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PubMed2001

Molecular cloning and evolution of lobster hemocyanin.

In the American lobster, Homarus americanus, oxygen is transported by a hemocyanin that is composed 2 x 6 subunits. N-terminal sequencing show the presence of three distinct subunit types (alpha, beta and gamma). We cloned the cDNA of one of these subunits that belong to the alpha-type. It encodes a hemocyanin subunit of 654 amino acids with a molecular mass of 84.8 kDa, which is synthesized in the hepatopancreas. Phylogenetic analyses of the crustacean hemocyanin sequences show two well-separated clades, which correspond to the alpha and gamma-type subunits. Sequences of beta-type subunits are still unknown. The gamma-sequences have evolved about 15% faster than the alpha-subunits, consistent with the proposed conservative function of the latter. Under the assumption of a molecular clock we calculated that alpha- and gamma-subunits split about 214 +/- 14 million years ago, suggesting their divergence only in the decapod Crustacea.

Amino Acid Sequence↗
PubMed2001

Effects of implantation of hypertrophied androgenic glands on sexual characters and physiology of the reproductive system in the female red claw crayfish, Cherax quadricarinatus.

The role of the androgenic gland (AG), an organ unique to male Crustacea, in the development of sex characters and physiology of the reproductive system has not been fully documented in the red claw crayfish, Cherax quadricarinatus. To investigate the role of the AG in this species, the effect of implanting hypertrophied AGs into immature female animals was followed. Of the female animals with AG implants, 91.6% developed male-like propodi, including the red patch characteristic of males of this species. The development of female secondary sex characteristics such as a wider abdomen, a wider endopod, and simple setation was inhibited. At the end of the experiment, the ovaries of the AG-implanted females contained mostly lipid-stage oocytes, with a small number of oocytes at the early yolk stage. The gonadosomatic index of the AG-implanted females was significantly lower than that of the control (sperm duct-implanted or sham-operated) females, which had mature oocytes with a well-defined perinuclear zone and yolk globules. An immunohistochemical test using an antibody developed against a 106-kDa secondary vitellogenic polypeptide showed only slight immunoreactivity in the oocytes of AG-implanted females compared with abundant immunoreactivity in control ovaries. In the polypeptide profile of the high-density lipoprotein (HDL) from the hemolymph of AG-implanted females, the 206- and 79-kDa secondary vitellogenesis-specific polypeptides were not found, whereas they were present in the profile of control females. In contrast, the female-specific 177-kDa polypeptide was present in the polypeptide profile of hemolymph HDL of both AG-implanted females and control females. It seems therefore that while secondary sex characters were masculinized under the influence of the implanted AG, the process of vitellogenesis was suppressed but not fully eliminated in the AG-implanted females.

Androgens↗
PubMed1994

Methyl farnesoate levels in male spider crabs exhibiting active reproductive behavior.

The concentration of methyl farnesoate (MF) in the hemolymph and its synthesis by the mandibular organs (MOs) were investigated to determine whether this compound is related to the differences in the size of the reproductive system and the mating behavior among male morphotypes of the spider crab, Libinia emarginata. Large-claw abraded males displayed mating behavior under competitive conditions. They have the largest reproductive systems, their MOs synthesize large amounts of MF in vitro, and the concentration of MF in their hemolymph is high. Small-claw abraded males displayed mating behavior with receptive females only when isolated. These smaller crabs have intermediate-sized reproductive systems, their MOs synthesize the most MF, and they have the highest circulating level of MF relative to their body size. The unabraded males did not display mating behavior; their reproductive systems are smaller; their MO activity is low, as is their circulating level of MF. The strong relationship between MF levels and the intensity of reproductive behavior suggests that MF may be one of the driving forces behind mating behavior in Crustacea.

Animals↗
PubMed1996

The Role of Chirocephalus diaphanus in the Early Recognition of Environmental Pollution by Trace Elements

A study was undertaken to evaluate whether the fairy shrimp Chirocephalus diaphanus (Crustacea: Anostraca) could be considered as a species capable of biomonitoring environmental contamination by accumulating trace elements. This preliminary investigation aimed at assessing the levels of As, Cd, Cu, Hg, Pb, Se, and Zn in natural waters as well as in adults and cysts of fairy shrimps sampled from five different sites in central Italy. Trace elements were determined by inductively coupled plasma (ICP)-based spectrometry. In particular, ICP-mass spectrometry (ICP-MS) was resorted to in most cases due to its high detection power. Pretreatment of samples (adults and cysts) consisted basically in the HNO3-assisted microwave digestion of the material. The highest levels of trace elements were found in the anostracan cysts, with concentration ranges (μg/g) of 0.464-3.11 for As, up to 0.820 for Cd, 2.62-13.0 for Cu, 0.011-0.213 for Hg, 0.966-8.46 for Pb, 0.295-2.45 for Se, and 16.4-50.4 for Zn. On the other hand, the lowest concentrations were found in natural waters, with values close to the ICP-MS detection power for some elements. These data are probative of a bioconcentration process, the extent of which is proportional to the environmental level of each element. This may turn out to be useful in planning biological monitoring and assessing ecotoxicological consequences which might ensue from the diffusion of the exploitation of such organisms in aquaculture.

Journal Article↗
PubMed2001

One-step PCR amplification of complete arthropod mitochondrial genomes.

A new PCR primer set which enables one-step amplification of complete arthropod mitochondrial genomes was designed from two conserved 16S rDNA regions for the long PCR technique. For this purpose, partial 16S rDNAs amplified with universal primers 16SA and 16SB were newly sequenced from six representative arthropods: Armadillidium vulgare and Macrobrachium nipponense (Crustacea), Anopheles sinensis (Insecta), Lithobius forficatus and Megaphyllum sp. (Myriapoda), and Limulus polyphemus (Chelicerata). The genomic locations of two new primers, HPK16Saa and HPK16Sbb, correspond to positions 13314-13345 and 12951-12984, respectively, in the Drosophila yakuba mitochondrial genome. The usefulness of the primer set was experimentally examined and confirmed with five of the representative arthropods, except for A. vulgare, which has a linearized mitochondrial genome. With this set, therefore, we could easily and rapidly amplify complete mitochondrial genomes with small amounts of arthropod DNA. Although the primers suggested here were examined only with arthropod groups, a possibility of successful application to other invertebrates is very high, since the high degree of sequence conservation is shown on the primer sites in other invertebrates. Thus, this primer set can serve various research fields, such as molecular evolution, population genetics, and molecular phylogenetics based on DNA sequences, RFLP, and gene rearrangement of mitochondrial genomes in arthropods and other invertebrates.

Animals↗
PubMed2001

Elongation factor-2: a useful gene for arthropod phylogenetics.

Robust resolution of controversial higher-level groupings within Arthropoda requires additional sources of characters. Toward this end, elongation factor-2 sequences (1899 nucleotides) were generated from 17 arthropod taxa (5 chelicerates, 6 crustaceans, 3 hexapods, 3 myriapods) plus an onychophoran and a tardigrade as outgroups. Likelihood and parsimony analyses of nucleotide and amino acid data sets consistently recovered Myriapoda and major chelicerate groups with high bootstrap support. Crustacea + Hexapoda (= Pancrustacea) was recovered with moderate support, whereas the conflicting group Myriapoda + Hexapoda (= Atelocerata) was never recovered and bootstrap values were always <5%. With additional nonarthropod sequences included, one indel supports monophyly of Tardigrada, Onychophora, and Arthropoda relative to molluscan, annelidan, and mammalian outgroups. New and previously published sequences from RNA polymerase II (1038 nucleotides) and elongation factor-1alpha (1092 nucleotides) were analyzed for the same taxa. A comparison of bootstrap values from the three genes analyzed separately revealed widely varying values for some clades, although there was never strong support for conflicting groups. In combined analyses, there was strong bootstrap support for the generally accepted clades Arachnida, Arthropoda, Euchelicerata, Hexapoda, and Pycnogonida, and for Chelicerata, Myriapoda, and Pancrustacea, whose monophyly is more controversial. Recovery of some additional groups was fairly robust to method of analysis but bootstrap values were not high; these included Pancrustacea + Chelicerata, Hexapoda + Cephalocarida + Remipedia, Cephalocarida + Remipedia, and Malaocostraca + Cirripedia. Atelocerata (= Myriapoda + Hexapoda) was never recovered. Elongation factor-2 is now the second protein-encoding, nuclear gene (in addition to RNA polymerase II) to support Pancrustacea over Atelocerata. Atelocerata is widely cited in morphology-based analyses, and the discrepancy between results derived from molecular and morphological data deserves greater attention.

Amino Acid Sequence↗
PubMed1994

The complete mitochondrial DNA sequence of the crustacean Artemia franciscana.

The complete mitochondrial DNA (mtDNA) sequence of the brine shrimp Artemia franciscana has been determined. It extends the present knowledge of mitochondrial genomes to the crustacean class and supplies molecular markers for future comparative studies in this large branch of the arthropod phylum. Artemia mtDNA is 15,822 nucleotides long, and when compared with its Drosophila counterpart, it shows very few gene rearrangements, merely affecting two tRNAs placed 3' downstream of the ND 2 gene. In this position a stem-loop secondary structure with characteristics similar to the vertebrate mtDNA L-strand origin of replication is found. This suggests that, associated with tRNA changes, the diversification of the mitochondrial genome from an ancestor common to crustacea and insects could be explained by errors in the mtDNA replication process. Although the gene content is the same as in most animal mtDNAs, the sizes of the protein coding genes are in some cases considerably smaller. Artemia mtDNA uses the same genetic code as found in insects, ATN and GTG are used as initiation codons, and several genes end in incomplete T or TA codons.

Animals↗
PubMed1995

Phylogenetic study of bisexual Artemia using random amplified polymorphic DNA.

Study of polymorphisms in the eukaryotic genome is an important way to discover the evolutionary relationships between species. Artemia (Crustacea, Anostraca) offers a very interesting model for evolutionary studies. In fact the genus, distributed all over the world in hundreds of known biotopes, comprises both bisexual sibling species and parthenogenetic populations easily available from the Artemia Reference Center of Ghent. In spite of great interest in it and its extensive use in aquaculture, little is known about relationships between the different species and intraspecific populations. Recently it has been demonstrated that polymorphisms in genomic fingerprints generated by arbitrarily primed polymerase chain reaction (PCR) can distinguish between strains in many organisms. We have used this technique to estimate the phylogenetic relationships existing between 14 populations living in the American continent, in the Mediterranean area, and in China. The principal coordinate analysis (PCO) obtained from 86 random amplified polymorphic DNA (RAPD) markers indicates that the populations analyzed can be divided into homogeneous clusters representing the four known bisexual species--the American A. franciscana and A. persimilis, the Mediterranean A. salina, and the A. species from China.

Animals↗
PubMed1983

Phagocytosis of rhabdomeral membrane by crab photoreceptors (Leptograpsus variegatus).

Many crabs possess fused rhabdoms which are partly broken down at dawn and re-synthesised at dusk. The cross-sectional area of the rhabdom is therefore smaller during the day than at night. The only previously described mechanism of membrane removal from the rhabdomere in Crustacea involves the formation of pinocytotic vesicles at the bases of the microvilli. The geometry of the rhabdom is such that uniform pinocytosis across the base of each rhabdomere would result in a stack of orthogonally oriented rectangles. In the process described here, microvilli from the outer edges of the rhabdomeres are engulfed by adjacent retinula cells, reducing the number as well as the length of the microvilli and maintaining the smooth longitudinal profile needed for optimal functioning of the rhabdom.

Animals↗
PubMed1977

The organisation of the lamina ganglionaris of the crabs Scylla serrata and Leptograpsus variegatus.

The gross structure and neuronal elements of the first optic ganglion of two crabs, Scylla serrata and Leptograpsus variegatus, are described on the basis of Golgi (selective silver) and reduced silver preparations. Of the eight retinula cells of each ommatidium, seven end within the lamina, while the eighth cell sends a long fibre to the external medulla. Five types on monopolar neurons are described, three types of large tangential fibres, and one fibre which may be centrifugal. The marked stratification of the lamina is produced by several features. The main synaptic region, the plexiform layer, is divided by a band of tangential fibres; the short retinula fibres end at two levels in the plexiform layer; and two types of monopolar cells have arboriasations confined to the distal or proximal parts of the plexiform layer. The information presently available concerning the retina-lamina projection in Crustacea is examined. Some of the implications of retina and lamina structure are discussed in conjunction with what is known about their electrophysiology.

Animals↗
PubMed1989

Comparative study of invertebrate actins: antigenic cross-reactivity versus sequence variability.

The immunological similarities or differences between various actins from muscles of invertebrates were investigated. We elicited antibodies to actin purified from the adductor muscle of the mollusc, Pecten maximus. The antiserum comprised a major population that was specific for the N-terminal extremity of this isoform. This antibody population was used together with other antibodies specific for various domains on the surface of skeletal muscle actin to compare seven actins from invertebrate muscles. The N-terminal extremity showed close homologies between invertebrates (except for Crustacea) but differences were observed with vertebrate skeletal and cytoplasmic actins. In contrast, sequence 18-28, which constitutes part of the myosin interaction domain, appears to be conserved in all actins studied. Other regions (sequences 40-113, 168-226 and 285-375) presented a variable behaviour depending on the particular invertebrate species.

Actins↗
PubMed1991

Highly repetitive DNA sequence in parthenogenetic Artemia.

The study of the structural organization of the eukaryotic genome is one of the most important tools for disclosing the evolutionary relationships between species. Artemia (Crustacea, Phyllopoda) offers a very interesting model for speciation studies. The genus, distributed all over the world, comprises both bisexual sibling species and parthenogenetic populations, exhibiting different chromosome numbers (diploidy, polyploidy, and heteroploidy). Digestion of genomic DNA of the parthenogenetic Artemia sp. from Tsing-Tao (China) with the restriction enzymes Eco RI and Alu I reveals that a highly repetitive sequence of 133 bp is present. The Eco RI fragment has been cloned and characterized by genomic organization. The distribution of the Eco RI family of repeats was also studied in several bisexual and parthenogenetic Artemia populations and compared with an Alu I repetitive fragment previously identified in Artemia franciscana.

Animals↗
PubMed1988

An evolutionary tree for invertebrate globin sequences.

A phylogenetic tree was constructed from 245 globin amino acid sequences. Of the six plant globins, five represented the Leguminosae and one the Ulmaceae. Among the invertebrate sequences, 7 represented the phylum Annelida, 13 represented Insecta and Crustacea of the phylum Arthropoda, and 6 represented the phylum Mollusca. Of the vertebrate globins, 4 represented the Agnatha and 209 represented the Gnathostomata. A common alignment was achieved for the 245 sequences using the parsimony principle, and a matrix of minimum mutational distances was constructed. The most parsimonious phylogenetic tree, i.e., the one having the lowest number of nucleotide substitutions that cause amino acid replacements, was obtained employing clustering and branch-swapping algorithms. Based on the available fossil record, the earliest split in the ancestral metazoan lineage was placed at 680 million years before present (Myr BP), the origin of vertebrates was placed at 510 Myr BP, and the separation of the Chondrichthyes and the Osteichthyes was placed at 425 Myr BP. Local "molecular clock" calculations were used to date the branch points on the descending branches of the various lineages within the plant and invertebrate portions of the tree. The tree divided the 245 sequences into five distinct clades that corresponded exactly to the five groups plants, annelids, arthropods, molluscs, and vertebrates. Furthermore, the maximum parsimony tree, in contrast to the unweighted pair group and distance Wagner trees, was consistent with the available fossil record and supported the hypotheses that the primitive hemoglobin of metazoans was monomeric and that the multisubunit extracellular hemoglobins found among the Annelida and the Arthropoda represent independently derived states.

Amino Acid Sequence↗
PubMed1991

Cloning and characterization of Daphnia mitochondrial DNA.

The mitochondrial genome of Daphnia pulex (Crustacea, Cladocera) was cloned as a single fragment into the plasmid vector pUC12. The genome size, estimated from restriction endonuclease fragment lengths, is 15,400 +/- 200 base pairs. The GC content, estimated from thermal denaturation studies, is 42%. The positions of 39 cleavage sites were mapped for 14 restriction enzymes. The distribution of these sites within the genome is random (P = 0.44). Heterologous hybridizations with Drosophila sylvestris mitochondrial DNA (mtDNA) probes indicate that gene orders within Daphnia and Drosophila mtDNAs are similar.

Animals↗
PubMed1975

Sterols of the lobster (Homarus americanus) and the shrimp (Pandalus borealis).

In this study we have analyzed the sterol compositions of two continental shelf species of crustacea, the lobster (Homarus americanus) and the shrimp (Pandalus borealis). Cholesterol was found to be the most abundant sterol in these two species with smaller amounts of desmosterol, 24-methylcholesterol, 24-ethylcholesterol, 24-methylenecholesterol and 22-dehydrocholesterol.

Animals↗
PubMed1994

Site of synthesis and phylogenetic distribution of a hemolymph trophic factor of the tobacco hornworm, Manduca sexta.

Identification of fifth instar larval Manduca sexta fat body and epidermis as sites of synthesis of a hemolymph protein (hemolymph trophic factor or HTF) was achieved using in vitro 3H-leucine incorporation into protein and subsequent immunoprecipitation of tissue homogenates. Fat body is the primary site of HTF synthesis with a maximal rate on Day 1; epidermis is a secondary site with peak synthesis on Day 0. In vitro radiolabelling followed by TCA precipitation of general protein of fat body and epidermal homogenates suggest that fat body actively elaborates protein on Days 0-5 with peak rates on Days 1 and 4, while epidermis is active on Days 0-5 with a peak rate on Day 3. Based on Anti-HTF ELISA estimates, HTF [500 to 1000 micrograms/ml] was found in the hemolymph of representatives of the insect orders Blattodea, Hemiptera, Orthoptera, and Lepidoptera and in the class Crustacea, but not in the class Merostomata. These studies suggest a possible fundamental role for HTF among modern arthropods in cuticular deposition involving both epidermis and fat body. The physiological role of HTF is undetermined.

Animals↗
PubMed2000

Cell proliferation, carcinogenesis and diverse mechanisms of telomerase regulation.

Replication of linear genomes is incomplete and leaves terminal gaps. Solutions to this 'end replication' problem can be traced back to the prebiotic RNA world: 'fossils' of the presumptive archetypes of telomere structure and of the telomerase enzyme are retained in the terminal structures of some RNA viruses. Telomerase expression in mammals is ubiquitous in embryonic tissues but downregulated in somatic tissues of adults. Exceptions are regenerative tissues and, notably, tumor cells. Telomerase activation is controlled by cellular proliferation, and it is an early step in the development of many tumors. In contrast to mammals, indeterminately growing multicellular organisms, such as fish and crustaceae, maintain telomerase competence in all somatic tissues. In human tumor diagnostics, detection of proliferation markers with monoclonal antibodies is well established, and in this review, the significance of additional telomerase assays is evaluated. Telomerase inhibitors are attractive goals for application in tumor therapy, and telomerase knockout mice have proven that telomere erosion limits the lifespan of cells in vivo. In contrast, telomerase stimulation can be used to expand the potential of cellular proliferation in vitro, with possible applications for transplantation of in vitro expanded human cells, for immortalizing primary human cells as improved tissue models and for the isolation of otherwise intractable products, such as genuine human monoclonal antibodies.

Adult↗
PubMed1997

engrailed duplication events during the evolution of barnacles.

Barnacles (Cirripedia) are a subclass of Crustacea. Their peculiar segmentation pattern (few segments, absence of abdominal segments, and, in the parasitic rhizocephalan, loss of segmentation at the adult stage) prompted us to study the engrailed gene, which encodes a homeodomain transcription factor and is expressed in arthropods in the posterior half of each segment. We searched for engrailed genes by PCR in a representative cross section of the Cirripedia. Eight unambiguous engrailed genes were cloned from five species, three genes belonging to the same species (Elminius modestus). This implies two duplication events. Molecular phylogenies were constructed and a cladistic approach was applied to the most informative sites. The results indicate that the older duplication (en.a/en.b) is probably very ancient and concerns the whole cirripedean subclass, whereas the other (en.a1/en.a2) is specific to the Elminius lineage.

Amino Acid Sequence↗