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When food is presented to P. paulensis, as well as to several other decapods, the animals exhibit a typical feeding behavior. The possible inducer of this response was determined by observing and analyzing their behavior when different chemicals were placed in the water, especially substances normally found in their food. L-isoleucine seems to be the most important substance in inducing such reactions.
Hepatopancreal tissue of the crab Ucides cordatus was investigated by light and electron microscopy. The observed epithelial cells were: E-cells (embryonic), located in the distal portion of the hepatopancreal tubules, R-cells (resorptive) F-cells (fibrillar) and B-cells (blister or secretory), found in its intermediate and proximal regions. Two types of electron-dense granules (EDGs) were found frequently in the cells of the proximal portion of the hepatopancreal tubule. Both types of EDGs presented alternating concentric electron-dense and electron-lucent layers. In order to better characterize these granules, energy dispersive X-ray analysis (EDXA) and glucose-6-phosphatase (G6Pase) cytochemistry were performed. One type of spherical granule was seen inside vacuoles surrounded by an association of myelin-like membranes as well as some small membrane-bound vesicles. This type of granule neither presented detectable Ca and P on EDXA spectra nor G6Pase cytochemical reaction products. The second type of granule had O, P and Ca characteristic peaks. G6Pase cytochemical products were observed inside these structures and showed that this mineralized type was surrounded by endoplasmic reticulum membranes. This result suggests that in U. cordatus the endoplasmic reticulum is associated with the genesis of mineralized EDGs. While amorphous mineral granules may be associated with a storage of Ca and P for the new carapace synthesis, EDGs covered by the non-mineralized spherical multi-layered membranes may be associated with late endosomes. No specific secretory pathway however was determined for the EDGs at the epithelial proximal portion.
The biological cycle of mercury in the terrestrial isopod Porcellio scaber was investigated. Testing the possibility of in vivo Hg(2+) methylation was divided into two methodologically different parts. Firstly, concentrations of total mercury and MeHg in isopods P. scaber and their environment from a Hg-unpolluted area were measured by the use of validated methods (CV AAS, CV AFS). The data obtained show that the percentage of MeHg in leaves, soil and faeces was less than 1%. In contrast, the percentage of MeHg in gut and hepatopancreas was increased to 14 and 77%, respectively, indicating methylation of Hg(2+) in the gut and its further accumulation in glands. To confirm this assumption, the second methodology was applied-a radiotracer technique with 203Hg(2+) of high specific activity. There are few radiotracer techniques for Hg-methylation assays; for our work we chose the method of Czuba et al. which includes alkaline leaching of Hg species, their extraction into dithizone-toluene, followed by specific separation of Hg dithizonates by thin-layer chromatography and gamma counting. All steps of the analytical protocol were checked and optimised by the use of aqueous solutions of 203Hg(2+) and Me(203)Hg(+). The most important finding was that cleaning-up the extract through a florisil column is not appropriate, because the column retains different percentages of Hg(2+) and MeHg(+) and consequently affects the accuracy of the final result. This optimised protocol was then applied to Hg transformation studies in the terrestrial isopod P. scaber. Leaching Hg species from P. scaber fed with 203Hg(2+) or Me(203)Hg(+) dosed food was completely efficient only at elevated temperatures. Preliminary results of methylation/demethlytion studies are rather variable but they show that both processes (Hg(2+)<-->MeHg(+)) take place in the isopod P. scaber. Additionally, an assessment of the mass balance of Hg in isopods P. scaber exposed to 203Hg(2+) indicates that volatile Hg species are also formed.
The gland cells located in the upper lip of the cladoceran Daphnia were studied by histochemical reactions to establish the chemical nature of some substances they synthesize. Neutral polysaccharides were found to be present, but acid glycosaminoglycans absent. Large amounts of proteins and ribonucleoproteins are also present, lipid substances were not detected. Immunohistochemical methods failed to reveal alpha-amylase in the labral gland cells, though the enzyme was detected in the cells of the intestine. The secretion products of the labral gland cells are probably glycoproteins. The results are discussed in terms of the possible roles of these substances in the animal's physiology.
Among the natural abiotic variables affecting the species density and composition of cladocerans, ammonia is important. Using population growth data as tool, we studied the competitive outcome of three cladoceran species (Ceriodaphnia dubia, Daphnia pulex, and Moina macrocopa) grown alone and together at three different concentrations (0, 25, and 100 mg L(-1)) of ammonia at one density (1 x 10(6)cells ml(-1)) of the microalga Chlorella vulgaris. Regardless of the presence or absence of competing species, C. dubia showed little population growth at 100 mg L(-1) of ammonia. At 25 mg L(-1) of ammonia, the population density of C. dubia increased but the value was much lower than that in control. D. pulex was able to grow at the ammonia concentration of 25 mg L(-1), comparable to controls. However, at 100 mg L(-1) of ammonia, the population declined and more rapidly when competed with other cladocerans. M. macrocopa showed decreased population density with increasing ammonia concentration in the medium. This trend was accelerated by the presence of competing cladocerans. M. macrocopa was benefited by the presence of D. pulex at 0 and 25 mg L(-1) of ammonia in the medium. The peak population density of C. dubia varied from 0.5 to 9 ind.ml(-1), whereas for D. pulex and M. macrocopa, these values varied from 1 to 3 ind.ml(-1) and 7 to 18 ind.ml(-1), respectively, depending on the ammonia concentration and the presence of competing species. The rates of population increase per day of C. dubia, D. pulex, and M. macrocopa ranged from -0.12+/-0.001 to 0.30+/-0.01, 0.04+/-0.001 to 0.22+/-0.002, and 0.201+/-0.028 to 0.235+/-0.019, respectively, depending on the ammonia concentration and the presence of competitors. Our study showed that while competition within the chosen cladoceran species caused suppression of one or two competing species, the presence of ammonia expedited this process based on the relative sensitivities of the tested zooplankton.
The occurrence, localization and response to environmental salinity of carbonic anhydrase (CA) activity were studied in all of the gills of the euryhaline crab Chasmagnathus granulata from Mar Chiquita coastal lagoon (Buenos Aires Province, Argentina). CA activity in all gills appeared to be dependent on salinity. The pattern of distribution of CA activity among gills was different upon transition of C. granulata from osmoionoconformity (more uniform distribution) to hyperregulation (highest activity in posterior gills 6-8). Upon abrupt salinity change a differential response of CA activity occurred among gills which could suggest a differential role of CA in ion transport process in different gills of this crab. Furthermore, CA activity in anterior and posterior gills was found in cytosolic and microsomal fractions, although highest activity appeared to be membrane-associated. Both pools of CA were also strongly influenced by salinity and very sensitive to sulfonamide acetazolamide. The results suggest a differential participation of branchial CA in ionoregulatory mechanisms of C. granulata.
The developing eggs of the crayfish Astacus leptodactylus are found to contain important carotenoid amounts. Free astaxanthin, mainly associated with protein and lutein represent the main pigments occurring in the yolk at the end of the embryonic period. Esterification processes affecting astaxanthin appear concomitantly with the resorption of vitellin reserves, the activation of the digestive gland and the rapid decrease of lutein amounts. Esterified astaxanthin contents increase significantly within the interval between the juvenile stages I and II. Nevertheless, free astaxanthin levels appear insufficient to balance the amounts of the esterified form, scored at the end of the endotrophic period.
Total RNA from shrimp hepatopancreas of Penaeus monodon showed three prominent bands that react with the shrimp ras probe, a 239-bp product, of approximately 4.8 kb (R1), 3.1 kb (R2) and 1.3 kb (R3) on the northern blot. The R1 is the least abundant. Analyses of total RNA from gill and heart were similar to each other. The highest expression of Ras was observed in the gill, while a negligible signal was detected with the Ras probe in muscle. Ras expression is developmentally regulated in embryonic stages of shrimp. Messenger RNA levels of ras were increased from a minimum in the nauplius stage to a maximum in the post-larvae stage for R1 and R2. R3 showed a maximum at the protozoea stage. On the other hand, the activity of protein geranylgeranyltransferase I was increased significantly in the early nauplius stage. No correlative increase of prenylation activity by protein geranylgeranyltransferase I was observed with the transcription activity of ras.
A cDNA was isolated from the shrimp Penaeus japonicus by homology cloning. The shrimp hepatopancreas cDNA encodes a 187-residue polypeptide whose predicted amino acid sequence shares 85% homology with mammalian K-Ras 4B protein and demonstrates identity in the guanine nucleotide binding domains. Expression of the shrimp cDNA in Escherichia coli yielded a 21-kDa polypeptide with a positive reactivity towards the monoclonal antibodies against mammalian Ras. The GTP binding of the shrimp ras-encoded fusion protein was approximated to be 30000units/mg of protein, whereas the binding for GDP was 5000units/mg of protein. Fluorography analysis demonstrated that the prenylation of both shrimp Ras GDP and shrimp Ras GTP by protein geranylgeranyltransferase I of shrimp Penaeus japonicus exceeded the shrimp Ras nucleotide-free form by 10-fold, and fourfold, respectively; that is, the shrimp protein geranylgeranyltransferase I prefers to react with the shrimp ras-encoded p25 fusion protein in the GDP-bound form.
The sequence of the mitochondrial DNA (mtDNA) of the branchiopod crustacean Daphnia pulex has been completed. It is 15333bp with an A+T content of 62.3%, and contains the typical complement of 13 protein-coding, 22 transfer RNA (tRNA) and two ribosomal RNA (rRNA) genes. Comparison of this sequence with the sequences of the other eight completely sequenced arthropod mtDNAs showed that gene order and orientation are identical to that of Drosophila but different from Artemia due to the rearrangement of two tRNA genes. Nucleotide composition, codon usage, and amino acid composition are very similar in the crustaceans, but divergent from insects and chelicerates which show a much higher bias towards A+T. However, with few exceptions, the mitochondrial proteins of Daphnia are more similar to those of the dipteran insects (Drosophila and Anopheles) than to those of Artemia, at both the nucleotide and amino acid levels, suggesting that Artemia mtDNA is evolving at an accelerated rate. These results also show that sequence evolution and the evolution of nucleotide composition can be decoupled. Analysis of nucleotide substitution patterns in COII showed that there has been an unbiased acceleration of the overall substitution rate in Artemia. In contrast, the accelerated substitution rate in Apis is due partly to extreme A+T mutation pressure. Secondary structures are proposed for the Daphnia tRNAs and rRNAs. The tRNAs are similar to those of other arthropods but tend to have TPsiC arms that are only 4bp long. The rRNA secondary structures are similar to those proposed for insects except for the absence of a small number of helices in Daphnia. Phylogenetic analysis of second codon positions grouped Daphnia with Artemia, as expected, despite the latter's accelerated divergence rate. In contrast, the unusual pattern of mtDNA divergence in Apis led to a topology in which the holometabolous insects (Anopheles, Drosophila, Apis) appeared to be paraphyletic with respect to the hemimetabolous insect, Locusta, due to the early branching of Apis.
Much effort has been put into developing vitellogenin antibodies against a wide variety of aquatic vertebrate species to study potential estrogen or anti-estrogen endocrine disrupters. Little work has been done on endocrine disruption in aquatic invertebrates. Although some antibodies have been produced against blue crab and penaeid shrimp lipovitellin, they have only poor cross-reactivity with the important estuarine grass shrimp, Palaemonetes pugio. Vitellin was purified from eggs, monoclonal antibodies were produced using standard techniques, and hybridoma supernatants were screened by ELISA. Western blots were done using extracts from male and female grass shrimp to verify specificity of the monoclonal antibodies. Two low molecular mass bands in the range of 68-85 kD and two high molecular mass bands in the range of 190-221 kD were found. In addition to grass shrimp, several other crustacean species were screened and cross-reactivity found, including blue crab (Callinectes sapidus), mud crab (Rhithropanopeus harrisii), red swamp crayfish (Procambarus clarkii ) and Daphnia magna. To further investigate the use of the antibody, we performed a chronic 6-week pyrene exposure study. We found that vitellin was upregulated in females after 6 weeks and that this may be a protective measure against lipophilic xenobiotics.
Histopathological effects of ammonia on the gills of the estuarine crab Chasmagnathus granulata (Dana, 1851) were evaluated after acute exposure to ammonia concentrations around LC(50) value (17.85 Mm). Disruption of pilaster cells and a subsequent collapse of gill lamellae were the main effects observed. Epithelial necrosis and hyperplasia were also detected. Significant (P<0.05) increases in pCO(2) and lactate, and significant decreases of pO(2) were detected in the haemolymph of ammonia-exposed crabs. These changes suggest that the observed histopathological damage affected gas exchange, possibly leading to death.
Terrestrial isopods store cuticular calcium in large sternal deposits composed of an amorphous CaCO(3) compound. A large part of the deposits consists of numerous small spherules that increase the exposed surface to facilitate resorption of CaCO(3) during cuticle mineralization. It is not known how these spherules are formed and how they are dissolved. This paper presents for the first time an analysis of ultrastructural changes occurring in the sternal CaCO(3) deposits of a terrestrial isopod during their formation and degradation. Our results indicate that formation of the spherules takes place in a specialized aggregation zone, in which 10- to 30-nm-thick granules form agglomerations that then increase in size to form spherules that reveal a concentric growth pattern. Degradation of the deposits occurs in a manner that exposes a maximum of surface area on all levels of their structural organization.
This contribution addresses two questions: which alignment patterns are causing non-monophyly of the Asellota and what is the phylogenetic history of this group? The Asellota are small benthic crustaceans occurring in most aquatic habitats. In view of the complex morphological apomorphies known for this group, monophyly of the Asellota has never been questioned. Using ssu rDNA sequences of outgroups and of 16 asellote species from fresh water, littoral marine habitats and from deep-sea localities, the early divergence between the lineages in fresh water and in the ocean, and the monophyly of the deep-sea taxon Munnopsidae are confirmed. Relative substitution rates of freshwater species are much lower than in other isopod species, rates being highest in some littoral marine genera (Carpias and Jaera). Furthermore, more sequence sites are variable in marine than in freshwater species, the latter conserve outgroup character states. Monophyly is recovered with parsimony methods, but not with distance and maximum likelihood analyses, which tear apart the marine from the freshwater species. The information content of alignments was studied with spectra of supporting positions. The scarcity of signal (=apomorphic nucleotides) supporting monophyly of the Asellota is attributed to a short stem-line of this group or to erosion of signal in fast evolving marine species. Parametric boostrapping in combination with spectra indicates that a tree model cannot explain the data and that monophyly of the Asellota should not be rejected even though many topologies do not recover this taxon.
Pigments and trophic behaviour of three species of Alvinocarididae from a Mid-Atlantic hydrothermal site were analysed. Carotenoid pigments are responsible for the more or less marked colouration of these animals. The carotenoid content of whole animals and different tissues were evaluated. Rimicaris exoculata exhibits an increased carotenoid level at the juvenile stage, while Chorocaris chacei and Alvinocaris markensis contain only few traces of pigment. Free and esterified astaxanthin, reported for most pelagic crustaceans, are present in these deep-sea shrimps. The origin of carotenoids of crustaceans living in the aphotic zone is discussed.
Energy metabolism in early life stages of the shrimp Farfantepenaeus paulensis subjected to temperature reduction (26 and 20 degrees C) was determined using the activities of citrate synthase (CS) and pyruvate kinase (PK). At both temperatures, weight-specific activity of CS decreased throughout the ontogenetic development from protozoea II (PZ II) to postlarva XII-XIV (PL XII-XIV). PK activity reached a pronounced peak in PL V-VI, followed by a further decrease in PL XII-XIV. Temperature reduction produced variation in oxygen consumption rates (QO(2)), ammonia-N excretion and in enzyme activities. Ammonia-N excretion was higher at 20 degrees C in mysis III (M III), PL V-VI and PL XII-XIV, resulting in substantially lower O:N ratios in these stages. QO(2) was increased in protozoea II (PZ II) and mysis I (M I) at 26 degrees C, while no difference in QO(2) was detected in the subsequent stages at either temperature. This fact coincided with higher CS and PK activities in M III, PL V-VI and PL XII-XIV at 20 degrees C compared with 26 degrees C. Regressions between individual enzyme activities and dry weight exhibited slope values of 0.85-0.92 for CS and 1.1-1.2 for PK and temperature reduction was reflected by higher slope values at 20 than at 26 degrees C for both enzymes. Weight-specific CS activity was positively correlated with QO(2) at 20 and 26 degrees C, and may thus be used as an indicator of aerobic metabolic rate throughout the early stages of F. paulensis. The variation in enzyme activities is discussed in relation to possible metabolic adaptations during specific ontogenetic events of the F. paulensis life cycle. Here, the catalytic efficiency of energy-metabolism enzymes was reflected in ontogenetic shifts in behaviour such as larval settlement and the adoption of a benthic existence in early postlarvae. In most cases, enhanced enzyme activities appeared to counteract negative effects of reduced temperature.
A new genus and species of a brachyuran crab, Allograea tomentosa n. gen. and n. sp., of the family Bythograeidae, collected at a hydrothermal vent locality south of the Easter Microplate (31 degrees 09'S) on the southern East Pacific Rise, is described. The genus Allograea is distinguished by lacking modifications of the fronto-orbital region, and by the absence of coloured fields and setal patches on the carapace and chelipeds. The body and legs are smooth but densely covered by a brown tomentum, the eyestalks are not reduced, but the cornea is unfaceted and unpigmented. Comparison of mitochondrial DNA sequences among all known bythograeid genera confirms the novelty of this taxon.
Two new species of Bythograea Williams, B. vrijenhoeki n. sp. and B. galapagensis n. sp., are described based on morphology and mitochondrial DNA comparisons. B. vrijenhoeki was collected on the southern East Pacific Rise, south of the Easter Microplate and B. galapagensis from the Galapagos Rift, from where B. intermedia de Saint Laurent was also described. Our analyses indicate that B. vrijenhoeki is the sister species of B. laubieri Guinot and Segonzac, and B. galapagensis is the sister species of B. thermydron Williams. Bythograea is now composed of six described species, all endemic to hydrothermal vents along the East Pacific Rise and from the Galapagos Rift.