Genetic screening for prey in the gut contents from a giant squid (Architeuthis sp.)

Giant squids (Architeuthis sp.) remain mysterious; they have evaded observation and are rarely taken from their deep sea habitat. Information on the diet of Architeuthis is scarce due to the limited number of specimens with morphologically recognizable remains in their digestive tracts. We explored the use of polymerase chain reaction (PCR)-based methods for detection of DNA in the prey remains and amorphous slurry from an Architeuthis gut sample. The DNA region amplified varied in size, allowing separation of fish and squid components. Sequence comparisons identified fish prey as Macruronus novaezelandiae. Isolation of Architeuthis DNA from an ingested tentacle and the presence of chitin fragments indicate cannibalism occurs in giant squid. Denaturing gradient gel electrophoresis was used to screen for less common DNA types, revealing a high frequency of PCR-generated false alleles, but no additional prey species.     

A unique advantage for giant eyes in giant squid

Giant and colossal deep-sea squid (Architeuthis and Mesonychoteuthis) have the largest eyes in the animal kingdom [1, 2], but there is no explanation for why they would need eyes that are nearly three times the diameter of those of any other extant animal. Here we develop a theory for visual detection in pelagic habitats, which predicts that such giant eyes are unlikely to evolve for detecting mates or prey at long distance but are instead uniquely suited for detecting very large predators, such as sperm whales. We also provide photographic documentation of an eyeball of about 27 cm with a 9 cm pupil in a giant squid, and we predict that, below 600 m depth, it would allow detection of sperm whales at distances exceeding 120 m. With this long range of vision, giant squid get an early warning of approaching sperm whales. Because the sonar range of sperm whales exceeds 120 m [3-5], we hypothesize that a well-prepared and powerful evasive response to hunting sperm whales may have driven the evolution of huge dimensions in both eyes and bodies of giant and colossal squid. Our theory also provides insights into the vision of Mesozoic ichthyosaurs with unusually large eyes.     

An encounter between a pelagic shark and giant cephalopod

An oceanic whitetip shark (Carcharhinus longimanus) was observed off the coast of Kona, Hawaii, with scars caused by the tentacles of a large cephalopod. While the exact species could not be confirmed, candidate species include the giant squid (Architeuthis dux) or species from the genera Thysanoteuthis (flying squids) and Megalocranchia (glass squids). Telemetry shows C. longimanus will dive within the mesopelagic zone and may interact with or even forage for large cephalopods.     

The giant squid Architeuthis in southern African waters

Three specimens of the rare giant squid (genus Architeuthis , 18 nominal species) are described from the waters around southern Africa. Consideration of their internal and external morphology, including beaks, radula, statocyst and statolith give no reason to suppose that the three specimens do not pertain to the same species. A standardized data collection form and a summary of existing morphometric data are presented for the first time and the morphology and systematics of the genus are discussed. The value of various systematic characters is examined and certain guidelines are proposed, with an appeal for a more scientific approach to Architeuthis studies.
Features of the internal anatomy, including the female reproductive tract, are described and illustrated and the beak dimensions are used to evaluate existing regression formulae for beak and body parameters. The statocyst and statolith are described and illustrated in full, the statolith for the first time, and are compared with those of other squids.
The value of recent data from trawl-caught specimens, as well as indirect evidence, is cited to replace speculation in discussions on distribution and habitat. Our specimens suggest that the waters off the south-western coast of Africa may be one of the natural habitats of Architeurhis , during both feeding and spawning periods of the life cycle.     

What does the giant squid Architeuthis dux eat?

Architeuthis dux diet has been analysed according to information available from literature and from the analysis of gut contents of five females and two males from Mediterranean and Atlantic Iberian waters (20 specimens in total). This is the first time that A. dux diet from Atlantic and Mediterranean waters is described. Body weight of specimens ranged from 22.5 to 200 kg. In order to infer common patterns in giant squid diet according to its geographic distribution range, size and sex, available data on their diet composition structure were joined and examined with multivariate techniques. No significant differences in the trophic level on which A. dux prey on were found, considering size, sex and location. Thus, A. dux seems to play the same role in the trophic webs throughout the distribution range examined in this paper, which takes up a very wide geographic area. The trophic level estimated from the diet composition is 4.7. Obtained results show that this species preys mainly on pelagic fast swimmers and shoaling fishes and cephalopods as an opportunistic ambushing hunter.     

Stable isotopes document the trophic structure of a deep-sea cephalopod assemblage including giant octopod and giant squid

Although deep-sea cephalopods are key marine organims, their feeding ecology remains essentially unknown. Here, we report for the first time the trophic structure of an assemblage of these animals (19 species) by measuring the isotopic signature of wings of their lower beaks, which accumulated in stomachs of stranded sperm whales. Overall, the species encompassed a narrow range in δ¹³C values (1.7‰), indicating that they lived in closely related and overlapping habitats. δ¹³C values can be interpreted in terms of distribution with the more ¹³C-depleted species (e.g. Stigmatoteuthis arcturi, Vampyroteuthis infernalis) having a more pelagic habitat than the more ¹³C-enriched, bathyal species (e.g. Todarodes sagittatus and the giant squid Architeuthis dux). The cephalopods sampled had δ¹⁵N values ranging 4.6‰, which is consistent with the species spanning approximately 1.5 trophic levels. Neither the giant octopod (Haliphron atlanticus) nor the giant squid reached the highest trophic position. Species δ¹⁵N was independent of body size, with large squids having both the highest (Taningia danae) and lowest (Lepidoteuthis grimaldii) δ¹⁵N values. Their trophic position indicates that some species share the top of the food web, together with other megacarnivores such as the sperm whale.     

Light-limitation on predator-prey interactions: consequences for metabolism and locomotion of deep-sea cephalopods

The present study attempts to correlate the metabolism and locomotory behavior of 25 species of midwater Cephalopoda from California and Hawaii with the maximal activities of key metabolic enzymes in various locomotory muscle tissues. Citrate synthase (CS) and octopine dehydrogenase (ODH) activities were used as indicators of aerobic and anaerobic metabolic potential respectively. CS activity in mantle muscle is highly correlated with whole-animal rates of oxygen consumption, whereas ODH activity in mantle muscle is significantly correlated with a species' ability to buffer the acidic end-products of anaerobic metabolism. Both CS and ODH activities in mantle muscle declined strongly with a species' habitat depth. For example, CS and ODH activities ranged respectively from 0.04 units g(-1) and 0.03 units g(-1) in the deep-living squid Joubiniteuthis portieri, to 8.13 units g(-1) and 420 units g(-1) in the epipelagic squid Sthenoteuthis oualaniensis. The relationships between enzymatic activities and depth are consistent with similar patterns observed for whole-animal oxygen consumption. This pattern is believed to result from a relaxation, among deep-living species, in the need for strong locomotory abilities for visual predator/prey interactions; the relaxation is due to light-limitation in the deep sea. Intraspecific scaling patterns for ODH activities may, for species that migrate ontogenetically to great depths, reflect the counteracting effects of body size and light on predator-prey detection distances. When scaled allometrically, enzymatic activities for the giant squid, Architeuthis sp., suggest a fairly active aerobic metabolism but little burst swimming capacity. Interspecific differences in the relative distributions of enzymatic activities in fin, mantle, and arm tissue suggest an increased reliance on fin and arm muscle for locomotion among deep-living species. We suggest that, where high-speed locomotion is not required, more efficient means of locomotion, such as fin swimming or medusoid arm propulsion, are more prevalent.     

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.     

The rate of action of tetrodotoxin on sodium conductance in the squid giant axon.

When tetrodotoxin is applied to or washed away from the squid giant axon, the rates at which the sodium conductatnce is blocked and unblocked are an order of magnitude smaller than those reported for the isolated node of Ranvier. This slowing is to be expected if in squid the tetrodotoxin binding sites act as a saturable sink in series with the barrier to free diffusion imposed by the presence of the Schwann cell. A comparison has been made between the rates observed experimentally and those calculated for a computer model of the system, in order to estimate the apparent density in the membrane of both specific and non-specific tetrodotoxin binding sites. The figure thus obtained for the number of sodium channels in the squid giant axon, several hundred per square micrometre, agrees well with those derived from other lines of argument.     

The presence of transfer RNA in the axoplasm of the squid giant axon

Previous work has revealed that 4S RNA is the primary species of RNA in the axoplasm from the giant axons of the squid and Myxicola. This study shows that axoplasmic 4S RNA from the squid giant axon has the functional properties of tRNA. Axoplasmic RNA was charged with amino acids by aminoacyl-tRNA synthetases prepared from squid brain. The aminoacylation was prevented by incubating the RNA with RNase prior to running the reaction. The amino acid-RNA complex was labile at pH 9, which is characteristic of the acyl linkage between an amino acid and its tRNA. Aminoacyl-tRNA synthetase activity was also present in the axoplasm, primarily in the soluble fraction.     

ACETYLCHOLINE CONTENT OF THE SCHWANN CELL AND AXON IN THE GIANT NERVE FIBRE OF THE SQUID

Abstract— Acetylcholine and choline were identified and their concentrations measured, by means of gas chromatography/mass spectrometry, in extracts obtained from nerve fibers of the hindmost stellar nerve of the squid Sepioteuthis sepioidea. These compounds were quantitated in samples of stellar nerve devoid of giant fiber, intact giant nerve fiber, extruded axoplasm, and axoplasm-free giant nerve fiber sheaths. In 11 samples of stellar nerve devoid of giant fiber, weighing an average of 20.8 ± 2.3 mg ( s.e.m. ), 756 ± 91 pmol ACh and 8.65 ± 0.62 nmol of choline were found. The total ACh content of the largest fibre in this group (10 μ m in diameter), for a 5 cm length of nerve, is in the order of 0.16 pmol. The average wet weights of a single giant nerve fiber (270-420 μ m in diameter) and its separate components ( s.e.m .; in mg; number of fibers in parentheses) were: intact fiber, 4.58 ± 0.19 (25); extruded axoplasm, 3.38 ± 0.13 (20); sheaths, 1.21 ± 0.11 (16). The average ACh content per unit weight of sample was about 2-3 times higher in the sheaths (5-13 pmol-mg⁻¹) than in the axoplasm (2-4 pmol mg⁻¹), whereas the ACh concentrations estimated per unit volume of cellular water were about 40 times higher in the Schwann cell (107-222 μ m ) than in the axon (2-5 μ m ). These experimental findings establish the presence of ACh in the giant nerve fiber of S. sepioidea. They also indicate the Schwann cells themselves as the main source for the release of ACh, responsible for their long-lasting hyperpolarizations following the conduction of nerve impulse trains by the axon.     

THE SYNTHESIS OF CHOLINE PHOSPHOGLYCERIDES IN THE GIANT FIBRE SYSTEM OF THE SQUID

The mechanisms and pathways of synthesis of phosphatidylcholine in the giant fibre system of the squid (Loligo vulgaris) have been examined by incubating the stellate ganglion-nerve preparation or its separated compartments in an artificial bathing solution with labelled choline. Other experiments were done by dissecting the whole stellate ganglion into axoplasm, axon sheath, giant fibre lobe, small fibres and ganglion residue, after incubation. The initial rate of choline incorporation into choline phosphoglycerides was severalfold higher in the lobe than in the axon. Higher lipid radioactivity was recovered in the axon sheath as compared to the axoplasm, and in the small fibres as compared to the ganglion residue which contains its cell bodies. The production of phosphorylcholine and CDP-choline in the intact ganglion-nerve preparation during incubation with choline points to the occurrence of the net synthesis pathway for phosphatidylcholine in this material. Base-exchange activity was also observed in the axon and giant fibre lobe preparations in vitro, but no indication can yet be given whether it also takes place in intact preparations. Electrical stimulation and‘depolarizing’conditions enhance choline phosphorylation in the squid axon and lobe, but decrease phosphatidylcholine labelling.     

Squid Giant Axon Contains Neurofilament Protein mRNA but does not Synthesize Neurofilament Proteins

When isolated squid giant axons are incubated in radioactive amino acids, abundant newly synthesized proteins are found in the axoplasm. These proteins are translated in the adaxonal Schwann cells and subsequently transferred into the giant axon. The question as to whether any de novo protein synthesis occurs in the giant axon itself is difficult to resolve because the small contribution of the proteins possibly synthesized intra-axonally is not easily distinguished from the large amounts of the proteins being supplied from the Schwann cells. In this paper, we reexamine this issue by studying the synthesis of endogenous neurofilament (NF) proteins in the axon. Our laboratory previously showed that NF mRNA and protein are present in the squid giant axon, but not in the surrounding adaxonal glia. Therefore, if the isolated squid axon could be shown to contain newly synthesized NF protein de novo, it could not arise from the adaxonal glia. The results of experiments in this paper show that abundant 3H-labeled NF protein is synthesized in the squid giant fiber lobe containing the giant axon’s neuronal cell bodies, but despite the presence of NF mRNA in the giant axon no labeled NF protein is detected in the giant axon. This lends support to the glia–axon protein transfer hypothesis which posits that the squid giant axon obtains newly synthesized protein by Schwann cell transfer and not through intra-axonal protein synthesis, and further suggests that the NF mRNA in the axon is in a translationally repressed state.     

Periaxonal K+ regulation in the small squid Alloteuthis. Studies on isolated and in situ axons.

A novel giant axon preparation from the squid Alloteuthis is described. Properties of in situ and isolated axons are similar. Periaxonal K+ accumulation is a function of the physiological state of the animal and of the axon and its sheathing layers. Carefully dissected isolated axons, and axons in situ in a healthy mantle, show much less K+ accumulation than previously reported in squid. It is suggested that the Schwann cells are involved in the observed K+ regulation.     

Axolinin Localization in the Nervous Tissue of Squid Revealed by Monoclonal Antibodies Specific for Axolinin: Cellular and Subcellular Localization of Axolinin in the Squid Neuron

Cellular and subcellular distributions of axolinin, the 260-kilodalton (kD) microtubule-associated glycoprotein originally purified from squid axons, in various squid tissues such as optical lobes, bundles of small nerve fibers (fin nerves), giant stellate ganglia, skin, muscle, liver, and gill, were immunologically studied using monoclonal antibodies specifically recognizing the polypeptide chain of axolinin. The following results were obtained: (1) Axolinin is confined to squid neurons and skin; (2) axolinin is localized in the axon whereas another 260-kD microtubule-associated protein, MAP B, is localized in the cell bodies; and (3) axolinin is localized mainly in the peripheral part of the axoplasm of the squid giant axon. The last result has confirmed our previous conclusion obtained using polyclonal antisera against axolinin, which contain antibodies recognizing not only axolinin-specific epitopes but also nonspecific epitopes. The physiological importance of the localization of axolinin in axons and the skin is discussed based on its possible relationship to excitability function.     

Demonstration of two stable potential states in the squid giant axon under tetraethylammonium chloride

1. Intracellular injection of tetraethylammonium chloride (TEA) into a giant axon of the squid prolongs the duration of the action potential without changing the resting potential (Fig. 3). The prolongation is sometimes 100-fold or more. 2. The action potential of a giant axon treated with TEA has an initial peak followed by a plateau (Fig. 3). The membrane resistance during the plateau is practically normal (Fig. 4). Near the end of the action potential, there is an apparent increase in the membrane resistance (Fig. 5D and Fig. 6, right). 3. The phenomenon of abolition of action potentials was demonstrated in the squid giant axon treated with TEA (Fig. 7). Following an action potential abolished in its early phase, there is no refractoriness (Fig. 8). 4. By the method of voltage clamp, the voltage-current relation was investigated on normal squid axons as well as on axons treated with TEA (Figs. 9 and 10). 5. The presence of stable states of the membrane was demonstrated by clamping the membrane potential with two voltage steps (Fig. 11). Experimental evidence was presented showing that, in an "unstable" state, the membrane conductance is not uniquely determined by the membrane potential. 6. The effect of low sodium water was investigated in the axon treated with TEA (Fig. 12). 7. The similarity between the action potential of a squid axon under TEA and that of the vertebrate cardiac muscle was stressed. The experimental results were interpreted as supporting the view that there are two stable states in the membrane. Initiation and abolition of an action potential were explained as transitions between the two states.     

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.     

Gating currents in the node of Ranvier: voltage and time dependence.

Like the axolemma of the giant nerve fibre of the squid, the nodal membrane of frog myelinated nerve fibres after blocking transmembrane ionic currents exhibits asymmetrical displacement currents during and after hyperpolarizing and depolarizing voltage clamp pulses of equal size. The steady-state distribution of charges as a function of membrane potential is consistent with Boltzmanns law (midpoint potential minus 33.7 mV; saturation value 17200 charges/mum-2). The time course of the asymmetry current and the voltage dependence of its time constant are consistent with the notion that due to a sudden change in membrane potential the charges undergo a first order transition between two configurations. Size and voltage dependence of the time constant are similar to those of the activation of the sodium conductance assuming m-2h kinetics. The results suggest that the presence of ten times more sodium channels (5000/mum-2) in the node of Ranvier than in the squid giant axon with similar sodium conductance per channel (2-3 pS).     

Effects of external ions on membrane potentials of a crayfish giant axon

Transmembrane potentials in the crayfish giant axon have been investigated as a function of the concentration of normally occurring external cations. Results have been compared with data already available for the lobster and squid giant axons. The magnitude of the action potential was shown to be a linear function of the log of the external sodium concentration, as would be predicted for an ideal sodium electrode. The resting potential is an inverse function of the external potassium concentration, but behaves as an ideal potassium electrode only at the higher external concentrations of potassium. Decrease in external calcium results in a decrease in both resting potential and action potential; an increase in external calcium above normal has no effect on magnitude of transmembrane potentials. Magnesium can partially substitute for calcium in the maintenance of normal action potential magnitude, but appears to have very little effect on resting potential. All ionic effects studied are completely reversible. The results are in generally good agreement with data presently available for the lobster giant axon and for the squid giant axon.