarvae to adults, all life stages demonstrate flexibility in escape jet responses and produce escape jets of surprisingly high propulsive efficiency.This article has an associated First Person interview with the first author of the paper.

Hawaiian Bobtail Squid Symbionts Inhibit Marine Bacteria via Production of Specialized Metabolites, Including New Bromoalterochromides BAC-D/D'.

The Hawaiian bobtail squid, Euprymna scolopes, has a symbiotic bacterial consortium in the accessory nidamental gland (ANG), a female reproductive organ that protects eggs against fouling microorganisms. To test the antibacterial activity of ANG community members, 19 bacterial isolates were screened for their ability to inhibit Gram-negative and Gram-positive bacteria, of which two strains were inhibitory. These two antibacterial isolates, Leisingera sp. ANG59 and Pseudoalteromonas sp. JC28, were subjected to further genomic characterization. Genomic analysis of Leisingera sp. ANG59 revealed a biosynthetic gene cluster encoding the antimicrobial compound indigoidine. The genome of Pseudoalteromonas sp. JC28 had a 14-gene cluster with >95% amino acid identity to a known bromoalterochromide (BAC) cluster. Chemical analysis confirmed production of known BACs, BAC-A/A' (compounds 1a/1b), as well as two new derivatives, BAC-D/D' (compounds 2a/2b). Extensive nuclear magnetic resonance (NMR) analyses allowed complete structural elucidation of compounds 2a/2b, and the absolute stereochemistry was unambiguously determined using an optimized Marfey's method. The BACs were then investigated for in vitro antibacterial, antifungal, and nitric oxide (NO) inhibitory activity. Compounds 1a/1b were active against the marine bacteria Bacillus algicola and Vibrio fischeri, while compounds 2a/2b were active only against B. algicola Compounds 1a/1b inhibited NO production via lipopolysaccharide (LPS)-induced inflammation in RAW264.7 macrophage cells and also inhibited the pathogenic fungus Fusarium keratoplasticum, which, coupled with their antibacterial activity, suggests that these polyketide-nonribosomal peptides may be used for squid egg defense against potential pathogens and/or fouling microorganisms. These results indicate that BACs may provide Pseudoalteromonas sp. JC28 an ecological niche, facilitating competition against nonsymbiotic microorganisms in the host's environment.IMPORTANCE Animals that deposit eggs must protect their embryos from fouling and disease by microorganisms to ensure successful development. Although beneficial bacteria are hypothesized to contribute to egg defense in many organisms, the mechanisms of this protection are only recently being elucidated. Our previous studies of the Hawaiian bobtail squid focused on fungal inhibition by beneficial bacterial symbionts of a female reproductive gland and eggs. Herein, using genomic and chemical analyses, we demonstrate that symbiotic bacteria from this gland can also inhibit other marine bacteria in vitro One bacterial strain in particular, Pseudoalteromonas sp. JC28, had broad-spectrum abilities to inhibit potential fouling bacteria, in part via production of novel bromoalterochromide metabolites, confirmed via genomic annotation of the associated biosynthetic gene cluster. Our results suggest that these bacterial metabolites may contribute to antimicrobial activity in this association and that such defensive symbioses are underutilized sources for discovering novel antimicrobial compounds.

Squid sucker teeth and cocoons of a terrestrial flatworm: amino acid content of two nano-structurally identical tissues in phylogenetically unrelated taxa.

An extraordinary micro-structural similarity between squid sucker teeth and the egg shell of a terrestrial planarian worm has been reported, but to date only the amino acid content of the squid sucker tooth has been available. This prompted us to analyse the amino acid content of the planarian egg shell. Although both share an absence of detectable chitin and metal ions and both possess relatively high amounts of the amino acids GLY and HIS, the planarian egg shell is considerably richer in GLU, LYS and ASP. Most dramatic was the difference in TYR, which was the second most abundant amino acid in the squid, but hardly featured at all in the planarian egg shell. In the light of these new findings the different functional roles that the structures in question play in the lives of the two taxa are discussed. An EDS-analysis revealed clear C, N, and O peaks and additional very small peaks less than 0.1w% suggesting the presence of one or both S and Cl.

New bobtail squid (Sepiolidae: Sepiolinae) from the Ryukyu islands revealed by molecular and morphological analysis.

Bobtail squid are emerging models for host-microbe interactions, behavior, and development, yet their species diversity and distribution remain poorly characterized. Here, we combine mitochondrial and transcriptome sequences with morphological analysis to describe three species of bobtail squid (Sepiolidae: Sepiolinae) from the Ryukyu archipelago, and compare them with related taxa. One Ryukyuan type was previously unknown, and is described here as Euprymna brenneri sp. nov. Another Ryukyuan type is morphologically indistinguishable from Sepiola parva Sasaki, 1913. Molecular analyses, however, place this taxon within the genus Euprymna Steenstrup, 1887, and additional morphological investigation led to formal rediagnosis of Euprymna and reassignment of this species as Euprymna parva comb. nov. While no adults from the third Ryukyuan type were found, sequences from hatchlings suggest a close relationship with E. pardalota Reid, 2011, known from Australia and East Timor. The broadly sampled transcriptomes reported here provide a foundation for future phylogenetic and comparative studies.

Close-up observations on the spawning behavior of a captive Japanese flying squid (Todarodes pacificus).

The spawning behavior of a Japanese flying squid (Todarodes pacificus) is described based on up-close observation of a captive female. The squid was first transferred from a 10-ton tank to a polystyrene plastic box containing 45 liters of seawater. About one hour later, the mantle-contraction rate increased rapidly, followed by a brief convulsion of the mantle and arms and a whitening of the body. The mantle contractions become shallow and rapid, and several seconds later, semitransparent jelly presumably from the nidamental glands emerged from the funnel and passed between the ventral pair of arms. Approximately 90 seconds after the egg mass first emerged, the female began ejecting oocytes through the funnel into the egg mass using rapid, powerful mantle contractions. Soon after the oocytes were ejected, translucent strands (presumably sperm) emanated from the buccal membrane. The female continued to eject oocytes for approximately two minutes, after which the mantle convulsed, and the mantle-contraction rate decreased slowly for about one minute until the contractions stopped. The squid died soon afterwards.

Surface functionalization of cuttlefish bone-derived biphasic calcium phosphate scaffolds with polymeric coatings.

Cuttlefish bone (CB) has been explored as biomaterial in the bone tissue-engineering field due to its unique porous structure and capacity of the aragonite mineral to be hydrothermally converted into calcium phosphates (CaPs). In the present study, undoped and ion (Sr2+, Mg2+ and/or Zn2+) doped biphasic calcium phosphate (BCP) scaffolds were prepared by hydrothermal transformation (HT, 200 °C, 24 h) of CB. The obtained scaffolds were sintered and then coated with two commercial polymers, poly(ε-caprolactone) (PCL) or poly(DL-lactide) (PDLA), and with two synthesized ones, a poly(ester amide) (PEA) or a poly(ester urea) (PEU) in order to improve their compressive strength. The scaffolds were characterized by X-ray diffraction (XRD) coupled with structural Rietveld refinement, Fourier transform infrared (FTIR) spectroscopy, and scanning electron microscopy (SEM). The results demonstrate that CB could be entirely transformed into BCPs in the presence or absence of doping elements. The initial CB structure was preserved and the polymeric coatings did not jeopardize the interconnected porous structure. Furthermore, the polymeric coatings enhanced the compressive strength of the scaffolds. The in vitro bio-mineralization upon immersing the scaffolds into simulated body fluid (SBF) demonstrated the formation of bone-like apatite surface layers in both uncoated and coated scaffolds. Overall, the produced scaffolds exhibit promising properties for bone tissue engineering applications.

Construction and Composition of the Squid Pen from Doryteuthis pealeii.

The pen, or gladius, of the squid is an internalized shell. It serves as a site of attachment for important muscle groups and as a protective barrier for the visceral organs. The pen's durability and flexibility are derived from its unique composition of chitin and protein. We report the characterization of the structure, development, and composition of pens from Doryteuthis pealeii. The nanofibrils of the polysaccharide ß-chitin are arranged in an aligned configuration in only specific regions of the pen. Chitin is secreted early in development, enabling us to characterize the changes in pen morphology prior to hatching. The chitin and proteins are assembled in the shell sac surrounded by fluid that has a significantly different ionic composition from squid plasma. Two groups of proteins are associated with the pen: those on its surface and those embedded within the pen. Only 20 proteins are identified as embedded within the pen. Embedded proteins are classified into six groups, including chitin associated, protease, protease inhibitors, intracellular, extracellular matrix, and those that are unknown. The pen proteins share many conserved domains with proteins from other chitinous structures. We conclude that the pen is one of the least complex, load-bearing, chitin-rich structures currently known and is amenable to further studies to elucidate natural construction mechanisms using chitin and protein.

Development of a contrast-enhanced micro computed tomography protocol for the oval squid (Sepioteuthis lessoniana) brain.

Coleoid cephalopods (squid, cuttlefish, and octopus) have a well-developed and complex central nervous system. Its absolute size is the largest among invertebrates, and the brain-to-body mass ratio is larger than that of fish and reptiles and equivalent to that of birds and mammals. Although a number of histological studies have been conducted on the brains of cephalopods, most of them used a light microscope or an electron microscope, which show the microstructure of the brain, but often cannot image the whole brain instantaneously. Of late, micro computed tomography (CT) has gained popularity for imaging animal brains because it allows for noninvasive three-dimensional (3D) reconstruction and preprocessing that are not cumbersome. To perform micro-CT on cephalopod brains, we first tested conditions suitable for preprocessing, paying special attention to staining conditions that would provide high contrast images. Four agents, iodine in 99.5% ethanol, iodine potassium iodide in water (IKI), phosphotungstic acid in 70% ethanol, and nonionic iodinated contrast agent in water, were tested at various concentrations and durations on brain of juvenile oval squid. To evaluate the quality of staining, we calculated the contrast ratio of the two-dimensional (2D) images and compared 3D segmentation of the best and worst 2D images. We concluded that 3% IKI staining for 7 days was the best combination to enhance the images contrast of the oval squid brain, in which each brain lobe was clearly detected and 3D segmentation of the whole brain was possible. The wider applicability of this preprocessing method for micro-CT of the brains of other cephalopods is discussed.

A proteomic analysis of the statocyst endolymph in common cuttlefish (Sepia officinalis): an assessment of acoustic trauma after exposure to sound.

Recent studies, both in laboratory and sea conditions, have demonstrated damage after sound exposure in the cephalopod statocyst sensory epithelium, which secretes endolymph protein. Here, the proteomic analysis of the endolymph was performed before and after sound exposure to assess the effects of exposure to low intensity, low frequency sounds on the statocyst endolymph of the Mediterranean common cuttlefish (Sepia officinalis), determining changes in the protein composition of the statocyst endolymph immediately and 24 h after sound exposure. Significant differences in protein expression were observed, especially 24 h after exposure. A total of 37 spots were significantly different in exposed specimens, 17 of which were mostly related to stress and cytoskeletal structure. Among the stress proteins eight spots corresponding to eight hemocyanin isoforms were under-expressed possible due to lower oxygen consumption. In addition, cytoskeletal proteins such as tubulin alpha chain and intermediate filament protein were also down-regulated after exposure. Thus, endolymph analysis in the context of acoustic stress allowed us to establish the effects at the proteome level and identify the proteins that are particularly sensitive to this type of trauma.

Predatory behaviour and taphonomy of a Jurassic belemnoid coleoid (Diplobelida, Cephalopoda).

We describe four complete specimens of the early squid-like cephalopod Clarkeiteuthis conocauda from the Toarcian Posidonienschiefer (Jurassic) each preserved with the bony fish Leptolepis bronni in its arms. Based on the arrangement of prey and predator, we suggest that the cephalopods caught and killed the fishes while still in well-oxygenated waters and then descended into oxygen-depleted water layers (distraction sinking) where the cephalopod suffocated. This explains the exceptional preservation, for which the Posidonienschiefer is famed. This association raises the question for the hunting behaviour of belemnoid Coleoidea. Using the proportions of soft and skeletal body parts of diplobelids and belemnitids, we estimated their body mass and buoyancy and determined the centres of mass and buoyancy. These two points were very close to each other in belemnitids, implying a low hydrodynamic stability (when ignoring the fins), while in diplobelids, the distance between those centres was greater. This suggests that diplobelids usually assumed an oblique to vertical orientation of the body axis while belemnitids could effortlessly achieve a horizontal orientation of their body. Presuming larger fins were attached to the bigger belemnitid rostra, belemnitids were better swimmers and perhaps pursuit predators while diplobelids rather ambushed their prey.

Effects of channel blocking on information transmission and energy efficiency in squid giant axons.

Action potentials are the information carriers of neural systems. The generation of action potentials involves the cooperative opening and closing of sodium and potassium channels. This process is metabolically expensive because the ions flowing through open channels need to be restored to maintain concentration gradients of these ions. Toxins like tetraethylammonium can block working ion channels, thus affecting the function and energy cost of neurons. In this paper, by computer simulation of the Hodgkin-Huxley neuron model, we studied the effects of channel blocking with toxins on the information transmission and energy efficiency in squid giant axons. We found that gradually blocking sodium channels will sequentially maximize the information transmission and energy efficiency of the axons, whereas moderate blocking of potassium channels will have little impact on the information transmission and will decrease the energy efficiency. Heavy blocking of potassium channels will cause self-sustained oscillation of membrane potentials. Simultaneously blocking sodium and potassium channels with the same ratio increases both information transmission and energy efficiency. Our results are in line with previous studies suggesting that information processing capacity and energy efficiency can be maximized by regulating the number of active ion channels, and this indicates a viable avenue for future experimentation.

Morphological changes of the optic lobe from late embryonic to adult stages in oval squids Sepioteuthis lessoniana.

The optic lobe is the largest brain area within the central nervous system of cephalopods and it plays important roles in the processing of visual information, the regulation of body patterning, and locomotive behavior. The oval squid Sepioteuthis lessoniana has relatively large optic lobes that are responsible for visual communication via dynamic body patterning. It has been observed that the visual behaviors of oval squids change as the animals mature, yet little is known about how the structure of the optic lobes changes during development. The aim of the present study was to characterize the ontogenetic changes in neural organization of the optic lobes of S. lessoniana from late embryonic stage to adulthood. Magnetic resonance imaging and micro-CT scans were acquired to reconstruct the 3D-structure of the optic lobes and examine the external morphology at different developmental stages. In addition, optic lobe slices with nuclear staining were used to reveal changes in the internal morphology throughout development. As oval squids mature, the proportion of the brain making up the optic lobes increases continuously, and the optic lobes appear to have a prominent dent on the ventrolateral side. Inside the optic lobe, the cortex and the medulla expand steadily from the late embryonic stage to adulthood, but the cell islands in the tangential zone of the optic lobe decrease continuously in parallel. Interestingly, the size of the nuclei of cells within the medulla of the optic lobe increases throughout development. These findings suggest that the optic lobe undergoes continuous external morphological change and internal neural reorganization throughout the oval squid's development. These morphological changes in the optic lobe are likely to be responsible for changes in the visuomotor behavior of oval squids from hatching to adulthood.

Self-coacervation of modular squid beak proteins - a comparative study.

The beak of the Humboldt squid is a biocomposite material made solely of organic components - chitin and proteins - which exhibits 200-fold stiffness and hardness gradients from the soft base to the exceptionally hard tip (rostrum). The outstanding mechanical properties of the squid beak are achieved via controlled hydration and impregnation of the chitin-based scaffold by protein coacervates. Molecular-based understanding of these proteins is essential to mimic the natural beak material. Here, we present detailed studies of two histidine-rich beak proteins (HBP-1 and -2) that play central roles during beak bio-fabrication. We show that both proteins have the ability to self-coacervate, which is governed intrinsically by the sequence modularity of their C-terminus and extrinsically by pH and ionic strength. We demonstrate that HBPs possess dynamic structures in solution and achieve maximum folding in the coacervate state, and propose that their self-coacervation is driven by hydrophobic interactions following charge neutralization through salt-screening. Finally, we show that subtle differences in the modular repeats of HBPs result in significant changes in the rheological response of the coacervates. This knowledge may be exploited to design self-coacervating polypeptides for a wide range of engineering and biomedical applications, for example bio-inspired composite materials, smart hydrogels and adhesives, and biomedical implants.

Characterization of the cell polarity gene crumbs during the early development and maintenance of the squid-vibrio light organ symbiosis.

The protein Crumbs is a determinant of apical-basal cell polarity and plays a role in apoptosis of epithelial cells and their protection against photodamage. Using the squid-vibrio system, a model for development of symbiotic partnerships, we examined the modulation of the crumbs gene in host epithelial tissues during initiation and maintenance of the association. The extracellular luminous symbiont Vibrio fischeri colonizes the apical surfaces of polarized epithelia in deep crypts of the Euprymna scolopes light organ. During initial colonization each generation, symbiont harvesting is potentiated by the biochemical and biophysical activity of superficial ciliated epithelia, which are several cell layers from the crypt epithelia where the symbionts reside. Within hours of crypt colonization, the symbionts induce the cell death mediated regression of the remote superficial ciliated fields. However, the crypt cells directly interacting with the symbiont are protected from death. In the squid host, we characterized the gene and encoded protein during light organ morphogenesis and in response to symbiosis. Features of the protein sequence and structure, phylogenetic relationships, and localization patterns in the eye supported assignment of the squid protein to the Crumbs family. In situ hybridization revealed that the crumbs transcript shows opposite expression at the onset of symbiosis in the two different regions of the light organ: elevated levels in the superficial epithelia were attenuated whereas low levels in the crypt epithelia were turned up. Although a rhythmic association in which the host controls the symbiont population over the day-night cycle begins in the juvenile upon colonization, cycling of crumbs was evident only in the adult organ with peak expression coincident with maximum symbiont population and luminescence. Our results provide evidence that crumbs responds to symbiont cues that induce developmental apoptosis and to symbiont population dynamics correlating with luminescence-based stress throughout the duration of the host-microbe association.