Identification of a female spawn‐associated Kazal‐type inhibitor from the tropical abalone Haliotis asinina

Abalone (Haliotis) undergoes a period of reproductive maturation, followed by the synchronous release of gametes, called broadcast spawning. Field and laboratory studies have shown that the tropical species Haliotis asinina undergoes a two‐week spawning cycle, thus providing an excellent opportunity to investigate the presence of endogenous spawning‐associated peptides. In female H. asinina, we have isolated a peptide (5145 Da) whose relative abundance in hemolymph increases substantially just prior to spawning and is still detected using reverse‐phase high‐performance liquid chromatography chromatograms up to 1‐day post‐spawn. We have isolated this peptide from female hemolymph as well as samples prepared from the gravid female gonad, and demonstrated through comparative sequence analysis that it contains features characteristic of Kazal‐type proteinase inhibitors (KPIs). Has‐KPI is expressed specifically within the gonad of adult females. A recombinant Has‐KPI was generated using a yeast expression system. The recombinant Has‐KPI does not induce premature spawning of female H. asinina when administered intramuscularly. However it displays homomeric aggregations and interaction with at least one mollusc‐type neuropeptide (LRDFVamide), suggesting a role for it in regulating neuropeptide endocrine communication. This research provides new understanding of a peptide that can regulate reproductive processes in female abalone, which has the potential to lead to the development of greater control over abalone spawning. The findings also highlight the need to further explore abalone reproduction to clearly define a role for novel spawning‐associated peptide in sexual maturation and spawning. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Induction of larval settlement and metamorphosis in the donkey-ear abalone,Haliotis asinina Linnaeus,by chemical cues

The effects of the chemical inducers, gamma-aminobutyric acid (GABA) and potassium chloride (KCl), on the larval settlement and metamorphosis of the donkey-ear abalone, Haliotis asinina, was investigated. H. asinina larvae (5–6 h post-hatch) were exposed to a range of GABA (0.125–2.00 μM) and KCl (1.00–12.00 mM) concentrations for 72 h. Results of the dose response experiments showed that settlement and metamorphosis vary according to the dose levels of the inducer compounds. Under controlled laboratory conditions, 0.45–0.50 μM and 6.0 mM seemed to be the optima for GABA and KCl, respectively, as these concentrations elicited the greatest number of postlarvae that metamorphosed, settled or survived. However, GABA generally promoted better attachment and metamorphic response as well as survival than KCl in H. asinina postlarvae.     

Uncovering the metabolic response of abalone (<Emphasis Type="Italic">Haliotis midae)</Emphasis> to environmental hypoxia through metabolomics

Introduction

Oxygen is essential for metabolic processes and in the absence thereof alternative metabolic pathways are required for energy production, as seen in marine invertebrates like abalone. Even though hypoxia has been responsible for significant losses to the aquaculture industry, the overall metabolic adaptations of abalone in response to environmental hypoxia are as yet, not fully elucidated.

Objective

To use a multiplatform metabolomics approach to characterize the metabolic changes associated with energy production in abalone (Haliotis midae) when exposed to environmental hypoxia.

Methods

Metabolomics analysis of abalone adductor and foot muscle, left and right gill, hemolymph, and epipodial tissue samples were conducted using a multiplatform approach, which included untargeted NMR spectroscopy, untargeted and targeted LC–MS spectrometry, and untargeted and semi-targeted GC-MS spectrometric analyses.

Results

Increased levels of anaerobic end-products specific to marine animals were found which include alanopine, strombine, tauropine and octopine. These were accompanied by elevated lactate, succinate and arginine, of which the latter is a product of phosphoarginine breakdown in abalone. Primarily amino acid metabolism was affected, with carbohydrate and lipid metabolism assisting with anaerobic energy production to a lesser extent. Different tissues showed varied metabolic responses to hypoxia, with the largest metabolic changes in the adductor muscle.

Conclusions

From this investigation, it becomes evident that abalone have well-developed (yet understudied) metabolic mechanisms for surviving hypoxic periods. Furthermore, metabolomics serves as a powerful tool for investigating the altered metabolic processes in abalone.

     

A new Perkinsus species (Apicomplexa,Perkinsea) from the abalone Haliotis ruber

A new protozoan of the genus Perkinsus is described from the muscle and hemolymph of the blacklip abalone, Haliotis ruber, from South Australia. It occurs in the muscle of the adductor and mantle, and free and in brownish masses in the hemolymph. Cells cultured in thioglycollate medium produced a prezoosporangium which stained blue-black with iodine. The parasite differs from Perkinsus marinus, the only other member of the class, in having a much larger trophozoite, an eosinophilic vacuoplast when present, a short discharge tube, and appears to be uninfective to oysters.     

First characterisation of the populations and immune-related activities of hemocytes from two edible gastropod species,the disk abalone,Haliotis discus discus and the spiny top shell,Turbo cornutus

The disk abalone Haliotis discus discus and the spiny top shell Turbo cornutus are edible gastropod species of high economic value, mainly in Asia. Mortality outbreaks and variations in worldwide stock abundance have been reported and suggested to be associated, at least in part, with pathogenic infections. Ecology, biology and immunology of both species are currently not well documented. The characterisation of the immune systems of these species is necessary to further assess the responses of H. discus discus and T. cornutus to environmental, chemical and disease stresses. In the present study, we investigated the morphology and immune-related activities of hemocytes in both species using light microscopy and flow cytometry. Two types of hemocytes were identified in the disk abalone hemolymph, blast-like cells and hyalinocytes; whereas four main hemocyte types were distinguished in the spiny top shell, blast-like cells, type I and II hyalinocytes, and granulocytes. Flow cytometric analysis also revealed differences between cell types in immune-related activities. Three subsets of hemocytes, defined by differing lysosomal characteristics, were observed in the hemolymph of the spiny top shell, and only one in the disk abalone. Phagocytic activity was higher in H. discus discus hemocytes than in T. cornutus hemocytes, and the kinetics of PMA-stimulated oxidative activity was different between hemocytes of the disk abalone and the spiny top shell. Finally our results suggest for the first time a predominant mitochondrial origin of oxidative activity in gastropod hemocytes.     

Skeletal muscle metabolic and ionic adaptations during intense exercise following sprint training in humans.

The effects of sprint training on muscle metabolism and ion regulation during intense exercise remain controversial. We employed a rigorous methodological approach, contrasting these responses during exercise to exhaustion and during identical work before and after training. Seven untrained men undertook 7 wk of sprint training. Subjects cycled to exhaustion at 130% pretraining peak oxygen uptake before (PreExh) and after training (PostExh), as well as performing another posttraining test identical to PreExh (PostMatch). Biopsies were taken at rest and immediately postexercise. After training in PostMatch, muscle and plasma lactate (Lac(-)) and H(+) concentrations, anaerobic ATP production rate, glycogen and ATP degradation, IMP accumulation, and peak plasma K(+) and norepinephrine concentrations were reduced (P<0.05). In PostExh, time to exhaustion was 21% greater than PreExh (P<0.001); however, muscle Lac(-) accumulation was unchanged; muscle H(+) concentration, ATP degradation, IMP accumulation, and anaerobic ATP production rate were reduced; and plasma Lac(-), norepinephrine, and H(+) concentrations were higher (P<0.05). Sprint training resulted in reduced anaerobic ATP generation during intense exercise, suggesting that aerobic metabolism was enhanced, which may allow increased time to fatigue.     

Short Communication

Anaerobic metabolism and oxygen carrying-capacity of white shrimp ( Penaeus vannamei ) exposed to short term (three days) and long term (two weeks) moderate hypoxia (2-2.6 mg/L) was investigated. Glucose and lactate levels in hemolymph increased under both hypoxic conditions, indicating an activation of anaerobic pathways during the two-weeks exposure period. In muscle, no differences of glucose and lactate levels were observed between the control group and the exposed groups. In animals exposed to hypoxia for two weeks, hemocyanin and copper in hemolymph were higher than in animals under normoxic conditions or exposed for three days. These results indicate that an increase in oxygen carrying-capacity in shrimp is evident only after a sustained condition of hypoxia. Copper levels in the hepatopancreas decreased in both hypoxic conditions, suggesting a mobilization of copper stores for hemocyanin synthesis. These results indicate that penaeid shrimp can tolerate moderate hypoxic conditions by physiological adaptations, such as anaerobic metabolism and increased oxygen carrying-capacity. These adaptations require an adequate dietary supply of proteins and copper for hemocyanin synthesis and of carbohydrates for anaerobic metabolism.     

Short Communication

Anaerobic metabolism and oxygen carrying-capacity of white shrimp (Penaeus vannamei) exposed to short term (three days) and long term (two weeks) moderate hypoxia (2-2.6 mg/L) was investigated. Glucose and lactate levels in hemolymph increased under both hypoxic conditions, indicating an activation of anaerobic pathways during the two-weeks exposure period. In muscle, no differences of glucose and lactate levels were observed between the control group and the exposed groups. In animals exposed to hypoxia for two weeks, hemocyanin and copper in hemolymph were higher than in animals under normoxic conditions or exposed for three days. These results indicate that an increase in oxygen carrying-capacity in shrimp is evident only after a sustained condition of hypoxia. Copper levels in the hepatopancreas decreased in both hypoxic conditions, suggesting a mobilization of copper stores for hemocyanin synthesis. These results indicate that penaeid shrimp can tolerate moderate hypoxic conditions by physiological adaptations, such as anaerobic metabolism and increased oxygen carrying-capacity. These adaptations require an adequate dietary supply of proteins and copper for hemocyanin synthesis and of carbohydrates for anaerobic metabolism.     

Walking performance and aerobic and anaerobic metabolism of Carcinus maenas (L.) in sea water at 15°C

Walking performance of the shore crab Carcinus maenas (L.) in sea water at 15 °C was assessed. In large crabs there was an inverse relationship between fatigue time and speed; crabs ran for $\text{\$}\text{?}$10 min at 3.2 m·min^?1 and for only 2 min at 14 m·min^?1. There were linear relationships between oxygen consumption and walking speeds for small and large animals walking at up to 4 m·min^?1 Estimates of maximum oxygen consumption were proportional to W^0.13 whereas inactive consumption is proportional to W^0.44 this resulted in aerobic scope (i.e. the difference between inactive and maximal rates of oxygen consumption) remaining almost constant across a weight range of animals whereas the aerobic expansibility (maximal rates/inactive rates) declined from 7- to 4-fold with increasing size. After a 12-h period without handling (settled animals) the animals could immediately become active and reach maximal rates of oxygen consumption similar to those of animals handled 1 h before the experiment. The aerobic expansibility of these settled animals could range from 21 to 8 times their inactive rates of oxygen consumption in small and large animals respectively. After 10 min of exercise oxygen consumption and whole body lactate levels returned to pre-exercise values within 5 to 25 min. The net oxygen debts range from 16 to 64% of the net oxygen consumption increase during exercise in small and large animals respectively.Calculations of the energy gained from lactate accumulation indicated that the net aerobic energy production during walking was supplemented from 4 to 71 % by anaerobic metabolism in small and large animals respectively. With increasing animal size the decline in aerobic expansibility was offset by an increased capacity for lactate production so that the overall maximum energy production during sustained activity remained almost constant at around seven times the inactive rate. The cost of transport (the net increase in oxygen consumption per g per m) falls with increased walking speed and increased animal size.     

Oxygen uptake and the potentiating effects of increased hemolymph lactate on oxygen transport during exercise in the blue crab,Callinectes sapidus

Summary At the onset of moderate swimming activity,Callinectes sapidus rapidly increased branchial ventilation, heart rate, and oxygen uptake, reaching steady state values in 2–3 min, with a half-time of 30 sec. Although O₂ extraction efficiency decreased slightly (50% to 43%) upon reaching steady state, O₂ uptake was increased 2.6 fold over resting (routine) levels. HemolymphP _{O 2} did not change during sustained (30–60 min) exercise, but a marked decrease in pH (7.60 to 7.10), associated with a 14-fold increase in hemolymph lactate concentration, caused decreases in both pre-and postbranchial O₂ content due to a large hemocyanin Bohr shift. The effect of the Bohr shift on O₂ binding, however, was minimized by an increase in hemocyanin O₂ affinity induced by lactate ions; the influence of lactate on hemocyaninP ₅₀ was shown to be the same in vivo and in vitro. As a result of the interaction between the Bohr and lactate effects, only slight increases were observed in the a-v O₂ difference (13%) and the quantitative role of hemocyanin in oxygen transport (11%) during exercise. The increase in O₂ delivery was therefore attributed primarily to a 2.3 fold increase in cardiac output (Fick estimate), resulting from increases in both heart rate (1.61 X) and stroke volume (1.42X). During exercise hemocyanin remained 21% oxygenated upon leaving the tissues, thus maintaining a substantial venous O₂ reserve which could be utilized to fuel more strenuous levels of exercise at least partly by aerobic pathways. The high hemolymph lactate levels, however, indicate that anaerobic metabolism makes a significant contribution to energy production even during moderate exercise. These results are similar to the respiratory and circulatory responses reported for other decapod crustaceans and fish during mild exercise.C. sapidus, however, appears to be highly resistant to fatigue, which correlates with its welldeveloped locomotor capabilities.     

Metabolic consequences of living in a wave-swept environment: Effects of simulated wave forces on oxygen consumption, heart rate, and activity of the shell adductor muscle of the abalone Haliotis iris

Animals in wave-exposed habitats must constantly contend with the hydrodynamic forces of lift and drag. In this study, we investigated aspects of the metabolic response of Haliotis iris to simulated wave forces varying in magnitude up to 9.6 N applied to the shell at 69° to horizontal, alternately from anterior and posterior directions, with a period of 10s. Shell adductor muscle activity (electromyogram, EMG), heart rate, and oxygen consumption were monitored during force application and during extended recovery. EMG spiking was absent at zero force, but increased markedly with increasing force, in synchrony with the wave cycle. In contrast, heart rate was unaffected by wave forces and varied by only 5% over the whole range of applied forces. During force application, oxygen consumption increased by 10-25% above resting rates and remained elevated throughout a 5-hour recovery period, indicating a switch to anaerobic metabolism. It is concluded that living in a wave-swept environment is metabolically costly for abalone although this may be compensated by improved food availability and more efficient ventilation induced by external flow.     

Active sodium extrusion reduces net efficiencies of oxidative phosphorylation in the strictly photoautotrophic cyanobacterium Anacystis nidulans

Efficiencies of oxidative phosphorylation ($\text{P}\text{O}$ ratios), intracellular high-energy phosphate pools (ATP and ADP) under aerobic and anaerobic dark conditions, and photosynthetic oxygen evolution measured with intact cells of Anacystis nidulans were found to be specifically depressed by NaCl, but not by KCl. A scheme is proposed which explains the deleterious effect of sodium on the energy metabolism of A. nidulans by competition for protons between ATP synthesis and active sodium extrusion.     

Effect of Modified Atmosphere Composition on the Metabolism of Glucose by Brochothrix thermosphacta

The influence of atmosphere composition on the metabolism of Brochothrix thermosphacta was studied by analyzing the consumption of glucose and the production of ethanol, acetic and lactic acids, acetaldehyde, and diacetyl-acetoin under atmospheres containing different combinations of carbon dioxide and oxygen. When glucose was metabolized under oxygen-free atmospheres, lactic acid was one of the main end products, while under atmospheres rich in oxygen mainly acetoin-diacetyl was produced. The proportions of the total consumed glucose used for the production of acetoin (aerobic metabolism) and lactic acid (anaerobic metabolism) were used to decide whether aerobic or anaerobic metabolism predominated at a given atmosphere composition. The boundary conditions between dominantly anaerobic and aerobic metabolisms were determined by logistic regression. The metabolism of glucose by B. thermosphacta was influenced not only by the oxygen content of the atmosphere but also by the carbon dioxide content. At high CO₂ percentages, glucose metabolism remained anaerobic under greater oxygen contents.     

Oxygen Demand and Energy Cost of Intense Muscular Activity in Humans

Changes in the kinetics of aerobic and anaerobic metabolism were studied in 26 highly profiled athletes performing bicycle ergometer exercise. The different intensity exercise sessions included those with a critical intensity corresponding to the maximum oxygen consumption up to value of the maximum anaerobic intensity of about 10 MMR units. The maximal aerobic metabolism was maintained in the exercises with a relative intensity of 1.0 to 2.5 MMR units. At the higher values of the exercise relative intensity, the oxygen current consumption exponentially decreased. An increase in the rate of anaerobic glycolytic energy production, which was first recorded at the threshold of anaerobic metabolism (W _AT = 0.5 MMR units), increased linearly with a further increase in the exercise relative intensity up to the level of the exhaustion intensity (W _ex = 4.7 MMR units). A sharp increase in the rate of an alactic anaerobic process was found at the relative intensity values of 2.5 MMR units, and this increase grew linearly up to values of the maximal anaerobic intensity (W _max = 9.5 MMR units).     

Environmental and functional limits to muscular exercise and body size in marine invertebrate athletes

Many similarities exist between the key characteristics of muscular metabolism in marine invertebrates and those found in vertebrate striated muscle, even though there are important phosphagens and glycolytic end products that differ between groups. Lifestyles and modes of locomotion also vary extremely among invertebrates thereby shaping the pattern of exercise metabolism. In accordance with the limited availability of integrated ecological and physiological information the present paper reports recent progress in the exercise physiology of cephalopods, which are characterized by high rates of aerobic and anaerobic energy turnover during high velocity hunts or escapes in their pelagic environment, and a sipunculid worm, which mostly uses anaerobic resources during extended marathon-like digging excursions in the hypoxic marine sediment. Particular attention is paid to how lifestyle and oxygen availability in various marine environments shapes the use and rates of aerobic and anaerobic metabolism and acidosis as they depend on activity levels and energy saving strategies. Whereas aerobic scope and, accordingly, use of ambient oxygen by blood oxygen transport and skin respiration is maximized in some squids, aerobic scope is very small in the worm and anaerobic metabolism readily used upon muscular activity. Until recently, it was widely accepted that the glycolytic end product octopine, produced in the musculature of these invertebrates, acted as a weak acid and so did not compromise acid-base balance. However, it has now been demonstrated that octopine does cause acidosis. Concomitant study of tissue energy and acid-base status allows to evaluate the contribution of glycolysis, pH and free ADP accumulation to the use of the phosphagen and to the delayed drop in the Gibb's free energy change of ATP hydrolysis. The analysis reveals species specific capacities of these mechanisms to support exercise beyond the anaerobic threshold. During high intensity anaerobic exercise observed in squid, the levels of ATP free energy change finally fall to critical minimum levels contributing to fatigue. Maintenance of sufficiently high energy levels is found at low but extended rates of anaerobic metabolism as observed in the long term digging sipunculid worm. The greatest aerobic and anaerobic performance levels are seen in squid inhabiting the open ocean and appear to be made possible by the uniform and stable physicochemical parameters (esp. high O(2) and low CO(2) levels) that characterize such an environment. It is suggested that at least some squid operate at their functional and environmental limits. In extremely different environments, both the worm and the squids display a tradeoff between oxygen availability, temperature, performance level and also, body size.     

The cDNA sequence of three hemocyanin subunits from the garden snail Helix lucorum

Hemocyanins are blue copper containing respiratory proteins residing in the hemolymph of many molluscs and arthropods. They can have different molecular masses and quaternary structures. Moreover, several molluscan hemocyanins are isolated with one, two or three isoforms occurring as decameric, didecameric, multidecameric or tubule aggregates. We could recently isolate three different hemocyanin isopolypeptides from the hemolymph of the garden snail Helix lucorum (HlH). These three structural subunits were named α_D-HlH, α_N-HlH and β-HlH. We have cloned and sequenced their cDNA which is the first result ever reported for three isoforms of a molluscan hemocyanin. Whereas the complete gene sequence of α_D-HlH and β-HlH was obtained, including the 5′ and 3′ UTR, 180 bp of the 5′ end and around 900 bp at the 3′ end are missing for the third subunit. The subunits α_D-HlH and β-HlH comprise a signal sequence of 19 amino acids plus a polypeptide of 3409 and 3414 amino acids, respectively. We could determine 3031 residues of the α_N-HLH subunit. Sequence comparison with other molluscan hemocyanins shows that α_D-HlH is more related to Aplysia californicum hemocyanin than to each of its own isopolypeptides. The structural subunits comprise 8 different functional units (FUs: a, b, c, d, e, f, g, h) and each functional unit possesses a highly conserved copper-A and copper-B site for reversible oxygen binding. Potential N-glycosylation sites are present in all three structural subunits. We confirmed that all three different isoforms are effectively produced and secreted in the hemolymph of H. lucorum by analyzing a tryptic digest of the purified native hemocyanin by MALDI-TOF and LC-FTICR mass spectrometry.     

Metabolic Acclimation to Anoxia Induced by Low (2-4 kPa Partial Pressure) Oxygen Pretreatment (Hypoxia) in Root Tips of Zea mays

Young intact plants of maize (Zea mays L. cv INRA 508) were exposed to 2 to 4 kilopascals partial pressure oxygen (hypoxic pretreatment) for 18 hours before excision of the 5 millimeter root apex and treatment with strictly anaerobic conditions (anoxia). Hypoxic acclimation gave rise to larger amounts of ATP, to larger ATP/ADP and adenylate energy charge ratios, and to higher rates of ethanol production when excised root tips were subsequently made anaerobic, compared with root tips transferred directly from aerobic to anaerobic media. Improved energy metabolism following hypoxic pretreatment was associated with increased activity of alcohol dehydrogenase (ADH), and induction of ADH-2 isozymes. Roots of Adh1⁻ mutant plants lacked constitutive ADH and only slowly produced ethanol when made anaerobic. Those that were hypoxically pretreated acclimated to anoxia with induction of ADH2 and a higher energy metabolism, and a rate of ethanol production comparable to that of nonmutants. All these responses were insensitive to the presence or absence of NO₃⁻. Additionally, the rate of ethanol production was about 50 times greater than the rate of reduction of NO₃⁻ to NO₂⁻. These results indicate that nitrate reductase does not compete effectively with ADH for NADH, or contribute to energy metabolism during anaerobic respiration in this tissue through nitrate reduction. Unacclimated root tips of wild type and Adhl⁻ mutants appeared not to survive more than 8 to 9 hours in strict anoxia; when hypoxically pretreated they tolerated periods under anoxia in excess of 22 hours.     

The effects of 5-hydroxytryptamine on the isolated and intact circulatory system of the pinto abalone,Haliotis kamtschatkana,and its presence in cardiac and non-cardiac tissues

1. The in vivo and in vitro effects of 5-hydroxytryptamine (5-HT) on the circulatory system of the pinto abalone, Haliotis kamtschatkana, were examined.2. In the isolated heart, 5-HT caused a dose-dependent increase in beat frequency.3. In the intact animal, however, this effect was accompanied by increases in aortic systolic, diastolic, and pulse pressures and, at very high doses, efferent ctenidial pressure.4. The effects of 5-HT were inhibited by several antagonists.5. Furthermore, high levels of 5-HT were found in the tissue samples taken from the auricles, and ventricle and in the hemolymph, and pericardial fluid.6. Therefore, 5-HT may have a role as an excitatory neurotransmitter in the control of circulation in H. kamtschatkana.