A membrane-bound hemoglobin from gills of the green shore crab Carcinus maenas

Most hemoglobins serve for the transport or storage of O(2). Although hemoglobins are widespread in "entomostracan" Crustacea, malacostracans harbor the copper-containing hemocyanin in their hemolymph. Usually, only one type of respiratory protein occurs within a single species. Here, we report the identification of a hemoglobin of the shore crab Carcinus maenas (Malacostraca, Brachyura). In contrast to the dodecameric hemocyanin of this species, C. maenas hemoglobin does not reside in the hemolymph but is restricted to the gills. Immunofluorescence studies and cell fractioning showed that C. maenas hemoglobin resides in the membrane of the chief cells of the gill. To the best of our knowledge, this is the first time that a membrane-bound hemoglobin has been identified in eukaryotes. Bioinformatic evaluation suggests that C. maenas hemoglobin is anchored in the membrane by N-myristoylation. Recombinant C. maenas hemoglobin has a hexacoordinate binding scheme at the Fe(2+) and an oxygen affinity of P(50) = 0.5 Torr. A rapid autoxidation rate precludes a function as oxygen carrier. We rather speculate that, analogous to prokaryotic membrane-globins, C. maenas hemoglobin carries out enzymatic functions to protect the lipids in cell membrane from reactive oxygen species. Sequence comparisons and phylogenetic studies suggested that the ancestral arthropod hemoglobin was most likely an N-myristoylated protein that did not have an O(2) supply function. True respiratory hemoglobins of arthropods, however, evolved independently in chironomid midges and branchiopod crustaceans.     

Larval development of Brachynotus gemmellari (Rizza, 1839) (Brachyura, Grapsidae) reared under laboratory conditions

The complete larval development of the grapsid crab Brachynorusgemmellari (Rizza, 1839) was obtained by culture in the Iabomtoiy.Five zoeal stages, the megalopa and the first crab stage aredescribed and illustrated. Larval development from hatchingto first crab took 26 days at 20C. The morphological charaeiarsof the larvae of B.gemmellari are compared with those of otherknown larvae of the genus Brachynosus.     

The influence of insect juvenile hormone agonists on metamorphosis and reproduction in estuarine crustaceans

Comparative developmental and reproductive studies were performedon several species of estuarine crustaceans in response to threejuvenile hormone agonists (pyriproxyfen, methoprene and fenoxycarb).Larval development of the grass shrimp, Palaemonetes pugio,was greater than two orders of magnitude more sensitive to disruptionby methoprene and fenoxycarb than was embryonic development.Developing larvae of the mud crab, Rhithropanopeus harrisii,exhibited reduced metamorphic success at lower concentrationsof methoprene and pyriproxyfen than grass shrimp larvae. Theseresponses suggest that the more rigidly controlled metamorphicprocess in crabs is more sensitive to compounds acting as endocrinedisruptors than is the more flexible metamorphic pattern inshrimp. The final crab larval stage, the megalopa, was moresensitive to methoprene and fenoxycarb exposure than earlierzoeal stages. Mud crab larvae exposed to fenoxycarb had reducedbiomass and lipid content, particularly triglycerides and sterols.Concentrations of fenoxycarb which reduced the reproductivecapacity in single life-cycle exposures of the estuarine mysid,Americamysis bahia, were similar to those concentrations whichinhibited metamorphosis in grass shrimp. Juvenile mysids releasedby exposed adults and reared through maturation without furtherexposure produced fewer young and had altered sex ratios (lowerpercentages of males) at lower parental-exposure concentrationsthan directly affected parental reproduction. These transgenerationalresponses may well be a product of irreversible effects duringdevelopmental exposures which become apparent following maturationand initiation of reproduction. These findings support usinga functional approach as an appropriate screening procedureto evaluate potential environmental endocrine-disrupting chemicalsin aquatic environments.     

Structure of arthropod hemocyanin

Hemocyanins are large multi-subunit copper proteins that transport oxygen in many arthropods and molluscs. The amino acid sequence of subunit a of Panulirus interruptus hemocyanin (657 residues) has been completed and fitted to the electron-density map (3.2 Å resolution). Comparison of amino acid sequence data for several different hemocyanin subunits of arthropod species indicated that the general features of the polypeptide architecture as found in spiny lobster hemocyanin occur in all arthropods. This structure must therefore be at least as old as the estimated time of divergence of crustaceans and chelicerates, 540–600 million years ago.     

Hemocyanins and the immune response: defense against the dark arts

The innate immune response is a conserved trait shared by invertebratesand vertebrates. In crustaceans, circulating hemocytes playsignificant roles in the immune response, including the releaseof prophenoloxidases. Activated phenoloxidase (tyrosinase) participatesin encapsulation and melanization of foreign organisms as wellas sclerotization of the new exoskeleton after wound-repairor molting. Hemocyanin functions as a phenoloxidase under certainconditions and thus also participates in the immune responseand molting. The relative contributions of hemocyte phenoloxidaseand hemocyanin in the physiological ratio at which they occurin hemolymph have been investigated in the crab Cancer magister.Differences in activity, substrate affinity, and catalytic abilitybetween the two enzymes indicate that hemocytes are the predominantsource of phenoloxidase activity in crabs. In contrast, hemocyaninis the primary source of phenoloxidase activity in isopods andchelicerates whose hemocytes show no phenoloxidase activity.Quantitative PCR studies on the distribution of prophenoloxidasemRNA in the tissues of Carcinus maenas showed little effectrelative to salinity stress. Phylogenetic analysis of hemocyanin,phenoloxidase, and other members of this arthropod gene familyare consistent with the possibility that a common ancestralmolecule had both phenoloxidase and oxygen-binding capabilities.     

Osmoregulation by Gills of Euryhaline Crabs: Molecular Analysis of Transporters

The physiological mechanisms by which aquatic animals regulatethe osmoconcentration of their body fluids remain unclear despitemany excellent studies of tissue and cell function. This reviewsummarizes the current status of an ongoing molecular biologicalapproach to investigating transporters and transport-relatedenzymes in ion-transporting gills of osmoregulating crustaceans.We have identified cDNAs coding for six candidate proteins ingills of the blue crab Callinectes sapidus and the green shorecrab Carcinus maenas, including a Na⁺ + K⁺-ATPase     

Ontogeny of hemocyanin in the ovoviviparous cockroach Blaptica dubia suggests an embryo-specific role in oxygen supply

For a long time it had been assumed that specific oxygen transport proteins are absent in insects. Only recently it has been demonstrated that hemocyanins occur in the hemolymph of many ametabolous and hemimetabolous insect taxa, but not in the Eumetabola (Hemiptera + Holometabola). Therefore, the loss of respiratory hemocyanin in insects is not correlated with the evolution of an efficient tracheal system. The specific contribution of hemocyanin to oxygen supply in insects, however, has remained uncertain. Here we investigate the stage-specific expression of hemocyanin in the ovoviviparous cockroach Blaptica dubia (Blattaria), which consists of two distinct subunit types (Hc1 and Hc2). Employing quantitative real-time RT-PCR and Western blotting, we showed that the expression of hemocyanin is restricted to late embryos, thus being detectable also in whole female extracts and oothecae. Hemocyanin protein is also present in 1st instar nymphs, but not in later developmental stages. The ontogeny of hemocyanin in cockroaches is distinct from that known from Zygentoma and Plecoptera, in which hemocyanin occurs in both nymphal and adult stages. Our findings suggest a specific role of hemocyanin in embryonic cockroaches, which may be related to an enhanced oxygen supply in the oothecae. For some reason, the fundamental physiological changes associated to the evolution of holometaboly have made hemocyanin unnecessary.     

Hemolymph Proteins and Molting in Crustaceans and Insects

The exoskeleton of crustaceans and insects is formed by cellsof the hypodermis, but several hemolymph proteins contributeto the synthesis of the new exoskeleton. These hemolymph proteinsshare a surprising degree of sequence similarity and are membersof the hemocyanin gene family. Copper-containing prophenoloxidasesof crustaceans and insects are directly involved in cross-linkingand hardening of the exoskeleton during molting and repair.Crustacean cryptocyanin and insect hexamerins lack copper andhave probably evolved from a copper-free product of an earlyhemocyanin gene duplication. These proteins have been implicatedin transport of hormones and phenols, and may be used directlyas structural components of the new exoskeleton. They are synthesizedelsewhere in the body, transported in the hemolymph, and probablytaken up by the hypodermis via specific receptors. Hemocyaninshave some residual phenoloxidase activity, in addition to theirprimary role of supplying oxygen to the metabolizing tissues.Thus multiple members of the hemocyanin gene family play vitalroles during molting, and a molecular phytogeny of these proteinswill contribute to our understanding of the evolution of formand function of these molecules from oxygen transport to molt-relatedactivities. Further studies on the expression of prophenoloxidase,cryptocyanin, hexamerins and hemocyanin, potential marker proteins,may extend our understanding of the relationship between othermolting animals in the proposed clade, Ecdysozoa.     

Evolution of the arthropod prophenoloxidase/hexamerin protein family

 Phylogenetic analysis of the prophenoloxidase/hexamerin family of arthropods revealed four well supported subfamilies: (1) the arylphorin subfamily, including arylphorins, storage proteins, and other proteins of uncertain function from insects; (2) the hemocyanins of branchiopod crustaceans, which are copper-binding proteins involved in oxygen transport; (3) the hemocyanins of chelicerates; and (4) the prophenoloxidases (proPO) of both insects and branchiopods, which are copper-binding molecules that play a role in sclerotization of cuticle and encapsulation of foreign particles. The phylogeny indicated that insect and branchiopod proPO constitute a monophyletic group but that branchiopod and chelicerate hemocyanins do not constitute a monophyletic group. Branchiopod hemocyanin and proPO diverged from each other prior to the divergence of insects from branchiopods and probably prior to the divergence of chelicerates from the insect-branchiopod lineage. Likewise, the insect arylphorin subfamily diverged from proPO prior to the divergence of insects from branchiopods and probably prior to the divergence of chelicerates; thus, the results did not support the hypothesis that insect arylphorins represent hemocyanins freed to assume a new function because the insect tracheal respiratory system removes the need for an oxygen-transport molecule. Nonetheless, reconstruction of ancestral sequences by the maximum parsimony method suggested that the ancestors of the arylphorin family were copper-binding. Regions corresponding to the copper-binding domains were found to have a faster rate of nonsynonymous evolution in arylphorin subfamily genes than in other hexamerin family genes; this presumably reflects a relaxation of purifying selection after the loss of copper-binding function. Received: 25 March 1998 / Revised: 3 July 1998     

Occurrence of Hemocyanin in Ostracod Crustaceans

Hemocyanin is a copper-containing protein that transports O₂ in the hemolymph of many arthropod species. Within the crustaceans, hemocyanin appeared to be restricted to Malacostraca but has recently been identified in Remipedia. Here, we report the occurrence of hemocyanin in ostracods, indicating that this respiratory protein is more widespread within crustaceans than previously thought. By analyses of expressed sequence tags and by RT-PCR, we obtained four full length and nine partial hemocyanin sequences from six of ten investigated ostracod species. Hemocyanin was identified in Myodocopida (Actinoseta jonesi, Cypridininae sp., Euphilomedes morini, Skogsbergia lerneri, Vargula tsujii) and Platycopida (Cytherelloidea californica) but not in Podocopida. We found no evidence for the presence of hemoglobin in any of these ostracod species. Like in other arthropods, we identified multiple hemocyanin subunits (up to six) to occur in a single ostracod species. Bayesian phylogenetic analyses showed that ostracod hemocyanin subunit diversity evolved independently from that of other crustaceans. Ostracod hemocyanin subunits were found paraphyletic, with myodocopid and platycopid subunits forming distinct clades within those of the crustaceans. This pattern suggests that ostracod hemocyanins originated from distinct subunits in the pancrustacean stemline.     

Complete larval and early juvenile development of the mangrove crab Perisesarma fasciatum (Crustacea: Brachyura: Sesarmidae) from Singapore, with a larval comparison of Parasesarma and Perisesarma

The complete larval development of the sesarmid crab Perisesarmafasciatum (Lanchester, 1900) from Singapore was obtained fromlaboratory culture. All four zoeal stages, the megalopa andthe first crab stage are described and illustrated. The morphologicalcharacteristics of the larvae of P. fasciatum are compared withthose of other known larvae of the genera Perisesarma and Parasesarma.The larval morphology of P. fasciatum clearly presents the typicalcombination of features that characterize sesarmid larvae. Overall,larval stages are very similar in Perisesarma and Parasesarmaand it is impossible to distinguish these two genera by larvalmorphology.     

Marine invertebrates of the Sea of Okhotsk as a new source of lypopolysaccharide-binding proteins

Invertebrates from the Sea of Okhotsk were studied as a source of proteins that are capable of binding to lipopolysaccharides (LPSs) of gram-negative bacteria. A DOT analysis using dansyl-labeled LPS that we developed revealed LPS-binding proteins in blood-cell lysates of 21 out of 33 investigated species of invertebrates. Most of the investigated species with positive LPS-binding activity were decapod crustaceans (class Malacostraca, phylum Arthropoda). Hemocyte lysates from the red king crab Paralithodes camtschaticus and the sculptured shrimp Sclerocrangon boreas contained several LPS-binding proteins with different molecular weights. LPS-binding proteins were found for the first time in echinoderms (classes Holothuroidea, Asteroidea, and Echinoidea), sipunculans, and brachiopods.     

Putative phenoloxidases in the tunicate<Emphasis Type="Italic"> Ciona intestinalis</Emphasis> and the origin of the arthropod hemocyanin superfamily

In addition to the respiratory copper-containing proteins for which it is named, the arthropod hemocyanin superfamily also includes phenoloxidases and various copperless storage proteins (pseudo-hemocyanins, hexamerins and hexamerin receptors). It had long been assumed that these proteins are restricted to the arthropod phylum. However, in their analysis of the predicted genes in the Ciona intestinalis (Urochordata:Tunicata) genome, Dehal et al. (Science 298:2157–2167) proposed that the sea squirt lacks hemoglobin but uses hemocyanin for oxygen transport. While there are, nevertheless, four hemoglobin genes present in Ciona, we have identified and cloned two cDNA sequences from Ciona that in fact belong to the arthropod hemocyanin superfamily. They encode for proteins of 794 and 775 amino acids, respectively. The amino acids required for oxygen binding and other structural important residues are conserved in these hemocyanin-like proteins. However, phylogenetic analyses and mRNA expression data suggest that the Ciona hemocyanin-like proteins rather act as phenoloxidases, possibly involved in humoral immune response. Nevertheless, the putative Ciona phenoloxidases demonstrate that the hemocyanin superfamily emerged before the Protostomia and Deuterostomia diverged and allow for the first time the unequivocal rooting of the arthropod hemocyanins and related proteins. Phylogenetic analyses using neighbor-joining and Bayesian methods show that the phenoloxidases form the most ancient branch of the arthropod proteins, supporting the idea that respiratory hemocyanins evolved from ancestors with an enzymatic function. The hemocyanins evolved in agreement with the expected phylogeny of the Arthropoda, with the Onychophora diverged first, followed by the Chelicerata and Pancrustacea. The position of the myriapod hemocyanins is not resolved.Abbreviations EST expressed sequence tags Communicated by G. Heldmaier     

Transport of Oxygen by the Blood of the Land Crab, Gecarcinus lateralis

Parameters relating to transport of oxygen were measured inthe pericardial blood and venous outflow from the first walkingleg of Gecarcinus lateralis. O₂-equilibrium curves of the hemocyaninof G. lateralis were found to be sigmoid and, at 27°C andpH 7.45, to have a half-saturation pressure of about 17 mm Hgoxygen. Average partial pressures of oxygen as measured by O₂-electrodewere 32 mm Hg in pericardial blood and 9 mm Hg in the venoussamples. Analysis of the O₂-content in corresponding samplesby the Van Slyke technique revealed an average of 2.17 volumes% O₂-capacity for whole blood, 1.45 volumes % for pericardialblood, and 0.61 volumes % for venous blood. Estimates basedon the Van Slyke analyses indicated an average pO₂ of 29 and14 mm Hg in pericardial and venous samples, respectively. Thesefigures agree fairly well with those obtained by means of O₂-electrodes.Of the oxygen carried to the tissues, about 94% is carried asoxyhemocyanin and about 6% is carried in physical solution.As the blood passes through the gills, the hemocyanin, on anaverage, becomes 80–85% saturated with oxygen and returnedfrom the tissues 18–45% saturated with oxygen. These resultsindicate that the blood of G. lateralis has a higher O₂-capacitythan the blood of most other decapod crustaceans for which similarinformation is available. In addition, the blood of G. lateralistransports more oxygen to the tissues per unit volume than doother crustacean bloods.     

Larval stages of Brachynotus atlanticus Forest, 1957 (Crustacea: Decapoda: Grapsidae) reared under laboratory conditions

The complete larval development of the Eastern Atlantic grapsidcrab, Brachynotus atlanticus, was obtained in the laboratory.Five zoeal stages, the megalopa and the first crab stage aredescribed and illustrated. Under laboratory conditions at 23°Cthe first crab appeared on the 25th day. This is the first speciesof the genus for which the complete larval development is known.Larval features are compared with those of other members ofthe subfamily Varuninae.