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.     

Collagen from diamondback squid (Thysanoteuthis rhombus) outer skin

Collagens (acid-solubilized and pepsin-solubilized collagens) were prepared from diamondback squid outer skin and partially characterized. The yields of acid-solubilized and pepsin-solubilized collagens were about 1.3 and 35.6%, respectively, on a dry weight basis. Pepsin-solubilized collagen was heterotrimer with a chain composition of ala2a3. The patterns of peptide fragments were different from that of porcine skin collagen. Denaturation temperature was 27.5 degrees C, about 10 degrees C lower than that of porcine collagen. The amino acid composition of pepsin-solubilized collagen from diamondback squid outer skin was similar to that from cuttlefish outer skin. This squid is big among squid species, and its skin is thick. It is clear that diamondback squid outer skin has a potential as an alternative source of collagen to bovine skin and bone. At present, collagen using aquatic materials such as skin (cod and a deep-sea fish) and scale (sea bream and anchovy) is the development stage in the related industries. Unless the problem of BSE infection in land animals is resolved aquatic materials as an alternative source of collagen will attract much attention in the cosmetic and medical fields.     

A new direct method of stock assessment of the loliginid squid

Hydroacoustic research conducted on chokka squid (Loligo reynaudi d’Orbigny, 1845), off the east coast of South Africa from 1994–2005, has led to the development of an innovative stock assessment technique, perhaps applicable to all loliginids that migrate inshore to spawn. This technique combines hydroacoustic biomass estimates made on the spawning concentrations inshore, and minimum biomass estimates made both inshore and offshore using demersal surveys employing the swept-area method. The hydroacoustic estimate uses an improved method to obtain target strength measurements, and squid concentrations are individually mapped from a small boat with a towed transducer. This method may be used even during intense fishing operations because of the manoeuvrability of the small boat inside a tight cluster of fishing vessels. Biomasses of the individual concentrations are then summed. The inshore biomass, also includes dispersed, mature squid migrating between concentrations, this is assessed using a concentration stability factor. The biomass of dispersed squid offshore is again calculated using the swept-area method, a well known demersal survey methodology. The biomass of concentrated (spawning) squid offshore is calculated using the same proportions between concentrated and dispersed squid which were found inshore. All four components are then summed to calculate the total biomass. The result obtained is subject to the effect of complex temporal dynamics, as new animals are recruited to the adult pool and those recently assessed migrate to other sectors of the distribution area.     

First-ever observations of a live giant squid in the wild

The giant squid, Architeuthis, is renowned as the largest invertebrate in the world and has featured as an ominous sea monster in novels and movies. Considerable efforts to view this elusive creature in its deep-sea habitat have been singularly unsuccessful. Our digital camera and depth recorder system recently photographed an Architeuthis attacking bait at 900m off Ogasawara Islands in the North Pacific. Here, we show the first wild images of a giant squid in its natural environment. Recovery of a severed tentacle confirmed both identification and scale of the squid (greater than 8m). Architeuthis appears to be a much more active predator than previously suspected, using its elongate feeding tentacles to strike and tangle prey.     

PRIMARY PEPTIDE SEQUENCES FROM SQUID MUSCLE AND OPTIC LOBE MYOSIN IIs: A STRATEGY TO IDENTIFY AN ORGANELLE MYOSIN

The squid giant axon provides an excellent model system for the study of actin-based organelle transport likely to be mediated by myosins, but the identification of these motors has proven to be difficult. Here the authors purified and obtained primary peptide sequence of squid muscle myosin as a first step in a strategy designed to identify myosins in the squid nervous system. Limited digestion yielded fourteen peptides derived from the muscle myosin which possess high amino acid sequence identities to myosin II from scallop (60–95%) and chick pectoralis muscle (31–83%). Antibodies generated to this purified muscle myosin were used to isolate a potential myosin from squid optic lobe which yielded 11 peptide fragments. Sequences from six of these fragments identified this protein as a myosin II. The other five sequences matched myosin II (50–60%, identities), and some also matched unconventional myosins (33–50%). A single band that has a molecular weight similar to the myosin purified from optic lobe copurifies with axoplasmic organelles, and, like the optic lobe myosin, this band is also recognized by the antibodies raised against squid muscle myosin II. Hence, this strategy provides an approach to the identification of a myosin associated with motile axoplasmic organelles.     

Development of Biomimetic Squid-Inspired Suckers

Biomechanical properties of squid suckers were studied to provide inspiration for the development of sucker artefacts for a robotic octopus.Mechanical support of the rings found inside squid suckers was studied by bending tests.Tensile tests were carried out to study the maximum possible sucking force produced by squid suckers based on the strength of sucker stalks,normalized by the sucking areas.The squid suckers were also directly tested to obtain sucking forces by a special testing arrangement.Inspired by the squid suckers,three types of sucker artefacts were developed for the arm skin of an octopus inspired robot.The first sucker artefact made of knitted nylon sheet reinforced silicone rubber has the same shape as the squid suckers.Like real squid suckers,this type of artefact also has a stalk that is connected to the arm skin and a ring to give radial support.The second design is a straight cylindrical structure with uniform wall thickness made of silicone rubber.One end of the cylinder is directly connected to the arm skin and the other end is open.The final design of the sucker has a cylindrical base and a concave meniscus top.The meniscus was formed naturally using the surface tension of silicone gel,which leads to a higher level of the liquid around the edge of a container.The wall thickness decreases towards the tip of the sucker opening.Sucking forces of all three types of sucker artefacts were measured.Advantages and disadvantages of each sucker type were discussed.The final design of suckers has been implemented to the arm skin prototypes.     

Calcium sensitivity of mantle muscle of squid.

Ca2+-sensitivity of squid muscle proteins was studied by competition tests between squid actin and carp myosin on squid myosin B and by cross combination tests between squid myosin and actin and between carp myosin and squid thin filament fraction. The results suggest that the regulatory system of the squid mantle muscle is actin-linked as in skeletal muscle rather than myosin-linked. This view is supported by the presence of troponin-like components on SDS-disc electrophoresis of squid thin filament fraction.     

Seafloor mapping and landscape ecology analyses used to monitor variations in spawning site preference and benthic egg mop abundance for the California market squid (Doryteuthis opalescens)

The California squid fishery is concentrated largely on nearshore squid spawning aggregations. Because of this practice a central concern for sustainable squid fisheries in California is to determine whether reproductive activities and subsequent egg laying occur at rates that are sufficient to support harvestable populations of this sub-annual species. Using high-resolution data collected via acoustic mapping methodology, we estimated a 99% decrease in egg mops abundance from 2005 to 2007. Sidescan sonar images from detailed benthic mapping suggest that although squids prefer a sandy substrate as their primary egg mop habitat, the depths across which egg mops were distributed differed significantly between surveys and spatial distribution of egg mops varied across years on this large spawning ground. Our results suggest that sidescan sonar surveys could serve as an important tool used to aid sustainable management of the California market squid fishery through the monitoring, designation and adaptive management of seasonally variable no-take spawning zones and can help in developing stock assessments of this commercially important species.     

Disintegration of a large concentration of loliginid squid as a response to predation

During hydroacoustic observations in November 2002, a large concentration of chokka squid (Loligo reynaudi) was monitored for three days during fairly constant wind speed and direction, that marginally improved during the period of observations. During this period, the concentration (estimated biomass 48 t) disintegrated into small, separate aggregations. Most often, such dispersal is weather-related, but obviously not in this case. Instead, a bottom trawl made adjacent to the concentration, as well as underwater camera observations revealed an unusually large number of predators, mostly bronze whaler sharks (Carcharhinus brachyurus). Most whaler sharks caught in the trawl had chokka in their stomachs. Therefore, one explanation for the break-up of this squid concentration was the unusual predator activity. Video observations revealed, that these attacks occurred on the bottom where squid spawn; while none were observed in the water column where squid pair, mate and swim in a circular motion preparing for descent to the egg bed. The disintegration of the whole concentration suggested that disrupted spawning affects the upper part of the typical mushroom-shaped structure as well, and squid subsequently disperse and/or move away as a result of predation by whaler sharks.     

A source of large axons for neurophysiology: the North Atlantic squid Illex illecebrosus.

A new axon preparation from the ommastrephid squid (Illex illecebrosusus (Lesueur)) is described. This squid is common in the Northwestern Atlantic and features a number of long, unbranched, and moderately large-diameter axons having no apparent 'twigging.' Although the diameters of these axons are somewhat smaller than those of 'giant' axons coming from some other species of squid, this axon preparation should be considered as an attractive alternative for neurobiologic research.     

Cloning and biochemical characterization of astacin-like squid metalloprotease

We have cloned four cDNAs encoding astacin-like squid metalloproteases (ALSMs)-I and -II from the Japanese common squid and ALSMs-I and -III from the spear squid. Analysis of the deduced amino acid sequences revealed that ALSMs possess a signal peptide and a pro-sequence followed by an astacin-like catalytic domain and an MAM (meprin, A5 protein, receptor protein-tyrosine phosphatase mu) domain. Phylogenetic analysis revealed that ALSM corresponds to a new cluster of astacins. To analyze the function of the MAM domain, wild-type ALSM and an MAM-truncated mutant were expressed in a baculovirus expression system. The expressed protein encoding full-length ALSM hydrolyzed myosin heavy chain as effectively as native ALSM, whereas the MAM-truncated mutant possessed no protease activity, suggesting that the MAM domain contributes to substrate recognition. ALSM has been isolated from squid liver and mantle muscle. However, analysis with a specific antibody generated against ALSM indicated the presence of ALSM in a wide variety of tissues. ALSM was located in the extracellular matrix of mantle muscle cells. Thus, ALSM is a secreted protease, as are other members of the astacin family. The extracellular localization raises the possibility of substrates other than myosin. The physiological role of ALSM remains unknown, at this time.     

Current-Voltage Relations in the Lobster Giant Axon Membrane Under Voltage Clamp Conditions

The sucrose-gap method introduced by Stämpfli provides a means for the application of a voltage clamp to the lobster giant axon, which responds to a variety of different experimental procedures in ways quite similar to those reported for the squid axon and frog node. This is particularly true for the behavior of the peak initial current. However, the steady state current shows some differences. It has a variable slope conductance less than that of the peak initial current. The magnitude of the steady state slope conductance is related to the length of the repolarization phase of the action potential, which does not have an undershoot in the lobster. The steady state outward current is maintained for as long as 100 msec.; this is in contrast to a decline of about 50 per cent in the squid axon. Lowering the external calcium concentration produces shifts in the current-voltage relations qualitatively similar to those obtained from the squid axon. On the basis of the data available, there is no reason to doubt that the Hodgkin and Huxley analysis for the squid giant axon in sea water can be applied to the lobster giant axon.     

Hydrogen sulfide as a precursor for the synthesis of isethionate in the squid giant axon.

Cysteine is taken up by the squid giant axon to about 200% of equivalent distribution, whereas sulfide is taken up (probably as hydrogen sulfide) to about 40% of equivalence. Thereafter, the squid axon synthesizes its major anion, isethionate, in about equal amounts from the sulfide, or from the sulfur of cysteine, but not at all from the carbons of cysteine. Squid nerve also contains rhodanese, an enzyme which transfers the outer (sulfane) sulfur of thiosulfate to cyanide to produce thiocyanate. It is speculated that, instead of “detoxifying cyanide,” as the reaction involving rhodanese is commonly described, the physiological role of this enzyme is the formation of a carbon-sulfur bond, leading finally, in the squid, to the formation of isethionate. This is the first evidence concerning the pathway for the synthesis of isethionate in squid nerve where this compound is normally present at a concentration of 150 mm.     

The potential impacts of climate change on inshore squid: biology, ecology and fisheries

Squid are important components of many marine ecosystems from the poles to the equator, serving as both important predators and prey. Novel aspects of their growth and reproduction mean that they are likely to play an important role in the changing oceans due to climate change. Virtually every facet of squid life-history examined thus far has revealed an incredible capacity in this group for life-history plasticity. The extremely fast growth rates of individuals and rapid rates of turnover at the population level mean that squid can respond quickly to environmental or ecosystem change. Their ‘life-in-the-fast-lane’ life-style allows them to rapidly exploit ‘vacuums’ created in the ecosystem when predators or competitors are removed. In this way, they function as ‘weeds of the sea’. Elevated temperatures accelerate the life-histories of squid, increasing their growth rates and shortening their life-spans. At first glance, it would be logical to suggest that rising water temperatures associated with climate change (if food supply remains adequate) would be beneficial to inshore squid populations and fisheries—growth rates would increase, life spans would shorten and population turnover would accelerate. However, the response of inshore squid populations to climate change is likely to be extremely complex. The size of hatchlings emerging from the eggs becomes smaller as temperatures increase and hatchling size may have a critical influence on the size-at-age that may be achieved as adults and subsequently, population structure. The influence of higher temperatures on the egg and adult stages may thus be opposing forces on the life-history. The process of climate change will likely result in squids that hatch out smaller and earlier, undergo faster growth over shorter life-spans and mature younger and at a smaller size. Individual squid will require more food per unit body size, require more oxygen for faster metabolisms and have a reduced capacity to cope without food. It is therefore likely that biological, physiological and behavioural changes in squid due to climate change will have far reaching effects.     

Role for cheR of Vibrio fischeri in the Vibrio-squid symbiosis

Upon hatching, the Hawaiian squid Euprymna scolopes is rapidly colonized by its symbiotic partner, the bioluminescent marine bacterium Vibrio fischeri . Vibrio fischeri cells present in the seawater enter the light organ of juvenile squid in a process that requires bacterial motility. In this study, we investigated the role chemotaxis may play in establishing this symbiotic colonization. Previously, we reported that V.?fischeri migrates toward numerous attractants, including N-acetylneuraminic acid (NANA), a component of squid mucus. However, whether or not migration toward an attractant such as squid-derived NANA helps the bacterium to localize toward the light organ is unknown. When tested for the ability to colonize juvenile squid, a V. fischeri chemotaxis mutant defective for the methyltransferase CheR was outcompeted by the wild-type strain in co-inoculation experiments, even when the mutant was present in fourfold excess. Our results suggest that the ability to perform chemotaxis is an advantage during colonization, but not essential.     

A BAYESIAN ANALYSIS TO ESTIMATE LOSS IN SQUID CATCH DUE TO THE IMPLEMENTATION OF A SEA LION POPULATION MANAGEMENT PLAN

This study estimates the effect of a sea lion ( Phocarctos hookeri ) population management plan on both the sea lion population and the associated squid ( Nototodarus sloanii ) fishery. The goal of the management plan is to rebuild the sea lion population and involves closing the squid fishery when a threshold level of sea lions have been caught. The threshold level is calculated from a generalized simulation analysis which conservatively allows for adequate population rebuilding for a number of different species and populations. Our analysis uses Bayesian theory to describe uncertainty in the sea lion population site and squid catch under the implementation of the management plan. The priors represent this particular sea lion population, and the analysis represents expectation rather than calculating conservative levels of safe fishing-related mortality. The results show that the squid catch is very sensitive to whether or not the squid fishery is closed when it exceeds the threshold level for sea lion bycatch. The sea lion population size is much less sensitive to the closure of the squid fishery. For an economically important fishery, the estimates of uncertainty in both loss of catch and increase in sea lion population are needed to allow informed decision-making about trade-offs between sea lion conservation and full exploitation of the fishery.     

Sensory, chemical and bacteriological changes during storage of iced squid (Todaropsis eblanae)

AIMS: To relate sensory shelf-life of iced whole and gutted squid to bacterial growth and chemical changes. METHODS AND RESULTS: Cooked mantles from whole and gutted individuals were rejected after 10 and 12 days of storage, respectively, due to ammoniacal off-odours. Rate of production of both ammonia and trimethylamine was highest in the whole lot. Agmatine, which was only present in trace amounts in freshly-caught squid, increased rapidly in both lots. The main microflora at the time of sensory rejection of iced whole squid included Gram-negative, motile and non-fermentative rods, which were psychrophilic and had a requirement for NaCl. 16S rDNA sequence analyses identified the strains as belonging to the genus Pseudoalteromonas. Shewanella putrefaciens, Pseudoalteromonas sp. and Pseudomonas sp. dominated in spoiled gutted squid. Identification of strains from the stomach and digestive gland of recently-captured squid showed that the main flora consisted of Photobacterium phosphoreum. CONCLUSIONS: Spoilage of iced squid is likely to result from a combination of autolytic and bacterial changes. Agmatine seems to be an excellent freshness indicator. Photobacterium phosphoreum may contribute to spoilage through activity in the digestive gland, followed by diffusion of volatile compounds and amines to the mantle. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to the psychrophilic nature of P. phosphoreum and Pseudoalteromonas sp., spread-plating and low temperature incubation are recommended for bacteriological evaluation of iced squid.     

Extreme plasticity in life‐history strategy allows a migratory predator (jumbo squid) to cope with a changing climate

Dosidicus gigas (jumbo or Humboldt squid) is a semelparous, major predator of the eastern Pacific that is ecologically and commercially important. In the Gulf of California, these animals mature at large size (>55 cm mantle length) in 1–1.5 years and have supported a major commercial fishery in the Guaymas Basin during the last 20 years. An El Niño event in 2009–2010, was accompanied by a collapse of this fishery, and squid in the region showed major changes in the distribution and life‐history strategy. Large squid abandoned seasonal coastal‐shelf habitats in 2010 and instead were found in the Salsipuedes Basin to the north, an area buffered from the effects of El Niño by tidal upwelling and a well‐mixed water column. The commercial fishery also relocated to this region. Although large squid were not found in the Guaymas Basin from 2010 to 2012, small squid were abundant and matured at an unusually small mantle‐length (<30 cm) and young age (approximately 6 months). Juvenile squid thus appeared to respond to El Niño with an alternative life‐history trajectory in which gigantism and high fecundity in normally productive coastal‐shelf habitats were traded for accelerated reproduction at small size in an offshore environment. Both small and large mature squid, were present in the Salsipuedes Basin during 2011, indicating that both life‐ history strategies can coexist. Hydro‐acoustic data, reveal that squid biomass in this study area nearly doubled between 2010 and 2011, primarily due to a large increase in small squid that were not susceptible to the fishery. Such a climate‐driven switch in size‐at‐maturity may allow D. gigas to rapidly adapt to and cope with El Niño. This ability is likely to be an important factor in conjunction with longerterm climate‐change and the potential ecological impacts of this invasive predator on marine ecosystems.     

A mantle length structured stock assessment model for the jumbo squid,Dosidicus gigas,fishery of the Ecuadorian Pacific: a limited data approach

The jumbo squid Dosidicus gigas is a new fishery resource in Ecuador; the species is harvested from January to December every year. Biological sampling was carried out each month during March–December (2013) and January–December (2014) in different coastal zones off Ecuador, and the monthly mantle length (ML) composition was obtained from field data in the coastal areas. The catch-at-mantle length data were analysed based on the mantle length structured model (CAMLA). The results showed that the biomass of D. gigas in Pacific Ecuadorian waters was 641,915 t (2013) and 1,866,857 t (2014). For 2013, for squid smaller than 30?cm ML the biomass was greater than 30,000 t, while individuals between 35 and 48?cm ML had an estimated biomass varying between 35,000 and 45,000 t. During 2014, the biomass estimated for squid smaller than 30?cm ML was low (40,000 t or less); however, squid between 38 and 48?cm ML showed a change in biomass compared with the previous fishing season, varying from 100,000 to 190,000 t. The harvested rate-at-mantle length for squid larger than 35?cm ML was higher than for individuals between 12 and 30?cm ML. The CAMLA model was found to be appropriate for modelling catch-at-mantle length of jumbo squid in Ecuadorian waters, and the model can be used to obtain harvest management quantities, as well as quantitative outputs useful for Ecuadorian stakeholders.     

Two calcium regulation systems in squid (Ommastrephes sloani pacificus) muscle. Preparation of calcium-sensitive myosin and troponin-tropomyosin.

The Ca-regulatory system in squid mantle muscle was studied. The findings were as follows. (a) Squid mantle myosin B (squid myosin B) was Ca-sensitive, and its Ca-sensitivity was unaffected by addition of a large amount of rabbit skeletal myosin (skeletal myosin) or rabbit skeletal F-actin (skeletal F-actin). (b) Squid myosin was prepared from the mantle muscle. It showed a heavy chain component and two light chain components in the SDS-gel electrophoretic pattern: the molecular weights of the latter two were 17,000 and 15,000. Actomyosin reconstituted from squid myosin and skeletal (or squid) actin showed Ca-sensitivity in superprecipitation and Mg-ATPase assays. EDTA- treatment had no effect on the Ca-sensitivity of squid myosin. (c) Squid mantle actin (squid actin) was prepared by the method of Spudich and Watt. Hybrid actomyosin reconstituted by using the pure squid actin preparation with skeletal myosin showed no Ca-sensitivity in Mg-ATPase assay, whereas that reconstituted using crude squid actin showed marked Ca-sensitivity. The crude squid actin contained four protein components which were capable of associating with F-actin in 0.1 M KCl, 1 mM MgCl2 and 20 mM Tris-maleate (pH7.5). (d) Native tropomyosin was prepared from squid mantle muscle, and it conferred Ca-sensitivity on skeletal actomyosin as well as on a hybrid actomyosin reconstituted from squid actin and skeletal myosin. (e) Squid native tropomyosin was separated into troponin and tropomyosin fractions by placing it in 0.4 M LiCl at pH 4.7. The troponin fraction was further purified by DEAE-cellulose chromatography. Squid troponin thus obtained was different in mobility from rabbit skeletal or carp dorsal troponin; three bands of squid troponin corresponded to molecular weights of 52,000, 28,000, and 24,000 daltons. It could confer Ca-sensitivity in the presence of tropomyosin on skeletal actomyosin as well as on a hybrid reconstituted from squid actin and skeletal myosin. (f) Squid myosin B, and two hybrid actomyosins were compared as regards Ca and Sr requirements for their Mg-ATPase activities. The myosin-linked regulatory system rather than the thin-filament-linked regulatory system was predominant in squid myosin B. Squid myosin B required higher Ca2+ and Sr2+ concentrations for Mg-ATPase activity; half-maximal activation of Mg-ATPase was obtained at 0.8 micron Ca2+ and 28 micron Sr2+ with skeletal myosin B, and at 2.5 micron Ca2+ and 140 micron Sr2+ with squid myosin B.