Lolliguncula Brevis Research Articles

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.

Influence of temperature on the microstructure of statoliths of the thumbstall squid Lolliguncula brevis

A laboratory study investigating the influence of temperature on the microstructure of statoliths of Lolliguncula brevis is described. Groups of squid were subjected to various temperature regimes for periods in excess of 30 d. Statoliths extracted from 20 squid were examined using a confocal microscope in laser scanning mode. The parts of the statoliths deposited during the course of the experiments were identified using either putative daily increment counts or from checks produced in response to capture and handling. These checks appear to consist of a series of prominent increments rather than reflecting a period of interrupted statolith growth. Increments deposited during the experiment generally displayed reduced contrast and clarity in comparison to the “wild” parts of the statolith, presumably in response to the constant conditions imposed in the laboratory. Average statolith growth rates observed over the course of the experiment showed a strong positive relationship to ambient temperature. A significant sex effect was apparent, with statoliths of female squid generally growing faster than those of males. Observed statolith growth rates at 15 °C were generally below 1 μm d−1, suggesting that the widths of daily increments produced under these conditions may approach the resolution limits of a light microscope. The implications for studies using increment numbers to estimate age are discussed.

Electrogenic Na(+)/Ca(2+) exchange. A novel amplification step in squid olfactory transduction.

Olfactory receptor neurons (ORNs) from the squid, Lolliguncula brevis, respond to the odors l-glutamate or dopamine with increases in internal Ca(2+) concentrations ([Ca(2+)](i)). To directly asses the effects of increasing [Ca(2+)](i) in perforated-patched squid ORNs, we applied 10 mM caffeine to release Ca(2+) from internal stores. We observed an inward current response to caffeine. Monovalent cation replacement of Na(+) from the external bath solution completely and selectively inhibited the caffeine-induced response, and ruled out the possibility of a Ca(2+)-dependent nonselective cation current. The strict dependence on internal Ca(2+) and external Na(+) indicated that the inward current was due to an electrogenic Na(+)/Ca(2+) exchanger. Block of the caffeine-induced current by an inhibitor of Na(+)/Ca(2+) exchange (50-100 microM 2',4'-dichlorobenzamil) and reversibility of the exchanger current, further confirmed its presence. We tested whether Na(+)/Ca(2+) exchange contributed to odor responses by applying the aquatic odor l-glutamate in the presence and absence of 2', 4'-dichlorobenzamil. We found that electrogenic Na(+)/Ca(2+) exchange was responsible for approximately 26% of the total current associated with glutamate-induced odor responses. Although Na(+)/Ca(2+) exchangers are known to be present in ORNs from numerous species, this is the first work to demonstrate amplifying contributions of the exchanger current to odor transduction.

Oxidative stress and antioxidative defense in cephalopods: a function of metabolic rate or age?

Activities of the antioxidative enzymes superoxide dismutase (SOD), catalase, glutathione peroxidase (GPX) and glutathione reductase (GR) were measured in the cephalopods Sepia officinalis and Lolliguncula brevis. Maximal enzyme activities were higher in gill tissue than in the mantle musculature of both species. Activities were generally lower in tissues of L. brevis than in S. officinalis. Comparison with other ectothermic animals showed both cephalopod species to have a low enzymatic antioxidative status despite their high metabolic rate. Furthermore, changes in antioxidative enzyme activities were measured in the cuttlefish S. officinalis with increasing age. The concentrations of malondialdehyde (MDA) and lipofuscin were determined as indicators of lipid peroxidation. Investigated animals were between 1.5 months and over 12 months old. Changes of antioxidative enzyme activities with age were not uniform. SOD and GPX activities increased with age, while catalase activity declined. In contrast, GR activity remained almost unchanged in all age groups. The low level of antioxidative defense might allow for the significant age-induced rise in MDA levels in gills and mantle musculature and for the increase in lipofuscin levels in mantle and brain tissue. It might thereby contribute to increased oxidative damage and a short life span in these cephalopods.

Metabolic performance of the squid Lolliguncula brevis (Cephalopoda) during hypoxia: an analysis of the critical PO2

Brief squid Lolliguncula brevis are regularly exposed to fluctuating oxygen levels in their shallow coastal environment. To assess hypoxia resistance, animals were exposed for two hours to ambient oxygen partial pressures ( P O 2 ) between 19.3 kPa (normoxia) and 6.6 kPa (34.2% air saturation) at 20±1°C. In a second set of experiments, the animals were subjected to a low P O 2 of 2.8±0.3 kPa (14.5±1.6% air saturation) for 15 to 60 min. Subsequently, metabolic, energy and acid–base status were analysed in the mantle tissue. Onset of anaerobic metabolism was observed between 9.4 and 7.9 kPa (48.7 and 40.9% air saturation), reflecting the critical oxygen tension for this species. The formation of octopine and acetate indicates a simultaneous onset of anaerobic metabolism in both the cytosol and the mitochondria during progressive hypoxia. Concomitantly, an intracellular acidosis developed. During exposure to oxygen partial pressures between 19.3 and 6.6 kPa, aerobic and anaerobic processes were sufficient to maintain energy status in the mantle musculature. No significant changes in ATP and phospho- l -arginine (PLA) concentrations were observed. In contrast, both ATP and PLA levels declined significantly after 15 min at an ambient P O 2 of 2.8±0.3 kPa. Concomitantly, the Gibb’s free energy change of ATP hydrolysis fell to a minimum value of about −44 kJ·mol −1 , a level suggested to reflect limiting energy availability for cellular ATPases. These results indicate that hypoxia at 2.8 kPa (14.5% air saturation) rapidly takes Lolliguncula brevis to the limits of performance. However, it is probably capable of withstanding longer periods of moderate hypoxia close to 50% air saturation (9.7 kPa), enabling the squids to cope with oxygen fluctuations in their shallow estuarine environment or to dive into hypoxic waters by use of their economic jetting strategy. Nonetheless, the critical P O 2 is considered to be high compared to other hypoxia tolerant animals, an observation likely related to the high metabolic rate of these squids.

Unique proteins from the statoliths of Lolliguncula brevis (Cephalopoda: Loliginidae)

Information concerning the role of the organic matrix (OM) in statolith mineralization may contribute to resolving problems currently facing the use of increments in squid statoliths to estimate the age of individuals. A preliminary study aimed at purifying and characterizing the OM proteins from statoliths of the loliginid squid Lolliguncula brevis is described. Proteins extracted from the statoliths were separated into two fractions, insoluble and soluble in aqueous solutions. Gel electrophoresis indicated that the insoluble fraction is composed of at least eight major proteins, ranging in size from 25 to over 200 kDa. The five largest insoluble proteins were glycosylated, as shown by immunoassay. In contrast, the soluble fraction is composed of a single dominant protein of about 100 kDa, and two other major proteins of higher molecular weight. All three soluble proteins were glycosylated. Molecular weight and partial sequence data of peptides from five of the insoluble and one of the soluble proteins were used to search databases for possible homologs. No matches were found, suggesting that these proteins may belong to a class of hitherto undescribed OM compounds. Different proteins are involved in biomineralization processes in different organisms. We propose that the insoluble matrix proteins are responsible for regulating the appearance of increments in squid statoliths.

Characterization of a Na+-dependent betaine transporter with Cl- channel properties in squid motor neurons.

Most marine invertebrates, including squids, use transporters to accumulate organic osmolytes such as betaine, to prevent water loss when exposed to elevated salinity. Although a limited number of flux studies have shown the Na+ dependence of betaine transport, nothing is known about the electrogenic properties of osmolyte transporters. We used whole cell and perforated-patch voltage-clamp techniques to characterize the electrical properties of the betaine transporter in giant fiber lobe motor neurons of the squid Lolliguncula brevis. Betaine activated a large, Cl--selective current that was reversibly blocked by 100 microM niflumic acid (97 +/- 2% block after 40 s, SD; n = 7) and partially inhibited by 500 microM SITS (29 +/- 11%; n = 5). The Cl- current was Na+ dependent and was virtually eliminated by isotonic replacement of Na+ with Li+, NMDG+, or Tris+. Concentration-response data revealed an EC50 in a physiologically relevant range for these animals of 5.1 +/- 0.9 mM (n = 11). In vertebrates, the betaine transporter is structurally related to the GABA transporter, and although GABA did not directly activate the betaine-induced current, it reversibly reduced betaine responses by 34 +/- 14% (n = 8). Short-term changes in osmolality alone did not activate the Cl- current, but when combined with betaine, Cl- currents increased in hypertonic solutions and decreased in hypotonic solutions. Activation of the betaine transporter and Cl- current in hypertonic conditions may affect both volume regulation and excitability in L. brevis motor neurons. This study is the first report of a novel betaine transporter in neurons that can act as a Cl- channel.

Transient and persistent tetrodotoxin-sensitive sodium currents in squid olfactory receptor neurons.

Squid olfactory receptor neurons are primary bipolar sensory neurons capable of transducing water-born odorant signals into electrical impulses that are transmitted to the brain. In this study, we have identified and characterized the macroscopic properties of voltage-gated Na+ channels in olfactory receptor neurons from the squid Lolliguncula brevis. Using whole-cell voltage-clamp techniques, we found that the voltage-gated Na+ channels were tetrodotoxin sensitive and had current densities ranging from 5 to 169 pA pF-1. Analyses of the voltage dependence and kinetics revealed interesting differences from voltage-gated Na+ channels in olfactory receptor neurons from other species; the voltage of half-inactivation was shifted to the right and the voltage of half-activation was shifted to the left such that a "window-current" occurred, where 10-18% of the Na+ channels activated and did not inactivate at potentials near action potential threshold. Our findings suggest that in squid olfactory neurons, a subset of voltage-gated Na+ channels may play a role in generating a pacemaker-type current for setting the tonic levels of electrical activity required for transmission of hyperpolarizing odor responses to the brain.

An examination of the role of the digestive gland of two loliginid squids, with respect to lipid: storage or excretion?

The role of the digestive gland, with respect to non-structural lipid, was examined using proximal analysis, histochemistry and quantitative histological techniques in the tropical loliginid squids Sepioteuthis lessoniana (Lesson) and Photololigo sp. The digestive gland of both species was characterized by large and numerous lipid droplets in the apical portion of the digestive cells and very few in the basal portion. The apical lipid droplets were released into the lumen of the gland and subsequently rapidly removed. Despite the numerous large apical lipid droplets, the lipid concentration in the digestive glands of S. lessoniana and Photololigo sp. was lower than that reported for most squid species. There was no relationship between lipid concentration and stage of digestion, suggesting that lipid is not stored in the gland after a meal. There was also no relationship between lipid concentration and the sex of an individual or stage of reproductive maturity, suggesting that these squids are not storing lipid in the digestive gland for use in fuelling reproductive maturation or providing an energy source for oocytes. I believe this study is the first to combine proximal analysis and quantitative histological techniques to examine the role of the squid digestive gland with respect to non-structural lipids. The results indicate that the digestive gland of these tropical loliginid squids is excreting, not storing, excess dietary lipid.

Betaine activates a hyperpolarizing chloride conductance in squid olfactory receptor neurons.

Isolated olfactory receptor neurons from the squid Lolliguncula brevis respond to betaine, a repellent odorant, with hyperpolarizing receptor potentials. Using perforated-patch techniques, we determined that the hyperpolarizing conductance was selective for Cl- and could be reversibly blocked by the Cl- channel blockers 4-acetamido-4'-isothio-cyanatistilbene-2,2'disulfonic acid and niflumic acid. Gramicidin-patch recordings revealed that [Cl-]i in squid olfactory receptor neurons is normally very low compared to vertebrate olfactory receptor neurons, and that activating a Cl- conductance would hyperpolarize the cell in vivo. The lack of dependence on internal or external K+ or Na+ ruled out the possibility that the Cl- conductance was generated by a cation-dependent cotransporter or pump. Common G-protein-dependent signalling pathways, including phospholipase C, arachidonic acid, and cyclic nucleotides, do not appear to be involved. Ca2+ imaging experiments showed that betaine did not affect [Ca2+]i, suggesting that the Cl- current is not Ca2+ dependent. Our findings represent the first report of an odorant-activated, hyperpolarizing chloride conductance in olfactory receptor neurons.

Age, growth, and maturation of Lolliguncula brevis (Cephalopoda: Loliginidae) in the northwestern Gulf of Mexico with a comparison of length-frequency versus statolith age analysis

Over 2000 individuals of Lolliguncula brevis were collected from 1975 to 1979 in the northern Gulf of Mexico off the Texas coast; their growth and reproductive biology were analyzed by traditional fisheries methods (ELEFAN analysis of the length-frequency data). From 1994 to 1996, 112 squid were captured in Galveston Bay, near Galveston Island, Texas, and their age and growth determined by statolith increment analysis. The results were strikingly different between the two approaches. Length-frequency analysis of growth overestimated life span by a factor of three to seven times. Statolith increment counts, verified by laboratory growth validation experiments, indicate that this species is very short-lived, in the order of 100-200 days depending on temperature. A slight increase in temperature during the early stage of development can greatly shorten the life span. This study provides evidence that increased temperature during a squid's early growth period could markedly accelerate growth. Mature individuals occurred throughout the year, although there were many more mature males collected than females. Gonad growth and maturation in L. brevis appear to be associated more with size than with individual age. There is now compelling evidence that length-frequency analysis should be abandoned as a technique for determining squid growth.

Characterization of voltage- and Ca(2+)-activated K+ channels in squid olfactory receptor neurons.

We performed whole-cell voltage-clamp experiments on isolated olfactory neurons from the squid Lolliguncula brevis. Total outward currents were composed of three identifiable K+ currents: a delayed rectifier K+ current that showed slow inactivation and was sensitive to 5 mmol l-1 tetraethylammonium; a rapidly inactivating, 4-aminopyridine (4-AP)-sensitive, A-type K+ current and a Ca(2+)-sensitive K+ current that was blocked by 200 nmol l-1 charybdotoxin and 10 mmol l-1 Cd2+ but was insensitive to apamin. The proportion of each current type varied from cell to cell, suggesting that responses to a given odorant would depend of the complement of channels present. The kinetics of the K+ currents were affected by temperature, with Q10 values ranging from 2 to 6. The identification and characterization of these K+ currents will greatly aid our understanding of action potential generation in these cells and will facilitate modelling of how odor responses are transduced and coded in squid olfactory receptor neurons.

Age, growth, and maturation of Lolliguncula brevis (Cephalopoda: Loliginidae) in the northwestern Gulf of Mexico with a comparison of length-frequency versus statolith age analysis

Age, growth, and maturation of Lolliguncula brevis (Cephalopoda: Loliginidae) in the northwestern Gulf of Mexico with a comparison of length-frequency versus statolith age analysis

Metabolic and energy correlates of intracellular pH in progressive fatigue of squid (L. brevis) mantle muscle.

Squid (Lolliguncula brevis) were exercised at increasing swimming speeds to allow us to analyze the correlated changes in intracellular metabolic, acid-base, and energy status of the mantle musculature. Beyond a critical swimming velocity of 1.5 mantle lengths/s, an intracellular acidosis developed that was caused by an initial base loss from the cells, the onset of respiratory acidification, and, predominantly, octopine formation. The acidosis was correlated with decreasing levels of phospho-L-arginine and, thus, supported ATP buffering at the expense of the phosphagen. Monohydrogenphosphate, the actual substrate of glycogen phosphorylase accumulated, enabling glycogen degradation, despite progressive acidosis. In addition to octopine, succinate, and glycerophosphate accumulation, the onset of acidosis characterizes the critical velocity and indicates the transition to a non-steady-state time-limited situation. Accordingly, swimming above the critical velocity caused cellular energy levels (in vivo Gibbs free energy change of ATP hydrolysis) to fall. A minimal value was reached at about -45 kJ/mol. Model calculations demonstrate that changes in free Mg2+ levels only minimally affect ATP free energy, but minimum levels are relevant in maintaining functional concentrations of Mg(2+)-complexed adenylates. Model calculations also reveal that phosphagen breakdown enabled L. brevis to reach swimming speeds about three times higher than the critical velocity. Comparison of two offshore squid species (Loligo pealei and Illex illecebrosus) with the estuarine squid L.brevis indicates that the latter uses a strategy to delay the exploitation of high-energy phosphates and protect energy levels at higher than the minimum levels (-42 kJ/mol) characterizing fatigue in the other species. A more economical use of anaerobic resources and an early reduction in performance may enable L. brevis to tolerate more extreme environmental conditions in shallow estuarine waters and even hypoxic environments and to prevent a fatal depletion of energy stores.

Squid (Lolliguncula brevis) life in shallow waters: oxygen limitation of metabolism and swimming performance.

Squid (Lolliguncula brevis) were exercised in a tunnel respirometer during a stepwise increase in water velocity in order to evaluate the anaerobic threshold, i.e. the critical swimming speed above which anaerobic metabolism contributes to energy production. The average anaerobic threshold was found at speeds of 1.5-2 mantle lengths s-1. Above this velocity, alpha-glycerophosphate, succinate and octopine started to accumulate in the mantle tissue. ATP levels fell and phospho-L-arginine was progressively depleted, while the levels of glucose 6-phosphate and inorganic phosphate rose. The finding of a simultaneous onset of anaerobic metabolism in the cytosol and the mitochondria indicates that a limited oxygen supply to the mitochondria elicits anaerobic energy production. This finding is opposite to the situation found in many other vertebrate and invertebrate species, in which energy requirements in excess of aerobic energy production are covered by anaerobic metabolism, with mitochondria remaining aerobic. In L. brevis, swimming at higher speeds is associated with a small factorial increase in metabolic rate based on a high resting rate of oxygen consumption. Pressure recordings in the mantle cavity support this finding, indicating a high basal level of spontaneous activity at rest and a small rise in mean pressure at higher swimming velocity. Bursts of higher pressures from the jet support elevated swimming speeds and may explain the early transition to anaerobic energy production which occurs when pressure amplitudes exceed 1.2-1.5 kPa or when mean pressure rises above 0.22-0.25 kPa. The finding of mitochondrial hypoxia at a low critical speed in these squid is interpreted to be related to their life in shallow coastal and bay waters, which limits the necessity to maintain high swimming velocities. At increased swimming velocities, the animals oscillate between periods of high and low muscular activity. This behaviour is interpreted to reduce transport cost and to permit a longer-term net use of anaerobic resources when speed exceeds the critical value or when the squid dive into hypoxic waters. The simultaneous onset of anaerobic metabolism in the cytosol and the mitochondria emphasizes that squid generally make maximal use of available oxygen under resting conditions, when their energy requirements are the highest among marine invertebrates.

Proprioceptive hair cells on the neck of the squid Lolliguncula brevis :a sense organ in cephalopods for the control of head-to-body position

Decapod cephalopods, such as cuttlefishes and squids, have a distinct neck region that allows movements (roll, pitch and yaw) of the head relative to the body. This paper describes the structure, innervation and central pathways of proprioceptive hair cells on the neck of the squid Lolliguncula brevis that sense such movements and control head-to-body position. These hair cells exist on the dorsal side of the neck underneath the nuchal cartilage, close to the animal's midline on either side of the nuchal crest. On each side, the hair cells can be divided into an anterior and a posterior group of 25-35 and 70-80 cells, respectively. An individual hair cell carries up to 300 kinocilia of equal length (about 30 microns), arranged in up to seven rows. The hair cells of the left and right anterior group are morphologically polarized in the medial direction, whereas the hair cells of the left and right posterior group are polarized in the anterior direction. The hair cells are primary sensory cells. They are innervated by a branch of the postorbital nerve and project ipsilaterally into the ventral part of the ventral magnocellular lobe. Efferent synaptic contacts are present at the base of the hair cells. In behavioural tests the influence of the neck hair cells on head position control was investigated. During imposed body rolls, a unilateral deafferentation of the cells caused an asymmetric change of the compensatory head roll response and elicited a head roll offset to the operated side. Bilateral deafferentation of the cells elicited a downward head pitch offset. This offset was superimposed on the compensatory head pitch response during imposed body pitch. These morphological and behavioural findings show that the neck hair cells and the associated nuchal cartilage structures of Lolliguncula brevis form a neck receptor organ that, together with statocyst and visual inputs, controls the position of the animal's head and body.

A dorsal light reflex in a squid.

A dorsal light reflex is described in the squid Lolliguncula brevis. When illuminated from the side in visually homogeneous surroundings, a free-swimming squid rolls the dorsal side of its head and trunk 10-20 degrees towards the light. With the trunk restricted in a holder, the squid rolls its head 4-5 degrees towards the light; this reaction increases by about 50% when the statocysts are bilaterally removed and increases further when the neck receptor organ is also destroyed. The results indicate a multi-modal interaction of visual, statocyst and proprioceptive inputs during postural control.

Interactions among Some Dominant Estuarine Nekton Species

Interactions between pairs of numerically dominant species collected at inlet and creek shorezone and channel habitats within a high salinity estuary in northeastern South Carolina were examined using two-way contingency tables and binomial tests. Of the significant species interactions, over 71% were positive and these primarily occurred within shorezone habitats. The strongest positive interactions were between young-of-the-year spot (Leiostomus xanthurus) and blue crab (Callinectes sapidus) juveniles in both shorezone habitats, and between striped killifish (Fundulus majalis), white mullet (Mugil curema), and striped anchovies (Anchoa hepsetus) in the inlet shorezone habitat. One of the most positive species associations in channel habitats was between the bay anchovy (Anchoa mitchilli) and the Atlantic brief squid (Lolliguncula brevis). These positive relationships between species may be explained by one species enhancing the habitat for another, both species responding to similar environmental conditions, cooperative social interactions such as mixed schooling, or the attraction of predators to prey. Negative interactions were found between schools of Atlantic silversides (Menidia menidia) and striped killifish in the inlet shorezone and between schools of Atlantic silversides and bay anchovies in the creek shorezone. Schools of Atlantic silversides may either displace or compete with other common shorezone species. Positive and negative interactions suggest that relationships between some species pairs did not occur randomly within certain habitats and may have contributed to the organization of the estuarine nekton community. Differences in the strengths and direction of interactions of certain species pairs among habitats and seasons were probably related to the differences in the physical characteristics of those habitats and/or changes in the relative abundance of dominant species and life stages over time.

Salt sensitivity of the hemocyanin of eury- and stenohaline squids

1. The hemocyanin of the euryhaline squid Lolliguncula brevis is insensitive to changes in salinity in the range 11–35%. 2. In contrast the hemocyanin of the stenohaline Loligo paelei loses its cooperativity and pH dependence at 11%., and O 2 affinity increases at physiological pH by more than half. 3. Hemocyanin O 2 affinity in L. brevis, a rare example of a squid that survives shipping in small containers, is also fairly high and has little pH dependence regardless of salinity.

Brain pathways of the chromatophore system in the squid Lolliguncula brevis

Brain pathways controlling the chromatophores of the squid Lolliguncula brevis are described using cobalt iontophoresis. The results show several input and output pathways of the anterior and posterior chromatophore and lateral basal lobes. These connections allow coordination and modification of the chromatophore motor program throughout the motor pathway. Unlike other cephalopod species, there seems to be no direct input from the optic lobes to the lateral basal lobes in L. brevis. This species displays only a few simple patterns; therefore the underlying neural pathways for chromatophore control may be different from those of other cephalopods with more extensive patterning repertoires.