Haemolymph Copper Research Articles

Impact of metal accumulation pattern on the annual rhythmicity of antioxidants and their interrelationship to maintain the oxidative balance in mollusc

A chronological relationship between the annual profiles of oxidative stress markers, the key regulator of stress physiology has been sought in a terrestrial mollusc (Nerita articulata) under natural photothermal conditions. The hemolymph samples were collected at two different times in each month (from January to December) and the same was repeated for two consecutive years throughout an annual cycle. The fluctuations in the concentrations of certain heavy and trace metals (zinc, copper, cadmium, mercury, lead, and nickel) in both soil and hemolymph of Nerita are also estimated accordingly. Therefore, the present study aims to explore the rhythmic responses of oxidative stress marker to assess the impact of different trace and heavy metals on selected mollusc species. We tries to develop a realistic conceptual idea to analyze and predict the effect of changing environmental pollution on the possible shift in the rhythmicity of aforesaid antioxidants in terrestrial mollusc and their adaptive responses to thrive in such environment. Our results indicates that the amplitude of circannual rhythms of all the selected stress markers varied accordingly but the pattern of annual fluctuation is noted to be similar, and correlated with the metal accumulation. Therefore current information might help to frame the adaptive strategies for invertebrate species under similar toxic circumstances.

Keyhole limpet hemocyanin induces innate immunity via Syk and Erk phosphorylation.

Hemocyanin is an extracellular respiratory protein containing copper in hemolymph of invertebrates, such as Mollusk and Arthropod. Keyhole limpet hemocyanin (KLH) is one of hemocyanins and has many years of experience for vaccine developments and immunological studies in mammals including human. However, the association between KLH and the immune systems, especially the innate immune systems, remains poorly understood. The aim of this study is to clarify the direct effects of KLH on the innate immune systems. KLH activated an inflammation-related transcription factor NF-κB as much as lipopolysaccharide (LPS) in a human monocytic leukemia THP-1 reporter cell line. We have found that the KLH-induced NF-κB activation is partially involved in a spleen tyrosine kinase (Syk) pathway. We have also successfully revealed that an extracellular signal-regulated kinase (Erk), a member of mitogen-activated protein kinases, is located in an upstream of NF-κB activation induced by KLH. Furthermore, a Syk phosphorylation inhibitor partially suppressed the Erk activation in KLH-stimulated THP-1. These results suggest that both Syk and Erk associate with the KLH-induced NF-κB activation in the human monocyte.

Is Copper Supplementation Required in Formulated Feeds forOctopus vulgaris(Cuvier, 1797)?

The aim of this study was to test the effect of copper supplementation in a formulated feed on the growth, feed efficiency, and nutritional composition of subadults Octopus vulgaris, with particular reference to the differences in the content of copper in the tissues (muscle, digestive gland, and hemolymph). Two formulated feeds were supplied to subadults octopuses kept individually for 56 days: Basal diet [11.6 mgCu/kg dry weight (dw)], and another feed with a similar composition but including a copper supplementation (copper diet: 115.2 mgCu/kg dw). No significant differences (P > 0.05) were detected in the respective growth rates (0.88% body weight/day), FE (79.5%–82.1%), proximate composition, hemolymph hemocyanin (1.6–1.8 mmol/L), hemolymph copper (0.19–0.20 mgCu/mL), or muscle copper levels (22.7–23.2 mgCu/kg dw). No significant changes were detected on proximate composition, hemocyanin and hemolymph, and muscle copper levels between animals fed formulated feeds and an initial group fed natural diet based on crab and fish (P > 0.05). The only parameter which showed a significant difference (P < 0.05) was the copper level in the digestive gland: 1,797, 390, and 1,148 mgCu/kg dw for initial (natural diet), basal and copper diet, respectively (P < 0.05). Considering the experimental conditions of this study, copper supplementation is not effective in improving the performance of a formulated feed for octopus.

Dietary copper absorption and excretion in three semi-terrestrial grapsoid crabs with different levels of terrestrial adaptation

Three species of phylogenetically related semi-terrestrial crabs (Superfamily Grapsoidea — Sesarma rectum, Goniopsis cruentata and Neohelice granulata (formerly: Chasmagnathus granulatus) with different degrees of terrestriality were studied to quantify the accumulation of copper (Cu) in hemolymph, gills, hepatopancreas and antennal gland, and its excretion through the faeces. These crabs were fed for 15 days practical diets containing 0 (A), 0.5 (B), 1.0 (C), and 1.5% (D) of added CuCl 2 (corresponding to 0, 0.2, 0.5 and 0.7% of Cu 2+, respectively). The amount of food ingested was directly proportional to the degree of terrestriality: S. rectum, the most terrestrial species, ate around 2–3 times more than the other crabs, whereas G. cruentata ate 1.5–2 times more than N. granulata, the least terrestrial. The amount of Cu excreted in the feces was proportional to Cu ingestion, and was 76.8% and 64.2% higher for Sesarma fed diet D compared to G. cruentata and N. granulata, respectively. Sesarma also displayed higher Cu concentration in the haemolymph, gills and antennal glands, but not in the hepatopancreas. A detoxifying mechanism followed by elimination was probably present at this last organ, preventing Cu accumulation. More terrestrial crabs, such as Sesarma, may accumulate more Cu in hemolymph and tissues, showing a correlation between metal accumulation and increased terrestriality. In this aspect, contaminated feed sources with Cu may have more impact in conservation of terrestrial crabs.

Growth of tissues related to haemolymph copper throughout the moult cycle of the lobster Homarus gammarus

The relationship between tissue weights and haemolymph copper levels in field-collected European lobsters Homarus gammarus was investigated to assess whether haemolymph copper concentration can be used to reliably determine nutritional condition in lobsters. During the moult cycle, the soft tissue weight (mostly muscle) increased concomitantly with a decrease in haemolymph weight while exoskeleton weight increased from postmoult to intermoult. Haemolymph copper concentration gradually increased from postmoult to premoult with a range of 29.7 (Stage B) to 163.5 pg g -1 wet weight (ww) (Stage D 0 ). Our measurements suggest that the main processes that increase haemolymph copper concentration is a reduction in haemolymph space and an increase in haemolymph copper content, indicating synthesis of new haemocyanin for the oxygen supply of growing tissues. During the moult cycle, haemolymph copper content was proportional to soft tissue mass and amounted to 43.6 pg Cu g -1 of soft-tissue ww (95% confidence limits = 35.5 and 51.6 μg g -1 ). Models for estimating soft-tissue and haemolymph wet weights from the haemolymph copper concentration and moult stage are presented. The use of haemolymph copper concentration as an index of nutritional condition reflecting tissue growth and food quantity and quality is validated.

Trace metal concentrations and contents in the tissues of the shore crab Carcinus maenas : effects of size and tissue hydration

Water content in the tissues, tissue proportions, and content and concentrations of copper, zinc, iron, cadmium, manganese, and cobalt were determined in male shore crabs Carcinus maenas of different sizes. Average wet to dry weight ratios in midgut gland, gills, muscle, exoskeleton, stomach, heart, testes, hypodermis, and haemolymph were 5.3, 9.5, 4.1, 1.5, 4.8, 6.1, 3.8, 4.7, and 13, respectively. The water content in the soft tissues generally increased with increasing size of the crabs (except in testes). Dry midgut gland, gills, muscle, exoskeleton, stomach, heart, testes, hypodermis, and haemolymph constituted 4, 0.9, 20, 68, 0.5, 0.12, 0.5, 0.7 and 5.3%, respectively, of the body dry weight. Dry midgut gland, gills, stomach, hypodermis, and haemolymph constituted a larger proportion of the body weight in small than in large crabs, whereas the opposite was the case for exoskeleton. Protein concentrations in the haemolymph decreased with size whereas haemolymph volume increased. Seventy percent of the copper body burden was located in the haemolymph. The majority of zinc (61%) occurred in muscle, whereas the midgut gland was the major site of cadmium storage (89%). The exoskeleton contained the major part of the body burden of iron (71%) and manganese (95%). Most of the cobalt body burden was located in the exoskeleton (45%) and haemolymph (44%). As tissue hydration and tissue proportions changed with size, tissue metal concentrations did not necessarily vary identically with size when expressed on a dry weight compared to a wet weight basis. Tissue potassium concentrations were negatively correlated with tissue water contents, indicating that water in tissues with a high wet to dry weight ratio was of extracellular origin. However, comparison of tissue and haemolymph copper levels showed that this extracellular water could not be pure haemolymph as tissue copper concentrations would have been higher than the values actually found.

Changes in the tissue concentrations and contents of calcium, copper and zinc in the shore crab Carcinus maenas (L.) (Crustacea: Decapoda) during the moult cycle and following copper exposure during ecdysis

The tissue concentrations and contents of calcium, copper and zinc were determined at different stages in the moult cycle of the shore crab Carcinus maenas. The crabs were dissected into the following tissues: the midgut gland, the gill, the ‘rest tissue’ (the latter defined as the left over tissue, i.e. muscles and exoskeleton), and the haemolymph, and analysed as such. One group of crabs was maintained in clean seawater. In these crabs, calcium was lost from haemolymph during premoult and apparently stored in the midgut gland. At ecdysis approximately 90% of the whole body calcium content was lost with the exuviae while the haemolymph calcium content doubled. During early postmoult calcium stored in the midgut gland was used in combination with calcium absorbed from the surrounding seawater for a rapid calcification of the new exoskeleton. Both the copper and zinc concentrations declined in the ‘rest tissue’ compartment during premoult. At ecdysis the haemolymph copper and zinc contents increased 35 and 86%, respectively, from premoult to the newly moult stage. At the same time, however, the copper and zinc haemolymph concentrations decreased. During postmoult, copper (25%) and zinc (45%) were lost from haemolymph and mainly excreted but a minor fraction (5–10%) was stored in the midgut gland. A second group of crabs was maintained in copper-contaminated seawater (0.1 mgl −1) during ecdysis and postmoult. Following exposure of the animals to raised ambient levels of copper, the calcium and zinc contents of the midgut gland were reduced while the copper contents of the midgut gland and most other tissues were increased in comparison to crabs held in clean seawater.

Copper levels during osmotic stress in the shore crab Carcinus aestuarii

The total amount of copper in hemolymph and hepatopancreas of the shore crab Carcinus aestuarii remained unchanged when seawater-acclimated crabs (salinity 38 ppt) were exposed to diluted seawater. However, hemolymph copper, protein, and hemocyanin concentrations increased approximately 30% after 12 hr of exposure to salinities of 11 and 13 ppt. These changes were transitory, and within 96 hr the initial concentrations were restored. The hemolymph volume, determined as copper space, significantly decreased after 12 hr of exposure to diluted seawater. This change was also temporary, and the difference between the copper space of seawater-adapted crabs and crabs exposed to diluted seawater for 96 hr was not significant. When crabs acclimated to diluted seawater ( S = 11 ppt) were transferred to normal seawater, no changes in hemolymph protein and hemocyanin concentrations occurred. After hypo-osmotic stress, the blood volume decrease and the consequent increase of hemolymph copper, protein, and hemocyanin concentrations were provoked by transitory hydration of the soft tissues of the crabs.

Haemolymph protein composition and copper levels in decapod crustaceans

Variations in haemolymph protein composition and concentration, in copper content and copper distribution in the tissue of decapod crustaceans are reviewed. Haemocyanin is the major haemolymph constituent (> 60%); the remaining proteins (in order of concentration) include coagulogen, apohaemocyanin, hormones and antisomes. Moulting, nutritional state, infection, hypoxia and salinity fluctuations are the major factors affecting the relative proportions and total quantities of the haemolymph proteins. With regard to haemocyanin, the changes in concentration during the moult cycle are principally associated with changes in haemolymph volume, rather than with changes in total haemocyanin content due to synthesis or catabolism. The role of the midgut gland in regulating haemolymph copper and haemocyanin concentration has been re-evaluated. More than 50% of the whole body copper load is stored in the haemolymph. In contrast, less than 3% of the copper load resides in the midgut gland. The latter has little potential for regulating haemolymph copper levels, at least in the short term (hours to a few days), though it may be involved in regulating haemocyanin levels over longer periods (weeks to months). The total copper content of the haemolymph remains within a narrow range, except during starvation when levels may decrease. Consequently, variations in the copper content of soft tissues, which constitute only 20% of decapod dry weight, do not significanlty alter whole body copper concentrations. Evidence that copper released following haemocyanin catabolism becomes bound to metallothionein for later use in the resynthesis of haemocyanin is reviewed and found to be inconclusive. The amount of copper that can be stored in this way is trivial compared with the amount of copper required to permit significant changes in haemolymph haemocyanin concentration. Average tissue copper requirements, calculated during the present study, are approx. 4 times higher than previous theoretical estimates.

Mobilization of copper among tissues in the estuarine crabScylla serrata(Forskal) under imposed starvation

The quantitative changes in copper free and bound to proteins in haemolymph and different forms of copper in muscle and hepatopancreas under imposed starvation were studied in the estuarine mud crab Scylla serrata. During the course of starvation, both haemolymph copper free and bound to proteins significantly declined and the regression analyses of these data further revealed that the haemolymph copper-free proteins were more affected than copper-bound proteins. The multiple stress condition namely injury and exsanguination along with starvation resulted in an earlier release and/or degradation of both these proteins. Hepatopancreas periodically accumulates and releases copper during starvation. The copper levels in haemolymph and hepatopancreas during different days of starvation showed a close inverse relationship between these two tissues. These changes in hepatopancreas were predominantly reflected in the copper that exists in association with low molecular weight substances. It is found that the copper thus accumulated was partly released back into haemolymph and a fraction may be excreted. This study also indicates the major role played by the low molecular weight substances in accommodation, detoxification and mobilization of copper in the decapod hepatopancreas during imposed starvation.

Copper toxicity in the crab, Scylla serrata, copper levels in tissues and regulation after exposure to a copper-rich medium.

In the decapod crustaceans copper is distributed in various tissues. In these animals the tissue copper generally exists in four forms; ionic, bound to proteins, lipids and membrane. In the estuarine crab Scylla serrata, the haemolymph copper exists only in association with proteins, whereas in the hepatopancreas it exists in all the four forms and in gills it exists in all the forms except in combination with lipids. Although food is the major source of copper in decapod crustaceans evidence indicate that copper may be directly obtained from the environment. It was postulated earlier that in Scylla serrata the haemolymph and hepatopancreas may be involved in copper regulation. In the present work the authors have studied the nature and levels of copper in different tissues after exposing the crabs to copper-rich medium. The results indicate the relative importance of various tissues in accumulation an the possible mechanisms of regulation of the environmental copper. Besides, as a pre-requisite for studies of this kind, the toxic levels for different forms of copper were estimated since the form of toxicant is known to influence the toxicity to the decapod crustaceans.

Changes in the haemolymph protein and copper in the crabOcypoda macrocera Milne Edwards (Crustacea: Brachyura)

Wide fluctuations in the concentration of the haemolymph protein and its copper were observed during the moult cycle of the crabOcypoda macrocera. Blood protein and copper concentrations were more in the intermoult (C4) and premoult crabs (D1-D2) but decreased in the postmoult crabs (A1-B2). A direct relationship between the blood protein and copper concentration is observed during the moult cycle. More copper in the hepatopancreas is seen during the premoult stage (D1-D2) and its level increased further in the postmoult stages (A1-B2). Increase of hepatic copper with a decrease in haemolymph copper content during the post moult stages shows the transfer of copper liberated from the haemolymph to the hepatopancreas due to the breakdown of haemocyanin, the major blood protein, during starvation period of moult cycle.

Nature and distribution of copper in green lagoon crab Scylla serrata (Forskal)

In Scylla serrata (Forskal), copper is present in detectable quantities in all tissues except the central nervous system. 75% of animal's total copper is found in the haemolymph, hepatopancreas, and cuticle. Both the haemolymph and the hepatopancreas share equally ≈ 60% of the animal's total copper. Copper is found in the TCA-soluble and insoluble fractions of water-soluble tissue extracts and in the chloroform : methanol-soluble and insoluble fractions of water-insoluble residue from the hepatopancreas. No other tissue has all the fractions of copper. In haemolymph copper is present only in the TCA-insoluble fraction. The TCA-soluble fraction is also absent in muscles. The absence of any lipid-bound copper in other tissues and the presence of copper in all fractions from the hepatopancreas indicates that this tissue may play a major role in the mobilization, conservation and detoxication of copper.

Oxygen consumption in the shrimp, Palaemonetes pugio, exposed to fluctuating temperatures and food contaminated with the diaromatic petroleum hydrocarbon, dimethylnaphthalene

Oxygen consumption rates ( V̇o 2) in the grass shrimp Palaemonetes pugio were determined after a 32 day exposure to fluctuating temperatures (FT) (18–22°C) and/or dimethylnaphthalene (DMN)-contaminated food (0·24 μg DMN g wet wt −1) and again after a 16 day recovery period of stable temperatures (20°C) and uncontaminated food. Ingestion of DMN-contaminated food for 32 days resulted in elevated V̇>o 2 in shrimp exposed to declining oxygen concentrations. After the 32 day exposure period, FT had no significant effect on V̇o 2 at 15, 20 and 25°C, tissue V̇o 2 and V̇o 2 in declining oxygen. Hemolymph copper concentrations were significantly depressed in shrimp exposed to DMN-contaminated food. After the 16 day recovery period, shrimp from the FT regime exhibited depressed V̇o 2 when exposed to 25°C but not to 15°C. These depressed respiratory rates were offset by the stimulatory effect of DMN-contaminated food. These respiration studies were generally unproductive in explaining the previously reported effects of FT and DMN-contaminated food on the survival of P. pugio under hypoxic conditions.

Respiratory rate, haemolymph oxygen tension and haemocyanin level in the shrimp Palaemon adspersus: Rathke

The marine shrimp Palaemon adspersus Rathke, frequenting Zostera meadows, shows an oxygen consumption rate (MO 2) that is independent of water oxygen tension (P wO 2) as PO 2 decreases to a critical point (P cr) near 70 mm Hg. This respiratory independence is associated with maintenance of a relatively constant “arterial” (post-branchial) haemolymph tension (P aO 2) at 70–80 mm Hg. At lower P wO 2 values, both MO 2 and P aO 2 fall, reflecting, in contrast to the above independence, a direct dependence of MO 2 on internal PO 2. Haemolymph copper measurements demonstrate relatively high haemocyanin concentrations and oxygen-carrying capacities, but MO 2 levels reflect an insignificant rôle for the pigment as an oxygen store. The data are discussed as regards adaptations for aerobic metabolism.

EFFECTS OF DIMETHYLNAPHTHALENE AND FLUCTUATING TEMPERATURES ON ESTUARINE SHRIMP

ABSTRACT The biological effects of a naturally occurring perturbation were compared with those of a perturbation that may be induced by human activities in the coastal zone. The estuarine shrimp Palaemonetes pugio was exposed for 32 days to fluctuating (18 to 22°C) temperatures (FT), to food contaminated with 2,6-dimethylnaphthalene (DMN) at a concentration to wet weight food of 0.24 microgram per gram, or to both. Relative survival rates were evaluated in a matrix of environmental challenge tests. Resistance to hypoxia plus reduced salinity was significantly reduced by exposure to FT and DMN-contaminated food. The influence of FT was much stronger and obscured the effects of DMN-contaminated food when shrimp were exposed to both perturbations at the same time. The persistent, residual effects of FT and DMN-contaminated food in reducing resistance of Palaemonetes pugio to hypoxia was evaluated after a 16-day recovery period with stable temperatures and uncontaminated food. The harmful effects of FT appeared to be reduced by the period of stable temperatures. On the other hand, shrimp that had ingested DMN-contaminated food before the clean food showed increased resistance to hypoxia. A number of sublethal measurements related to aerobic metabolism were made in order to find an index that would be useful in discriminating the effects of FT from DMN-contaminated food and to determine the affected mechanism(s) that lead to altered survival in hypoxia. No measurement examined could satisfactorily explain the observed effects of FT on resistance to hypoxia, independent of diet regime. However, shrimp exposed to both FT and DMN-contaminated food had increased respiratory rates and oxygen to nitrogen rates decreased metabolic independence from external oxygen, and low hemolymph copper concentrations. Measurements that may be useful in detecting stress resulting from DMN-contaminated food, independent of temperature regime, include nitrogen excretion rates, hemolymph copper, and the relation, between oxygen consumption and external oxygen. After the shrimp had ingested contaminated food for 32 days, the concentration of DMN in their tissues was significantly greater than that in the food. Shrimp kept in FT had higher concentrations of DMN than shrimp held at stable temperatures. Feeding uncontaminated food to contaminated shirimp for 16 days resulted in a significant reduction of DMN in the tissues.

Significance of the variation in haemolymph copper-protein ratio in the crabScylla serrata (Forskal) during different hours of the day

InScylla serrata, haemolymph copper is bound to protein and it can be more reliably determined by the 2,2′ biquinoline method than by other spectrophotometric methods. Ionic or free copper is absent from the haemolymph. The lack of significant time-of-day variation in copper concentration and the occurrence of variation in total protein concentration and copper-protein ratio, indicate fluctuations in copper-free proteins, which may either be periodically sequestered or released by the tissues during different hours of the day.

The Uptake of Cadmium Into the Haemolymph of the Shore Crab Carcinus Maenas: The Relationship With Copper and Other Divalent Cations

ABSTRACT When Carcinus was exposed to 20 μ-mol 1-1 cadmium, the haemolymph cadmium level was initially dependent upon the salinity of the external medium. After 14 days the mean haemolymph cadmium level in 50% s.w. animals was nearly twice that of 100% s.w. animals. This trend was not sustained, however, and the situation was complicated by occasional inconsistent values. In both in vivo and in vitro conditions nearly all the haemolymph cadmium becomes bound to haemolymph protein within a few days. The relationship between haemolymph cadmium, copper and protein concentration has been investigated. Although the latter are highly correlated with each other, cadmium formed a significant positive relationship with haemolymph copper (r = 0·523) and protein (r = 0·533) only after 3−4 weeks uptaike. Exposure to 20 μ-mol 1-1 cadmium has no obvious effects on haemolymph protein and copper concentrations, which are clearly dependent on feeding status. Mortalities among experimental animals were often preceded by a rise in haemolymph cadmium concentration. This is usually seen before there are any obvious signs of tissue breakdown. Urine cadmium loss is probably unimportant as a pathway for the elimination of this metal. Urine cadmium concentrations often exceeded serum cadmium levels indicating that cadmium may sometimes be eliminated in bound form.

Zinc-hemocyanin binding in the hemolymph of Carcinus maenas (Crustacea, Decapoda)

1. 1. A quantitative and electrophoretic study of copper and zinc in hemolymph of the crustacea decapoda, Carcinus maenas, has been carried out. 2. 2. Most of the copper was linked with hemocyanin. 3. 3. All copper-binding proteins are hemocyanin. 4. 4. Zinc belongs to three different compartments: more than 68% is linked with hemocyanin; about 10% is shown in the fraction containing blood cells, lipids and carotenoids; 15% is present in the hemolymph under a soluble and dialysable form. 5. 5. Specific zinc-binding protein has not been found out in the serum of Carcinus maenas. 6. 6. Zinc linked with hemocyanin is found on high molecular weight protein fractions only.

Haemolymph and hepatopancreas copper in Porcellio laevis latreille (Porcellionidae, Peracarida)

1. 1. The nature of variations in copper stores of Porcellio laevis adults (tenth to thirteenth instar) has been investigated in relation to the age of the individual and under conditions of experimentally induced inanition. 2. 2. Relatively little copper was detected in tissues other than the haemolymph and the hepatopancreas in which the copper levels ranged from 9·0 to 42·1 μg/ml and 32·60–78·51 μg/g dry wt. respectively. Older individuals in general had higher copper concentrations than did the younger adults. 3. 3. Histochemical observations revealed copper granules to be localized inside the small ovoid S-cells of the hepatopancreas caecum. Wide individual variations were found in the size and the amount of copper granules and in the number of hepatopancreatic S-cells containing the copper. 4. 4. The moult-cycle, as well as successive bleeding in fully fed animals (on every tenth day for 30 days) did not affect significantly the copper contents in either the haemolymph or the hepatopancreas. However, a starvation period of up to 30 days resulted in a pronounced accumulation of copper in the hepatopancreas and a loss in haemolymph copper.