Role of aerobic and anaerobic circular mantle muscle fibers in swimming squid: electromyography

Circular mantle muscle of squids and cuttlefishes consists of distinct zones of aerobic and anaerobic muscle fibers that are thought to have functional roles analogous to red and white muscle in fishes. To test predictions of the functional role of the circular muscle zones during swimming, electromyograms (EMGs) in conjunction with video footage were recorded from brief squid Lolliguncula brevis (5.0-6.8 cm dorsal mantle length, 10.9-18.3 g) swimming in a flume at speeds of 3-27 cm s(-1). In one set of experiments, in which EMGs were recorded from electrodes intersecting both the central anaerobic and peripheral aerobic circular mantle muscles, electrical activity was detected during each mantle contraction at all swimming speeds, and the amplitude and frequency of responses increased with speed. In another set of experiments, in which EMGs were recorded from electrodes placed in the central anaerobic circular muscle fibers alone, electrical activity was not detected during mantle contraction until speeds of about 15 cm s(-1), when EMG activity was sporadic. At speeds greater than 15 cm s(-1), the frequency of central circular muscle activity subsequently increased with swimming speed until maximum speeds of 21-27 cm s(-1), when muscular activity coincided with the majority of mantle contractions. These results indicate that peripheral aerobic circular muscle is used for low, intermediate, and probably high speeds, whereas central anaerobic circular muscle is recruited at intermediate speeds and used progressively more with speed for powerful, unsteady jetting. This is significant because it suggests that there is specialization and efficient use of locomotive muscle in squids.     

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.     

Myosin-linked calcium regulation in squid mantle muscle. Light-chain components of squid myosin.

As reported by Kendrick-Jones et al. (1976), myosin from squid mantle muscle contains two types of light-chain components, different in size but similar in net charge. We were able to separate the two types of light chains by a five-step procedure, yielding LC-1 (17,000 daltons) and LC-2 (15,000 daltons). It was found that squid mantle LC-1 and LC-2 function exactly like SH-light chains and EDTA-light chains of scallop adductor myosin, respectively. In functional tests, we used "desensitized" myosin of scallop adductor muscle, simply because "EDTA washing" removed neither LC-1 nor LC-2 from squid mantle myosin. The removal and recombination of light chains were examined by gel electrophoresis, and Ca or Sr sensitivity was determined by measuring the Mg-ATPase activity of skeletal acto-scallop or squid myosin. It was found that EDTA washing readily released the EDTA-light chains of scallop myosin completely, and that the EDTA-washed scallop myosin was capable of regaining its full content of EDTA-LC as well as its full sensitivity to calcium. We also found that as regards combining with, and conferring calcium sensitivity on the EDTA-washed myosin of scallop adductor, squid mantle LC-2 could effectively replace scallop adductor EDTA-LC. In addition, calcium or strontium ions were found to induce changes in the UV absorption spectrum of scallop adductor EDTA-LC, although the apparent binding constants estimated from the difference spectrum were too low to account for the Ca or Sr sensitivity of scallop actomyosin-ATPase. The divalent cations also induced changes in the UV absorption spectrum of squid LC-2, and the apparent binding constants estimated from the difference spectrum were sufficiently high (1.5 X 10(5) M-1 for Ca binding, and 1.6 X 10(3) M-1 for Sr binding) to account for the Ca and Sr sensitivities of squid mantle myosin B-ATPase. The findings with scallop adductor myosin are in conflict with those reported by Kendrick-Jones et al., and must be accounted for in formulating the molecular mechanism of myosin-linked calcium regulation in molluscan muscles.     

Amino-acid sequence of LC-1 light chain of squid mantle muscle myosin

Tryptic and chymotryptic peptides of the LC-1 light chain of squid mantle muscle myosin were isolated and sequenced by conventional methods, so that the whole amino-acid sequence of the light chain was established. The light chain consisted of 159 amino-acid residues, and its N-terminal alpha-amino group is blocked. Comparing the established sequence with those of vertebrate muscle myosin light chains, only 35% sequence homology was recognized, but a conservative structure was observed in the third domain.     

Amino acid sequence of the regulatory light chain of squid mantle muscle myosin

The amino acid sequence of the regulatory light chain of mantle muscle myosin from squid (Todarodes pacificus) was determined by conventional methods. It was: xA-E-E-A-P-R-R-V-K-L-S-Q-R-Q-M-Q-E-L-K-E-A-F-T-M-I-D-Q-D-R-D-G-F-I-G-M- E-D-L-K-D-M-F-S-S-L-G-R-V-P-P-D-D-E-L-N-A-M-L-K-E-C-P-G-Q-L-N-F-T- A-F-L-T-L-F-G-E-K-V-S-G-T-D-P-E-D-A-L-R-N-A-F-S-M-F-D-E-D-G-Q-G-F-I-P- E-D-Y-L-K-D-L-L-E-N-M-G-D-N-F-S-K-E-E-I-K-N-V-W-K-D-A-P-L-K-N-K-Q-F- N-Y-N-K-M-V-D-I-K-G-K-A-E-D-E-D. The alpha-amino group of this light chain was blocked, and a typical calcium-binding structure was recognized at the sequence of residue 26 to residue 37, like those in other myosin regulatory light chains.     

Separation of cathepsin D-like proteinase and acid thiol proteinase of squid mantle muscle

When the proteinases of the squid mantle muscle were extracted in the presence of dithiothreitol (DTT), the acid proteinase activity increased, indicating that the squid mantle muscle contains a considerable amount of the acid thiol proteinase. The crude extract hydrolyzed neither alpha-N-benzoyl-D,L-arginine-p-nitroanilide (BAPA) nor azocasein, thus refuting the presence of cathepsins B and L in the mantle muscle. The cathepsin D-like proteinase and the acid thiol proteinase were separated by Sephadex A-50 column chromatography. Each of the above partially purified proteinases was able to degrade carp actomyosin at pH 2.5 and 5.0, respectively.     

Locomotory aspects of squid mantle structure

Morphological aspects of squid ( Loligo, Lolliguncula ) mantle relevant to locomotory function were studied. Methods used included polarized light microscopy of frozen sections of untreated tissue taken from animals immediately after death and electron microscopy.
The mantle consists of circular and radial muscles arranged in alternating rings along the whole length of the mantle. The muscle is obliquely striated. Connective tissue fibres are found in the body of the muscle and in the outer and inner tunics. The outer tunic consists of layers of large collagenous fibres. The fibres run in superimposed right- and left-handed helical courses that lie at an angle of 27° to the long axis of the animal. The tunics and the intramuscular connective fibres are thought to resist length changes in the mantle while permitting the changes in girth required for the jet power stroke. Both the intramuscular and the tunic fibre systems may provide elastic energy for the return phase of the jet cycle. Tunic fibres appear to be a geodesic tensile reinforcing system ensuring smooth shape changes in the mantle.     

Locomotory function of the squid mantle

A detailed kinematic analysis of the mantle movements of swimming Lolliguncula brevis was made. Some data were also obtained on Loligo pealei. The qualitative and quantitative data provided are of use in discussing mechanisms of squid mantle function.
Several possible mechanisms of squid mantle re-expansion were proposed and investigated. The inhalant phase of jet propulsion is probably effected by contraction of the radial muscles, since thinning of the mantle wall accompanies re-expansion. The radial muscles may cause mantle re-expansion by contracting alternately with the circular muscles in response to nerve impulses, or by contracting alternately with the circular muscles in response to stretch cycles effected by mantle wall thickening in the power stroke, or by contracting continuously through both power and recovery strokes. Elasticity of the mantle tissue may contribute to mantle re-expansion. Neither pressure pumps nor a Bernoulli effect mechanism are effectors of mantle re-expansion.     

How swimming fish use slow and fast muscle fibers: implications for models of vertebrate muscle recruitment

We quantified the intensity and duration of electromyograms (emgs) from the red and white axial muscles in five bluegill sunfish (Lepomis macrochirus) which performed three categories of behavior including steady swimming and burst and glide swimming at moderate and rapid speeds. Steady swimming (at 2 lengths/s) involved exclusively red muscle activity (mean posterior emg duration = 95 ms), whereas unsteady swimming utilized red and white fibers with two features of fiber type recruitment previously undescribed for any ectothermic vertebrate locomotor muscle. First, for moderate speed swimming, the timing of red and white activity differed significantly with the average onset time of white lagging behind that of red by approximately 40 ms. The durations of these white emgs were shorter than those of the red emgs (posterior mean = 82 ms) because offset times were effectively synchronous. Second, compared to steady and moderate speed unsteady swimming, the intensity of red activity during rapid unsteady swimming decreased while the intensity of white muscle activity (mean white emg duration = 33 ms) increased. Decreased red activity associated with increased white activity differs from the general pattern of vertebrate muscle recruitment in which faster fiber types are recruited in addition to, but not to the exclusion of, slower fiber types.     

Cloning and nucleotide sequence of a novel 28-kDa protein from the mantle muscle of the squid Todarodes pacificus with homology to tropomyosin

In recent studies, we found autodegradation of collagen from the mantle muscle of the squid Todarodes pacificus and also that the 28- and 25-kDa proteins are closely related to this phenomenon [Connect. Tissue Res. 45 (2004) 109-121]. We obtained partial sequences of three internal portions of this protein, which suggested that 25-kDa protein is a partially degraded form of the 28-kDa protein. We determined the full cDNA sequence of this protein by the degenerate polymerase chain reaction (PCR) using the information of amino acid sequences. The deduced amino acid sequence corresponding to the 212-bp cDNA contained all of the amino acid identified from the 28-kDa protein. Rapid amplification of cDNA ends (RACE) and squid mantle muscle RNA allowed cloning of the full 522-bp sequence, corresponding to a protein of 174 amino acids. A database search indicated that this is a new protein that shares 27-34% identity with tropomyosins from various animals. Structural prediction suggested that it possesses heptad repeats that form coiled-coil structures. We expressed a recombinant protein encoded by the 212-bp cDNA in Escherichia coli and used it to generate a polyclonal antibody. Western blotting with this antibody showed that the 28-kDa protein is expressed in fin, tentacle, and mantle muscle, but not in liver.     

The neural control of contraction in a fast insect muscle.

The wing muscles used in singing by the katydid, Neoconocephalus robustus, are extraordinarily fast. At 35 degrees C, the animal's thoracic temperature during singing, an isometric twitch lasts only five to eight msec (onset to 50% relaxation) and the fusion frequency of these muscles is greater than 400 Hz. Stimulating the motornerve to a singing muscle initiates a short (2.5 msec at 35 degrees C), sometimes overshooting depolarization of the muscle fibers. Despite their spike-like appearance, the electrical responses are largely synaptic potentials. The muscle membrane appears to be capable of only weak, electrically-excitable, depolarizing electrogenesis. The short synaptic potentials result in part from rapidly-developing delayed rectification, in part from a low resting membrane resistance (Rm = 162 omega cm2) and a concomitantly short membrane time constant (about 1.5 msec).     

Red muscle recruitment during steady swimming correlates with rostral-caudal patterns of power production in trout

Rainbow trout (Oncorhynchus mykiss) and brook trout (or charr, Salvelinus fontinalis) display different rostral-caudal patterns of power production by the red or aerobic muscle during steady swimming. The anterior muscle of rainbow trout produces much less power for swimming than the posterior, while in brook trout there is no variation in power output. To determine if red muscle recruitment is associated with anterior-posterior patterns of power production, electromyography (EMG) was used to record red muscle activity at three body positions across a range of swimming speeds in fish of each species. The initial recruitment of the anterior red muscle in swimming rainbow trout was predicted to lag behind, i.e. occur at higher speeds, that of the posterior due to the variation in power production, but no variation in recruitment was expected for brook trout. Burst of red muscle EMG activity occurring with each tailbeat was analyzed for frequency (tailbeat frequency), duty cycle (DC) (duration of burst relative to the period of the tailbeat) and burst intensity (BI) (magnitude of the measured EMG activity). Brook trout swam with higher tailbeat frequencies and longer values of DC than rainbow trout. Both species showed a pattern of longitudinal variation in DC, with longer DC values in the anterior red muscle. BI also differed significantly along the length of rainbow trout but not brook trout. In the former, BI of anterior muscle was significantly less than the posterior at lower steady swimming speeds. The EMG data suggest that power production and muscle recruitment are related. In rainbow trout, where there is longitudinal variation in muscle power output, there are also significant rostral-caudal differences in red muscle recruitment.     

Ocean acidification responses in paralarval squid swimming behavior using a novel 3D tracking system

The study presents the biomass and spatial distribution of Sardinella aurita in the Libyan, Maltese and southern Sicily waters, estimated during four acoustic surveys in summer 2008 and 2010. Strong differences in terms of both spatial distribution and total biomass between years and areas were found, with higher total biomass in 2010 than in 2008 in both southern and northern areas of the central Mediterranean. Habitat suitability and changes in the spatial dynamics of round sardinella among areas and years are then explored in relation to total biomass variation and environmental factors. The area of presence of S. aurita increased in the Maltese and Sicilian waters in 2010 with respect to 2008, while most of the total biomass observed in the two years occupied the same proportion of the continental shelf in Libyan waters. The link between environmental conditions and S. aurita area of presence and aggregation was investigated by means of generalized additive models (GAM). The application of GAM singled out the key role of depth and temperature in driving higher round sardinella aggregations, as they were able to explain in both years about the 48% of total deviance in the case of strictly positive values. Favourable habitat for round sardinella was found in waters shallower than 60 m depths, with a clear peak around 40 m depth. Favourable temperature values were above 22°C for presence/absence case and above 24°C when GAM was applied on strictly positive values. Overall S. aurita biomass and distribution were discussed in the light of the classical models proposed to describe the fish spatial dynamics in relation to an increase in total biomass.     

Purification and properties of squid mantle adenylate kinase. Role of NADH in control of the enzyme.

Adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) from the mantle muscle of the squid, Loligo pealeii, was purified over 170-fold to homogeneity as judged by polyacrylamide and starch gel electrophoresis. The tissue contains a single isozyme of adenylate kinase, the enzyme from cytoplasmic and mitochondrial compartments (90 and 10% of total activity, respectively) being identical in physical and kinetic properties. Molecular weight was found to be 27,000 +/- 400. The enzyme shows a pH optimum of 8.2 in the forward (APD utilizing) and 7.4 in the reverse direction. Michaelis constants for ADP, ATP, and AMP are 0.70, 0.13, and 0.15 mM, respectively, with optimal Mg2+:adenylate ratios being 1:2 for ADP and 1:1 for ATP. A comparison of mass action ratios with the equilibrium constant indicated that squid adenylate kinase is held out of equilibrium in resting, but not active, muscle. A search for metabolic modulators of adenylate kinase revealed that NADH (Ki of 0.1 mM) was the only modulator which exerted a significant effect within its in vivo concentration range. The data presented indicate that NADH inhibition is the factor maintaining adenylate kinase in a nonequilibrium state in resting muscle and that release of this inhibition can serve to integrate adenylate kinase into the known scheme of intermediary metabolism in this tissue. A sharp drop in NADH levels at the onset on muscular work co-ordinates that activation of aerobic metabolism in this tissue and allows adenylate kinase to return to equilibrium function. At equilibrium, the enzyme can function to ampligy the concentration of AMP, a potent activator and deinhibitor of key glycolytic and Krebs cycle enzymes. The effect of modulators of adenylate kinase in preventing denaturation by heat or proteolysis revealed that NADH and substrates induced conformational changes in the enzyme which rendered it less susceptible to denaturation. The conformation state induced by NADH differed from that induced by substrate.     

Purification and properties of glutamate dehydrogenase from the mantle muscle of the squid, Loligo pealeii. Role of the enzyme in energy production from amino acids.

1. The activity of glutamate dehydrogenase was measured in the tissues of the squid, Loligo pealeii. The enzyme occurs in high activity in digestive pouch, systemic heart, and all muscle tissues. 2. Glutamate dehydrogenase from mantle muscle is located intra-mitochondrially, has a molecular weight of 310,000, and is electrophoretically similar to the enzyme from all other squid tissues. 3. The enzyme from mantle muscle was purified 40-fold by elution from DEAE-cellulose and used for kinetic studies. The enzyme is NAD+-specific, activated by ADP, AMP, and leucine, and inhibited by GTP, GDP, ATP, and reaction products (in particular NADH). 4. Squid glutamate dehydrogenase shows an almost absolute dependence on ADP. The purified enzyme is activated over 100-fold by saturating concentrations of ADP (Ka = 0,75 7M); The pH optima are also altered significantly by ADP. 5. The enzyme appears to be kinetically adapted to favour glutamate oxidation in comparison to glutamate dehydrogenase from other resources. The evidence indicates that the primary role of glutamate dehydrogenase in squid mantle muscle is in regulating the catabolism of amino acids for energy production.     

Wrist stabilisation and forearm muscle coactivation during freestyle swimming.

The aim of this study was to evaluate the stabilisation of the wrist joint and the ad hoc wrist muscles activations during the two principal phases of the freestyle stroke. Seven male international swimmers performed a maximal semi-tethered power test. A swimming ergometer fixed on the start area of the pool was used to collect maximal power. The electromyography signal (EMG) of the right flexor carpi ulnaris (FCU) and extensor carpi ulnaris (ECU) was recorded with surface electrodes and processed using the integrated EMG (IEMG). Frontal and sagittal video views were digitised frame by frame to determine the wrist angle in the sagittal plane and the principal phases of the stroke (insweep, outsweep). Important stabilisation of the wrist and high antagonist muscle activity were observed during the insweep phase due to the great mechanical constraints. In outsweep, less stabilisation and lower antagonist activities were noted. Factors affecting coactivations in elementary movements, e.g. intensity and instability of the load, accuracy and economy of the movement were confirmed in complex aquatic movement.