Predicting the effects of spatiotemporal modifications of muscle activation on the tentacle extension in squid.

Squid use eight arms and two slender tentacles to capture prey. The muscular stalks of the tentacles are elongated approximately 80% in 20-40 ms towards the prey, which is adhered to the terminal clubs by arrays of suckers. Using a previously developed forward dynamics model of the extension of the tentacles of the squid Doryteuthis pealeii (formerly Loligo pealeii), we predict how spatial muscle-activation patterns result in a distribution of muscular power, muscle work, and kinetic and elastic energy along the tentacle. The simulated peak extension speed of the tentacles is remarkably insensitive to delays of activation along the stalk, as well as to random variations in the activation onset. A delay along the tentacle of 50% of the extension time has only a small effect on the peak extension velocity of the tentacle compared with a zero-delay pattern. A slight delay of the distal portion relative to the proximal has a small positive effect on peak extension velocity, whereas negative delays (delay reversed along stalk) always reduce extension performance. In addition, tentacular extension is relatively insensitive to superimposed random variations in the prescribed delays along the stalk. This holds in particular for small positive delays that are similar to delays predicted from measured axonal diameters of motor neurons. This robustness against variation in the activation distribution reduces the accuracy requirements of the neuronal control and is likely due to the non-linear mechanical properties of the muscular tissue in the tentacle.

One size does not fit all: diversity of length-force properties of obliquely striated muscles.

Obliquely striated muscles occur in 17+ phyla, likely evolving repeatedly, yet the implications of oblique striation for muscle function are unknown. Contrary to the belief that oblique striation allows high force output over extraordinary length ranges (i.e. superelongation), recent work suggests diversity in operating length ranges and length-force relationships. We hypothesize oblique striation evolved to increase length-force relationship flexibility. We predict that superelongation is not a general characteristic of obliquely striated muscles and instead that length-force relationships vary with operating length range. To test these predictions, we measured length-force relationships of five obliquely striated muscles from inshore longfin squid, Doryteuthis pealeii: tentacle, funnel retractor and head retractor longitudinal fibers, and arm and fin transverse fibers. Consistent with superelongation, the tentacle length-force relationship had a long descending limb, whereas all others exhibited limited descending limbs. The ascending limb at 0.6P0 was significantly broader (P<0.001) for the tentacle length-force relationship (0.43±0.04L0; where L0 is the preparation length that produced peak isometric stress, P0) than for the arm (0.29±0.03L0), head retractor (0.24±0.06L0), fin (0.20±0.04L0) and funnel retractor (0.27±0.03L0). The fin's narrow ascending limb differed significantly from those of the arm (P=0.004) and funnel retractor (P=0.012). We further characterized the tentacle preparation's maximum isometric stress (315±78 kPa), maximum unloaded shortening velocity (2.97±0.55L0 s-1) and ultrastructural traits (compared with the arm), which may explain its broader length-force relationship. Comparison of obliquely striated muscles across taxa revealed length-force relationship diversity, with only two species exhibiting superelongation.

Evidence that eye-facing photophores serve as a reference for counterillumination in an order of deep-sea fishes.

Counterillumination, the masking of an animal's silhouette with ventral photophores, is found in a number of mesopelagic taxa but is difficult to employ because it requires that the animal match the intensity of downwelling light without seeing its own ventral photophores. It has been proposed that the myctophid, Tarletonbeania crenularis, uses a photophore directed towards the eye, termed an eye-facing photophore, as a reference standard that it adjusts to match downwelling light. The potential use of this mechanism, however, has not been evaluated in other fishes. Here, we use micro-computed tomography, photography and dissection to evaluate the presence/absence of eye-facing photophores in three families of stomiiform fishes. We found that all sampled species with ventral photophores capable of counterillumination possess an eye-facing photophore that is pigmented on the anterior and lateral sides, thus preventing its use as a laterally directed signal, lure or searchlight. The two species that are incapable of counterillumination, Cyclothone obscura and Sigmops bathyphilus, lack an eye-facing photophore. After determining the phylogenetic distribution of eye-facing photophores, we used histology to examine the morphology of the cranial tissue in Argyropelecus aculeatus and determined that light from the eye-facing photophore passes through a transparent layer of tissue, then the lens, and finally strikes the accessory retina. Additionally, eight of the 14 species for which fresh specimens were available had an aphakic gap that aligned with the path of emitted light from the eye-facing photophore, while the remaining six had no aphakic gap. These findings, combined with records of eye-facing photophores from distantly related taxa, strongly suggest that eye-facing photophores serve as a reference for counterillumination in these fishes.

Specialization for rapid excitation in fast squid tentacle muscle involves action potentials absent in slow arm muscle.

An important aspect of the performance of many fast muscle fiber types is rapid excitation. Previous research on the cross-striated muscle fibers responsible for the rapid tentacle strike in squid has revealed the specializations responsible for high shortening velocity, but little is known about excitation of these fibers. Conventional whole-cell patch recordings were made from tentacle fibers and the slower obliquely striated muscle fibers of the arms. The fast-contracting tentacle fibers show an approximately 10-fold greater sodium conductance than that of the arm fibers and, unlike the arm fibers, the tentacle muscle fibers produce action potentials. In situ hybridization using an antisense probe to the voltage-dependent sodium channel present in this squid genus shows prominent expression of sodium channel mRNA in tentacle fibers but undetectable expression in arm fibers. Production of action potentials by tentacle muscle fibers and their absence in arm fibers is likely responsible for the previously reported greater twitch-tetanus ratio in the tentacle versus the arm fibers. During the rapid tentacle strike, a few closely spaced action potentials would result in maximal activation of transverse tentacle muscle. Activation of the slower transverse muscle fibers in the arms would require summation of excitatory postsynaptic potentials over a longer time, allowing the precise modulation of force required for supporting slower movements of the arms.

A Soft End Effector Inspired by Cephalopod Suckers and Augmented by a Dielectric Elastomer Actuator.

This article describes a soft suction cup end effector with squid-inspired suction generation and an octopus-inspired cup design that uses a dielectric elastomer actuator (DEA) to generate suction for adhesion. The fabrication process for the end effector is described in detail, and a mechanical model for generated pressure differential as a function of voltage is presented. When actuated, the DEA exerts an electrostatic stress on the walls of the end effector, resulting in pressure reduction in its water-filled cavity. The actuator is soft, flexible, and creates suction without a reliance on typical DEA elements such as rigid supporting structures and elastomer prestrain. It does not require net fluid flux out of the sucker, allowing faster attachment and easier release. It can be actuated underwater and has been validated with pull-off tests. The sucker generates a pressure differential of 3.63 ± 0.07 kPa (±SD) when driven at 10.75 kV in water and should reach a 4.90 kPa pressure differential when energized at its theoretical failure point of 12.4 kV. Data normalized by the input voltage show that 90% of the maximum pressure differential can be achieved within 50 ms of voltage application. Weighing less than 30 g in air, this elastomer end effector is capable of pulling with a force of 8.34 ± 0.10 N (±SD) and reversibly lifting 26.7 times its own mass underwater when actuated at 10.75 kV.

Shape, Size, and Structure Affect Obliquely Striated Muscle Function in Squid.

Hollow, cylindrical body plans, and obliquely striated muscles are characteristic of soft-bodied invertebrates, and both affect the biomechanics of movement in these diverse animals. We highlight two different aspects of functional heterogeneity in obliquely striated muscles, one driven by animal shape and size and the other by the intrinsic mechanical properties of the fibers. First, we show how a hollow, cylindrical shape in the mantle of cephalopod molluscs causes a significant difference in muscle strain (defined as the change in length divided by resting length) across the mantle wall, and describe the implications of such "transmural gradients of strain" for the length-tension relationship of the obliquely striated muscles that power movements in these animals. We show that transmural gradients of strain increase in magnitude as mantle wall proportions change during ontogeny, with the relatively thin mantle walls of newly hatched squid experiencing significantly smaller differences in strain than the thicker mantle walls of adults. Second, we describe how the length-tension relationship of obliquely striated mantle muscles varies with position to accommodate the transmural gradient of strain, with the result that circular muscle fibers near the inner and outer surfaces of the mantle are predicted to produce similar force during mantle contraction. The factors that affect the length-tension relationship in obliquely striated muscles are unknown, and thus we have not yet identified the mechanism(s) responsible for the transmural shift in the length-tension properties of the mantle circular fibers. We have, however, developed a mathematical model that predicts small changes in the oblique striation angle (which varies from 4 to 12° in adult squid) have a significant effect on the shape of the length-tension relationship, with lower angles predicted to result in a broader length-tension curve.

Tuning of shortening speed in coleoid cephalopod muscle: no evidence for tissue-specific muscle myosin heavy chain isoforms.

The contractile protein myosin II is ubiquitous in muscle. It is widely accepted that animals express tissue-specific myosin isoforms that differ in amino acid sequence and ATPase activity in order to tune muscle contractile velocities. Recent studies, however, suggested that the squid Doryteuthis pealeii might be an exception; members of this species do not express muscle-specific myosin isoforms, but instead alter sarcomeric ultrastructure to adjust contractile velocities. We investigated whether this alternative mechanism of tuning muscle contractile velocity is found in other coleoid cephalopods. We analyzed myosin heavy chain transcript sequences and expression profiles from muscular tissues of a cuttlefish, Sepia officinalis, and an octopus, Octopus bimaculoides, in order to determine if these cephalopods express tissue-specific myosin heavy chain isoforms. We identified transcripts of four and six different myosin heavy chain isoforms in S. officinalis and O. bimaculoides muscular tissues, respectively. Transcripts of all isoforms were expressed in all muscular tissues studied, and thus S. officinalis and O. bimaculoides do not appear to express tissue-specific muscle myosin isoforms. We also examined the sarcomeric ultrastructure in the transverse muscle fibers of the arms of O. bimaculoides and the arms and tentacles of S. officinalis using transmission electron microscopy and found that the fast contracting fibers of the prey capture tentacles of S. officinalis have shorter thick filaments than those found in the slower transverse muscle fibers of the arms of both species. It thus appears that coleoid cephalopods, including the cuttlefish and octopus, may use ultrastructural modifications rather than tissue-specific myosin isoforms to adjust contractile velocities.

The Musculature of Coleoid Cephalopod Arms and Tentacles.

The regeneration of coleoid cephalopod arms and tentacles is a common occurrence, recognized since Aristotle. The complexity of the arrangement of the muscle and connective tissues of these appendages make them of great interest for research on regeneration. They lack rigid skeletal elements and consist of a three-dimensional array of muscle fibers, relying on a type of skeletal support system called a muscular hydrostat. Support and movement in the arms and tentacles depends on the fact that muscle tissue resists volume change. The basic principle of function is straightforward; because the volume of the appendage is essentially constant, a decrease in one dimension must result in an increase in another dimension. Since the muscle fibers are arranged in three mutually perpendicular directions, all three dimensions can be actively controlled and thus a remarkable diversity of movements and deformations can be produced. In the arms and tentacles of coleoids, three main muscle orientations are observed: (1) transverse muscle fibers arranged in planes perpendicular to the longitudinal axis; (2) longitudinal muscle fibers typically arranged in bundles parallel to the longitudinal axis; and (3) helical or obliquely arranged layers of muscle fibers, arranged in both right- and left-handed helixes. By selective activation of these muscle groups, elongation, shortening, bending, torsion and stiffening of the appendage can be produced. The predominant muscle fiber type is obliquely striated. Cross-striated fibers are found only in the transverse muscle mass of the prey capture tentacles of squid and cuttlefish. These fibers have unusually short myofilaments and sarcomeres, generating the high shortening velocity required for rapid elongation of the tentacles. It is likely that coleoid cephalopods use ultrastructural modifications rather than tissue-specific myosin isoforms to tune contraction velocities.

Differences in scaling and morphology between lumbricid earthworm ecotypes.

Many soft-bodied invertebrates use a flexible, fluid-filled hydrostatic skeleton for burrowing. The aim of our study was to compare the scaling and morphology between surface-dwelling and burrowing earthworm ecotypes to explore the specializations of non-rigid musculoskeletal systems for burrowing locomotion. We compared the scaling of adult lumbricid earthworms across species and ecotypes to determine whether linear dimensions were significantly associated with ecotype. We also compared the ontogenetic scaling of a burrowing species, Lumbricus terrestris, and a surface-dwelling species, Eisenia fetida, using glycol methacrylate histology. We show that burrowing species are longer, thinner and have higher length-to-diameter ratios than non-burrowers, and that L. terrestris is thinner for any given body mass compared with E. fetida. We also found differences in the size of the musculature between the two species that may correlate with surface crawling or burrowing. Our results suggest that adaptations to burrowing for soft-bodied animals include a disproportionately thin body and strong longitudinal muscles.

The structure and function of a muscle articulation-type jaw joint of a polychaete worm.

The arrangement of the musculature and the fibers of the extracellular matrix (ECM) in the flexible jaw joint of the sandworm Alitta virens (Annelida, Polychaeta) was studied using dissection and histology. The jaws are capable of a wide range of motions principally related to defense and feeding. The left and right jaws are embedded in and moved by a compact pharyngeal bulb of muscle and ECM that also forms the mouth and esophagus. Eight pharyngeal bulbs were removed and dissected to document gross anatomical features or preserved and embedded in plastic for sectioning in multiple planes. The sections were stained with toluidine blue and basic fuchsin to differentiate muscle and ECM. The sections were then digitized and used to develop a three-dimensional computer illustration. We hypothesize that the muscle and fibers in the ECM are arranged as a muscular hydrostat to support the movement of the jaws. Four specimens were recorded using a digital video camera and a tank with an angled mirror to record lateral and ventral views of jaw movements during locomotion and biting associated with burrow guarding and feeding. Frame by frame kinematic analysis of this video showed that the jaws move symmetrically in a roughly horizontal plane. Although the angle between the jaws increases and then decreases after maximum gape has been reached, the jaws also translate relative to each other such that the axis of rotation is not fixed. Together, these functional morphological and behavioral data identify the jaw mechanism as a flexible joint known as a muscle articulation. As muscle articulations have been previously described only in the beaks of cephalopods and flatworms, this study implies that this type of joint is more common and important than previously recognized.

Scaling of the hydrostatic skeleton in the earthworm Lumbricus terrestris.

The structural and functional consequences of changes in size or scale have been well studied in animals with rigid skeletons, but relatively little is known about scale effects in animals with hydrostatic skeletons. We used glycol methacrylate histology and microscopy to examine the scaling of mechanically important morphological features of the earthworm Lumbricus terrestris over an ontogenetic size range from 0.03 to 12.89 g. We found that L. terrestris becomes disproportionately longer and thinner as it grows. This increase in the length to diameter ratio with size means that, when normalized for mass, adult worms gain ~117% mechanical advantage during radial expansion, compared with hatchling worms. We also found that the cross-sectional area of the longitudinal musculature scales as body mass to the ~0.6 power across segments, which is significantly lower than the 0.66 power predicted by isometry. The cross-sectional area of the circular musculature, however, scales as body mass to the ~0.8 power across segments, which is significantly higher than predicted by isometry. By modeling the interaction of muscle cross-sectional area and mechanical advantage, we calculate that the force output generated during both circular and longitudinal muscle contraction scales near isometry. We hypothesize that the allometric scaling of earthworms may reflect changes in soil properties and burrowing mechanics with size.

The length-force behavior and operating length range of squid muscle vary as a function of position in the mantle wall.

Hollow cylindrical muscular organs are widespread in animals and are effective in providing support for locomotion and movement, yet are subject to significant non-uniformities in circumferential muscle strain. During contraction of the mantle of squid, the circular muscle fibers along the inner (lumen) surface of the mantle experience circumferential strains 1.3 to 1.6 times greater than fibers along the outer surface of the mantle. This transmural gradient of strain may require the circular muscle fibers near the inner and outer surfaces of the mantle to operate in different regions of the length-tension curve during a given mantle contraction cycle. We tested the hypothesis that circular muscle contractile properties vary transmurally in the mantle of the Atlantic longfin squid, Doryteuthis pealeii. We found that both the length-twitch force and length-tetanic force relationships of the obliquely striated, central mitochondria-poor (CMP) circular muscle fibers varied with radial position in the mantle wall. CMP circular fibers near the inner surface of the mantle produced higher force relative to maximum isometric tetanic force, P0, at all points along the ascending limb of the length-tension curve than CMP circular fibers near the outer surface of the mantle. The mean ± s.d. maximum isometric tetanic stresses at L0 (the preparation length that produced the maximum isometric tetanic force) of 212 ± 105 and 290 ± 166 kN m(-2) for the fibers from the outer and inner surfaces of the mantle, respectively, did not differ significantly (P=0.29). The mean twitch:tetanus ratios for the outer and inner preparations, 0.60 ± 0.085 and 0.58 ± 0.10, respectively, did not differ significantly (P=0.67). The circular fibers did not exhibit length-dependent changes in contraction kinetics when given a twitch stimulus. As the stimulation frequency increased, L0 was approximately 1.06 times longer than LTW, the mean preparation length that yielded maximum isometric twitch force. Sonomicrometry experiments revealed that the CMP circular muscle fibers operated in vivo primarily along the ascending limb of the length-tension curve. The CMP fibers functioned routinely over muscle lengths at which force output ranged from only 85% to 40% of P0, and during escape jets from 100% to 30% of P0. Our work shows that the functional diversity of obliquely striated muscles is much greater than previously recognized.

The diversity of hydrostatic skeletons.

A remarkably diverse group of organisms rely on a hydrostatic skeleton for support, movement, muscular antagonism and the amplification of the force and displacement of muscle contraction. In hydrostatic skeletons, force is transmitted not through rigid skeletal elements but instead by internal pressure. Functioning of these systems depends on the fact that they are essentially constant in volume as they consist of relatively incompressible fluids and tissue. Contraction of muscle and the resulting decrease in one of the dimensions thus results in an increase in another dimension. By actively (with muscle) or passively (with connective tissue) controlling the various dimensions, a wide array of deformations, movements and changes in stiffness can be created. An amazing range of animals and animal structures rely on this form of skeletal support, including anemones and other polyps, the extremely diverse wormlike invertebrates, the tube feet of echinoderms, mammalian and turtle penises, the feet of burrowing bivalves and snails, and the legs of spiders. In addition, there are structures such as the arms and tentacles of cephalopods, the tongue of mammals and the trunk of the elephant that also rely on hydrostatic skeletal support but lack the fluid-filled cavities that characterize this skeletal type. Although we normally consider arthropods to rely on a rigid exoskeleton, a hydrostatic skeleton provides skeletal support immediately following molting and also during the larval stage for many insects. Thus, the majority of animals on earth rely on hydrostatic skeletons.

Muscular tissues of the squid Doryteuthis pealeii express identical myosin heavy chain isoforms: an alternative mechanism for tuning contractile speed.

The speed of muscle contraction is largely controlled at the sarcomere level by the ATPase activity of the motor protein myosin. Differences in amino acid sequence in catalytically important regions of myosin yield different myosin isoforms with varying ATPase activities and resulting differences in cross-bridge cycling rates and interfilamentary sliding velocities. Modulation of whole-muscle performance by changes in myosin isoform ATPase activity is regarded as a universal mechanism to tune contractile properties, especially in vertebrate muscles. Invertebrates such as squid, however, may exhibit an alternative mechanism to tune contractile properties that is based on differences in muscle ultrastructure, including variable myofilament and sarcomere lengths. To determine definitively whether contractile properties of squid muscles are regulated via different myosin isoforms (i.e. different ATPase activities), the nucleotide and amino acid sequences of the myosin heavy chain from the squid Doryteuthis pealeii were determined from the mantle, arm, tentacle, fin and funnel retractor musculature. We identified three myosin heavy chain isoforms in squid muscular tissues, with differences arising at surface loop 1 and the carboxy terminus. All three isoforms were detected in all five tissues studied. These results suggest that the muscular tissues of D. pealeii express identical myosin isoforms, and it is likely that differences in muscle ultrastructure, not myosin ATPase activity, represent the most important mechanism for tuning contractile speeds.

Morphology of the muscle articulation joint between the hooks of a flatworm (Kalyptorhynchia, Cheliplana sp.).

Schizorhynch kalyptorhynchs are meiofaunal turbellarian predators that possess an eversible proboscis that can be armed with two stout hooks. The hooks grasp and manipulate prey using a wide range of rotations and translations. These diverse motions are possible because the hook supports may function as a muscle articulation type joint-that is, a joint formed of muscle and connective tissue that connects, separates, and moves the microscopic hooks. We analyze the morphology of the flexible joint in a species of Cheliplana by using three types of microscopy: light, laser scanning confocal, and transmission electron. Radial myofilament bundles are present in the core of the hook supports, and lateral divaricator muscle fibers are located on their lateral surfaces. We develop a novel model for movements of the proboscis and describe the tensile function of the basement membrane that surrounds each hook support's medial glandular region. Contraction of divaricator muscle fibers antagonized by contraction of radial myofilaments causes the lateral bending of the hook supports and opening of the hook apparatus. Relaxation of the divaricator fibers and maximal contraction of the radial myofilaments, which put the medial basement membranes in tension, may cause medial bending in the hook supports and closing of the hook apparatus. During proboscis retraction, closure may also be aided by the compression of the hook apparatus as the proboscis is drawn through the rostral pore. The study provides new insights into the principles of support and movement in muscle articulations.

Venom kinematics during prey capture in Conus: the biomechanics of a rapid injection system.

Cone snails use an extensile, tubular proboscis as a conduit to deliver a potent cocktail of bioactive venom peptides into their prey. Previous studies have focused mainly on understanding the venom's role in prey capture but successful prey capture requires both rapid physiological and biomechanical mechanisms. Conus catus, a fish-hunting species, uses a high-speed hydraulic mechanism to inject its hollow, spear-like radular tooth into prey. We take an integrated approach to investigating the biomechanics of this process by coupling kinematic studies with morphological analyses. Taking advantage of the opaque venom and translucent proboscis of a mollusc-hunting juvenile cone snail, Conus pennaceus, we have determined that a high-speed prey capture mechanism is not unique to cone species that hunt fish prey. Two morphological structures were found to play crucial roles in this process. A constriction of the lumen near the tip of the proboscis, composed of tall epithelial cells densely packed with microfilaments, impedes forward movement of the radular tooth prior to its propulsion. Proximal to the constriction, a muscular sphincter was found to regulate venom flow and pressurization in the proboscis. In C. pennaceus, the rapid appearance and flushing of venom within the proboscis during prey capture suggests a mechanism involving the delivery of a discrete quantity of venom. The interplay between these elements provides a unique and effective biomechanical injection system for the fast-acting cone snail venom peptides.

Muscle specialization in the squid motor system.

Although muscle specialization has been studied extensively in vertebrates, less is known about the mechanisms that have evolved in invertebrate muscle that modulate muscle performance. Recent research on the musculature of squid suggests that the mechanisms of muscle specialization in cephalopods may differ from those documented in vertebrates. Muscle diversity in the development and the evolution of cephalopods appears to be characterized by modulation of the dimensions of the myofilaments, in contrast to the relatively fixed myofilament dimensions of vertebrate muscle. In addition, the arrangement of the myofilaments may also be altered, as has been observed in the extensor muscle fibres of the prey capture tentacles of squid and cuttlefish, which show cross-striation and thus differ from the obliquely striated pattern of most cephalopod locomotor muscle fibres. Although biochemical specializations that reflect differences in aerobic capacity have been documented previously for specific layers of the mantle muscle of squid, comparison of protein profiles of myofilament preparations from the fast cross-striated tentacle fibres and slow obliquely striated fibres from the arms has revealed remarkably few differences in myofilament lattice proteins. In particular, previous studies using a variety of SDS-PAGE techniques and peptide mapping of the myosin heavy chain were unable to resolve differences in the myosin light and heavy chains. Since these techniques cannot exclude the presence of a highly conserved variant that differs in only a few amino acids, in this study semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis of myosin heavy chain messenger RNAs (mRNAs) from the cross-striated tentacle and obliquely striated arm muscle fibres was conducted. This analysis showed that a previously reported alternatively spliced isoform of the squid myosin motor domain is present only in low abundance in both muscle types and therefore differential expression of the two myosins cannot explain the difference in contractile properties. It thus appears that modulation of the contractile properties of the musculature of squid and other cephalopods occurs primarily through variation in the arrangement and dimensions of the myofilaments.