The Muscle Arrangement and Functions of Cephalopod Tentacles

ABSTRACT

Structured models of movement involve the description of muscle specialization, which refers to the use of muscles to perform specific tasks such as locomotion or posture. Although the mechanics and arrangements of muscles in vertebrates have been extensively studied, there is a growing interest in exploring non-model organisms in the field of movement – specifically the cephalopod i.e. Loligo pealei. This paper explores the scaffolds and modulations of myofilament arrangements that give rise to unique movements and properties of the squid tentacles with the rate of change in dimensions and velocity occurring due to specific contractions of the different isoforms (or types) of myofilaments, including: (1) transverse muscle fibers (2) longitudinal muscle fibers and (3) oblique layers of muscle fibers. The dominant functional muscle types used in the different activities of the tentacles is explored in context of the underlying biomechanical functions of the myofilaments. The methodology used to obtain the results for the biomechanical and biochemical components to derive the current model of the cephalopod tentacle was described, drawing importance to the techniques parallel to the vertebrate models. Additionally, ontogeny of the developing sarcomeres from juvenile to mature tentacles, the functional implications of ultrastructural modifications, and the effect of ATPase activity on the peak contractile strength and velocity are discussed to further explore the current tentacle model. This paper combines the standard models of macro and ultra-structures, their movements, and its applications of force and velocity to examine, and best represent, the cephalopod tentacle.

BACKGROUND AND INTRODUCTION

Muscles are categorized as a collection of muscle fibers, as shown in fig. 1, which are composed of sarcomeres that have thin actin filaments that overlap and interact with the thick myosin filaments to produce contracted and relaxed lengths of the muscle, contributing to the change in velocity, force, and lengths of movements. All muscles operate under the conditions of the sliding filament principle (Squire J.M., 2016), where contraction occurs when bands of the sarcomere change in length.

Figure 1-  Hierarchy of muscle structures present in vertebrates (Squire J.M., 2016)

However, these models are studied in the context of the skeletal system, where a frame is provided for the support of the muscular system. In the cephalopod tentacle, there is a more unique muscular arrangement that produces more diverse and impressive movements and functions. Most of the research regarding the functional morphology and muscle arrangement has been conducted in 1982 (Kier W.M., 1982), mapping out the base components of the squid, Loligo pealei’s tentacle as shown in fig. 2. The cephalopod appendages consist of five pairs of tentacles that are used for locomotion, attacking prey, feeding, copulation, and manipulating and exploring the environment. These appendages were initially observed to be composed of longitudinal and transverse striated muscle fibers that can produce skeletal independent movements to elongate, shorten, turn, and twist the tentacles. Moreover, the tentacles have also been observed to display regenerative abilities and with the cephalopods being able to seemingly recover from damage to loss of whole limbs (Bello G., 1995). Better understanding of the morphology and contractile properties of the tentacles will provide an extensive description of its impressive abilities. This is developed through the discussion of the recent, most accurate anatomical model of the squid tentacle, the ultrastructures of the muscles, the functional significance of oblique striations, and the support provided by the funnel retractor muscles, highlighted in the following sections.

Figure 2- Cross-section of a loliginid squid – Ax (axon), L (longitudinal), T (transverse) (Kier W.M., 1982)

CURRENT CEPHALOPOD MUSCLE MODEL

Recently, research has shown that there are three main general groups of muscles present in the cephalopod tentacles that control the majority of the movements of the tentacles: Transverse muscles, Longitudinal muscles, and Oblique/Helical muscles (Kier W.M., 2016). As shown in fig.3, the core of the tentacle houses the artery and axial nerve core, which innervates and controls the appendage, covered by the transverse muscle mass. Surrounding the transverse muscle mass are groups of longitudinal muscle fibers, that run parallel to the axial length of the tentacle. Thin layers of helical (oblique) muscle fibers sheath the tentacle overlapping the circular muscles, which are the distal arrangements of the transverse muscles. Without a skeletal structure, the cephalopod tentacles rely on muscular hydrostat, which utilizes the incompressible properties of water and musculature for support (Chantler P.D., 1983). Bending movements of the tentacle are a product of the longitudinal and transverse muscle fibers contracting and relaxing, respectively, to overcome shortening of the arm while producing angular movements. Elongation of the tentacles occur when the transverse muscle mass contracts radially, reducing the circumferential volume of the muscle and therefore increasing the length of the muscle as the displacement of mass is productively increased. The cephalopod tentacle is also capable of bidirectional twisting when the helical (oblique) muscle layers, wrapped on top of the circular muscles, arranged at right-handed and left-handed helices contract and relax, depending on the direction of the turn.

Figure 3- Anatomical model of an oral tentacle, closest to the mouth – AN (axial nerve), AR (artery), TM (transverse), LM (longitudinal), CM (circular), HM (helical/oblique) (Kier W.M., 2016).

An issue that the tentacle faces because of its constant distortion of shape, length, and size, is the change in volume due to a lack of a rigid structure. The cephalopod tentacle utilizes its resistance to volume change in active bending of the arm (Smith et al. 1985). The orientation of the transverse muscle contractions change the medial-distal diameters of the tentacles, which allows it to retain a constant length while bending and twisting as shown in fig. 4.

Figure 4- Illustration of muscular hydrostat mechanism – Radial contractile properties give rise to motions with uniform length (Smith et al. 1985)

The relative contribution of the transverse and longitudinal muscle contractions varies according to the type of motion performed, the direction of the motion, and the amount of resistance experienced by longitudinal compression. Aside from the arrangements and varied contributive contractions of the tentacle muscles, ultrastructures provide an additional element of unique movements to the cephalopod.

ULTRASTRUCTURES OF THE TENTACLES

These movements of the tentacles are possible, largely due to the ultra structure properties of the (Kier W.M. 2016) Transmission electron micrographs sections of the transverse muscle mass, shown in fig. 5, reveals the cross striation patterns with remarkably short sarcomeres and extended thick filaments present in the tentacles. In mammalian myofibrils, thick filaments can be approximately 2-6 µm whereas those in the cross striated transverse muscles, they are 0.8 µm. Having short myofilaments and sarcomeres makes denser muscular elements over a unit length of fiber, which influences the contractile speed of the tentacles. This specialization contributes to the fast active movements of the tentacles, specifically in catching prey and locomotive agility.

Figure 5- Transverse muscle mass with cross striations and short sarcomeres (Kier W.M. 2016)

This importance of ultrastructure development is emphasized in both the development of juvenile cephalopods and in the ATPase activity of the muscles. The change in length and resultant increase in contractile velocity of the cephalopod was observed in the ontogeny of its musculature (Thompson et al. 2006). As the ultrastructures develop in the juvenile, the transverse muscles present at the radial end of tentacles, called circular muscles, begin to express shorter lengths of thick filaments due to the selection of performance during growth and senescent gene expression. Having shorter thick filaments would mean having smaller distances between myosin cross bridges which leads to increased velocity of contraction. Moreover, even though ATPase activity in myosin on the sarcomere level controls the contractile properties in muscles, it largely had no influence on the tentacle contractile velocity (Shaffer & Kier, 2012). The methodology used to obtain the results was by replicating the three major myosin heavy chain isoforms, or muscle fiber types, and testing for the presence of ATPase dependent activity, which was negative. This concludes that in invertebrates such as cephalopods, the ultrastructures and its isoforms provide an alternative to the ATPase dependent muscle-fiber contractile properties of muscles. The different muscle types and functional significances are less reliant on biochemistry and more so on arrangements.

FUNCTIONAL SIGNIFICANCE OF STRIATIONS

Further development and recorded data that confirmed the unique muscular arrangements not only allowing for different degrees of freedom of movement, but also enabling specialized biomechanical movements, such as increased relative velocity of elongation, and reduced tension produced by contractions was also observed (Curtin & Kier, 2002). Shown in fig. 6, the obliquely striated muscle fibers (A) of the tentacle were compared to the transverse muscle fibers (B), where the relative active force and passive force generated by each type of muscular arrangement was plotted. Because there are very short thick filaments present, there was an additive series of arrangement in the transverse striations which lead to increased speed of contraction and the oblique striations lead to an increased relative force production. These properties combined gives rise to the adapted mechanisms that allows the cephalopod to capture prey and maneuver around its surroundings.

Figure 6- Relative force comparison of transverse and oblique muscle mass – note: low force production of transverse (Curtin & Kier, 2002).

FUNNEL RETRACTOR MUSCLE

The biomechanics of the tentacles can be further elucidated by tracing the ultrastructure components of the superior musculature that indirectly but profoundly affects its movements. The funnel retractor muscle, located at the mantle of the cephalopod, acts as a muscular strut for propulsive movements involving the tentacles (Rosenbluth et al. 2010). Shown in fig. 7, the obliquely striated tissue present in the funnel retractor also has minimal elements of connective tissues present that contributes to its limited operating range. Despite the striations, it resembles a unitary smooth muscle without any conspicuous nerve innervations, gap-junctions, and collagen fibrils. These properties involves the
funnel retractor in isometric contractions that aid in the propulsion of the tentacles as suggested by the connective physiology of the cephalopod.

Figure 7- Electron micrograph showing striation patterns and lack of gap-junctions in the funnel retractor (Rosenbluth et al. 2010)

REGENERATIVE ABILITIES OF TENTACLES

The unique musculature of the tentacle does not, however, assist in the regenerative abilities of the tentacle – called myogenesis. The cephalopod utilizes established wound closure mechanisms with enhanced repairing and growth factors, including platelet-derived growth factor (PDGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) (Zullo et al. 2017). Moreover, in recent studies, it was revealed that the regenerative cell determinant transcription factor for the cephalopod muscle was an NK-2 class homeodomain, NK4 (Bonnaud-Ponticelli & Bassaglia, 2014). While usually reserved for cardiac muscle replication in some classes of vertebrates, there is an increased projection and recruitment of the growth factors in arm and tentacle myogenesis of the cephalopod. Another key factor is the expression of mature muscle cells at the site of regeneration that surrounds the main axial nerves in the tentacles and the assembly of the various muscle isoform lattices that has yet to be studied comparatively.

DISCUSSIONS AND FURTHER RESEARCH

It has been revealed that the cephalopod tentacle is in stark contrast with the vertebrate model that relies on both the skeletal frame for support, and on ATPase activity to perform specific muscle-fiber contractions. However, there still remains the biochemical factors that influence the arrangement of the tentacles, and the general phenomenon of the

Moreover, because of the large axial nerve that innervates the tentacle, there must be further research conducted regarding the downstream control of the arm, the different methods of feedback produced in the tentacle, and the complex motor functions involved in the controlled force production and shape manipulation.

There also remain some key factors that need to be explored regarding the efficacy of the tentacle model which includes the thick:thin filament ratio present in the tentacles, the arrangement of the cross-bridges, gap junction properties, etc. Not only does the cephalopod tentacle represent the immense diversity of muscle isoforms that exist but it is a gateway of the evolutionary development of one of the most fascinating life-forms on Earth. Studying the tentacle may also be key in developing a close imitation of the model as it is highly beneficial in terms of both speed and force production as well as in exploration of environments with various textures and terrains, and objects of different size, shapes, and surfaces.

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• Special appreciation for Kier W. M. who has conducted expansive research that has investigated most of the modern concepts of the cephalopod muscle arrangements.