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The cuttlefish Sepia officinalis expresses several hemocyanin isoforms with potentially different pH optima, indicating their reliance on efficient pH regulation in the blood. Ongoing ocean warming and acidification could influence the oxygen-binding properties of respiratory pigments in ectothermic marine invertebrates. This study examined whether S. officinalis differentially expresses individual hemocyanin isoforms to maintain optimal oxygen transport during development and acclimation to elevated seawater pCO(2) and temperature. Using quantitative PCR, we measured relative mRNA expression levels of three different hemocyanin isoforms in several ontogenetic stages (embryos, hatchlings, juveniles, and adults), under different temperatures and elevated seawater pCO(2). Our results indicate moderately altered hemocyanin expression in all embryonic stages acclimated to higher pCO(2), while hemocyanin expression in hatchlings and juveniles remained unaffected. During the course of development, total hemocyanin expression increased independently of pCO(2) or thermal acclimation status. Expression of isoform 3 is reported for the first time in a cephalopod in this study and was found to be generally low but highest in the embryonic stages (0.2% of total expression). Despite variable hemocyanin expression, hemolymph total protein concentrations remained constant in the experimental groups. Our data provide first evidence that ontogeny has a stronger influence on hemocyanin isoform expression than the environmental conditions chosen, and they suggest that hemocyanin protein abundance in response to thermal acclimation is regulated by post-transcriptional/translational rather than by transcriptional modifications., (2012 Wiley Periodicals, Inc)

In the estuarine amphipod Chaetogammarus marinus, differences in O(2) binding by haemocyanins (Hc) could be related to natural and salinity-related quantitative variation in just one polypeptide subunit (band 2) and not to variations in any of the other seven bands present. Band 2 was always present, irrespective of salinity treatment, and naturally makes up 6%-36% of the total Hc. However, low salinity exposure (S=4 per thousand for 48-49 h, T=15 degrees C) was accompanied by an increase in the prevalence of greater concentrations of band 2 (i.e., range, 20%-36% of total Hc present) and a concomitant increase in Hc-O(2) affinity (half saturation [P(50)] decreased from 1.38 to 1.12 kPa at pH=7.81, T=15 degrees C). A similar salinity-related mechanism (that is, one band altering) has been shown previously for the blue crab Callinectes sapidus, although the functional consequences were different. In contrast with C. marinus, an increase in the proportion of one polypeptide subunit in C. sapidus resulted in a decrease in Hc-O(2) affinity. This study has confirmed that between-individual variation (quantitative rather than qualitative) in just one Hc subunit may have functional consequences, although the significance of such variation is difficult to interpret.

Oxygen-transport proteins are multisubunit, circulating molecules that provide an efficient supply of oxygen to metabolically active metazoans. Hemoglobins, hemerythrins and hemocyanins have evolved in both structural and functional diversity and exhibit functional repertoires beyond that of simple, monomeric tissue myoglobins. Their phylogenetic distribution is intriguing, especially with respect to those organisms that express more than one type of oxygen-transport protein. An animal can modify the delivery of oxygen to its tissues by varying the rate of synthesis of these proteins or by selective expression of individual subunits and/or molecules. Changes in levels of allosteric modifiers that affect the protein's oxygenation properties will also modify oxygen delivery; some organisms have more ability than others to control concentrations of modulators. Hemoglobins have assumed functions in addition to oxygen transport, while hemocyanins have diversified through multiple gene duplications and functional specializations. Understanding the mechanisms of regulation of expression, synthesis and modulator levels is a key focus of current investigations.

The dependency of the Christmas Island blue crab, Cardisoma hirtipes, on fresh water for respiratory gas exchange and transport was investigated in laboratory simulations. The gas exchange rates of air-breathing C. hirtipes were similar to those of other land crabs but decreased to 20% in submerged crabs. Crabs with access to air maintained arterial and pulmonary O2 content (CO2) and partial pressure (PO2), while in submerged crabs the PO2 and CO2 rapidly decreased (by 50%). There was no anaerobiosis, but haemolymph glucose concentration and cardiac output decreased when crabs were submersed, which suggests a hypometabolic state. Submersion induced a metabolic rather than a respiratory alkalosis, and since respiratory gas exchange was low, CO2 excretion to water was unimportant. Cardisoma hirtipes haemocyanin (Hc) has high O2 affinity but low pH sensitivity, which facilitates O2 uptake from hypoxic environments. The high Hc-O2 affinity supports O2 loading but may prevent access to a venous O2 reserve. Calcium, magnesium, and urate, but not L-lactate, were effectors of Hc-O2 affinity. In submerged crabs increased circulating urate maintained haemolymph O2 content. The CO2 capacitance and nonbicarbonate buffering of the haemolymph were relatively low. A significant Haldane effect seemed important for CO2 excretion but would require CO2 and O2 exchange to occur at the same organ (gills or lungs). Submersion interferes with respiration and is not needed for haemolymph acid-base balance; thus; C. hirtipes is an air-breathing crab.

The oxygen affinity of the hemocyanin of the crab Carcinus maenas was studied in vitro by measuring the O2 partial pressure at 50% saturation, P50, at various conditions of pH, temperature and salt concentration. At alkaline pH, the hemocyanin exhibits a normal Bohr effect, inverted below pH=pH0=6.9 at 15 degrees C. A rise of temperature provokes an increase of both P50 and pH0. Within the physiological range, only Ca++ and Mg++ ions influence the oxygen affinity. Both ions decrease P50, and Mg++ increases the Bohr factor, deltalogP50/deltapH. The variations of P50 due to changes of pH, temperature (degrees C) and Ca++ and Mg++ concentrations (mEq-L-1) are satisfactorily described by the empirical quantitative expressions: see article. Long-term temperature adaptation of the animals results in changes of the parameter k' which appears to be dependent on the concentration of an unidentified dialysable substance in blood. As temperature rises, this phenomenon limits the in vivo increase of P50 induced by both the intrinsic temperature effect and the decrease of in vivo blood pH. When external salinity decreases, lower blood Ca++ and Mg++ concentrations entail an increase of P50, but without changes of k', an effect compensated for in vivo by an increase of the blood pH. The observations suggest that the functional oxygen affinity of the pigment is physiologically regulated to ensure an adequate oxygen transport by the blood in quite varied conditions of temperature and salinity.