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The effect of Zn on the digestive gland epithelium of Porcellio scaber (Isopoda, Crustacea).

The effect of zinc (Zn)-contaminated food on the shape of digestive gland epithelium was studied in the terrestrial isopod, Porcellio scaber. In animals fed with 5,000 micrograms Zn g-1 dry wt. of food, the epithelium was flattened in the anterior part of the gland tube. In the posterior part of the gland tube, the shape and size of cells did not change drastically, but they had folded apical surface. In animals fed with more Zn-contaminated food (10,000 micrograms Zn g-1 dry wt.) the epithelium was uniformly flattened and the basal lamina was intensively folded. In both cases, reduction of lipid bodies was evident. The possibility of using the shape of the gland epithelium as a biomarker of toxic chemicals is discussed.

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Ultrastructural analysis of the integument during the moult cycle in Ligia italica (Crustacea, Isopoda).

The formation of the cuticle was investigated during moulting of the isopod crustacean Ligia italica. The intermoult cuticle is a four-layered lamellar structure composed of chitin-protein fibrils and mineralized in its upper half. The distribution of calcium carbonate in cuticle during moult cycle was determined by cytochemical methods and X-ray microanalysis, Epi-and exocuticle are secreted during premoult. Calcium is resorbed from the old cuticle and accumulates in the ecdysal gap as calcium granules. Endocuticle is secreted after moult when the mineralization of exocuticle starts. The shape and ultrastructure of epithelial cells change during cuticle secretion and mineralization. Mitochondria, bundles of filaments, calcium granules and large amounts of glycogen accumulate in the apical cytoplasm of cells in premoult animals.

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Mitochondrial genes collectively suggest the paraphyly of Crustacea with respect to Insecta.

Complete sequences of seven protein coding genes from Penaeus notialis mitochondrial DNA were compared in base composition and codon usage with homologous genes from Artemia franciscana and four insects. The crustacean genes are significantly less A + T-rich than their counterpart in insects and the pattern of codon usage (ratio of G + C-rich versus A + T-rich codon) is less biased. A phylogenetic analysis using amino acid sequences of the seven corresponding polypeptides supports a sister-taxon status for mollusks-annelid and arthropods. Furthermore, a distance matrix-based tree and two most-parsimonious trees both suggest that crustaceans are paraphyletic with respect to insects. This is also supported by the inclusion of Panulirus argus COII (complete) and COI and COIII (partial) sequence data. From analysis of single and combined genes to infer phylogenies, it is observed that obtained from single genes are not well supported in most topologies cases and notably differ from that of the tree based on all seven genes.

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5,000 year-old spermatozoa in quaternary Ostracoda (Crustacea).

Fossil spermatozoa are recorded for the first time in freshwater ostracods (small bivalved crustaceans) from Holocene sediments at sites in the United Kingdom and Germany. Exceptional conditions at these sites have resulted in the preservation of chitinous "soft parts", including limbs, the remains of Zenker's Organs (sperm pumps that are part of the male reproductive apparatus in cypridoidean ostracods) and spermatozoa from eight different species. Comparisons are made with spermatozoa from living ostracods and the implications of these discoveries for evolutionary studies of reproductive modes are discussed.

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Acute effects of Microcystis aeruginosa from the Patos Lagoon estuary, southern Brazil, on the microcrustacean kalliapseudes schubartii (Crustacea: Tanaidacea).

Toxic blooms of the cyanobacterium Microcystis aeruginosa, a microcystin producer, have been observed in the past two decades in the Patos Lagoon estuary (southern Brazil). This cyanobacterium reaches the estuary from northern waters and accumulates as toxic blooms in the shallow margins of the environment. Microcystins are phosphatase (PP1 and PP2A) inhibitors and cause animal death via alteration of the liver cell cytoskeletons and intrahepatic hemorrhage. The massive accumulation of toxic material affects the survival of several benthonic estuarine local organisms. The tanaidacea Kalliapseudes schubartii is a benthonic estuarine species which occurs at high densities throughout the year in mixohaline areas of the Patos Lagoon. This microcrustacean is of high ecological relevance and plays an important role in the estuarine food web, as it is consumed on a large scale by estuarine fish. This work verifies the acute toxicity of aqueous extracts of M. aeruginosa RST9501 and of sediments spiked with lyophilized material of the same strain on K. schubartii; it also evaluates the sublethal effects on tanaidacean oxygen consumption rates and glycogen levels under acute exposure to M. aeruginosa aqueous extracts. The strain M. aeruginosa RST9501 was cultured in BGN/2 medium. The aqueous extracts were prepared using the lyophilized material from the strain cultures. Acute tests were performed over 96 h at a salinity of 15, at six toxic concentrations, and resulted in an average 96-h LC50 of 1.44 mg ml(-1). The spiked sediment tests were performed with a 10-day duration, using the lyophilized material in three proportions of powder/sediment and showed an average LC50 of 1.79 mg ml(-1). Oxygen consumption was determined after 24 and 48 h of incubation in adult organisms exposed to sublethal aqueous extract concentrations and showed a significant increase at the highest concentrations. This suggests alterations in the organism's metabolism by exposure to the cyanobacterium extract. The glycogen levels were determined with a commercial kit (Glicox 500; DOLES Ltd.); after 24 and 48 h the dosages were administered in the same organisms utilized in the oxygen consumption test and did not demonstrate significant differences. The results demonstrate the possible risks of intoxication to which the natural populations of K. schubartii were exposed in the environment and emphasize the importance of studies involving sublethal concentrations of M. aeruginosa to other organisms of the trophic web in this aquatic system.

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Comparative toxicity of dissolved metals to early larval stages of Palaemon serratus, Maja squinado, and Homarus gammarus (Crustacea:Decapoda).

The acute lethal toxicities of mercury, copper, and cadmium to the first larval stage of the prawn (Palaemon serratus), spider crab (Maja squinado), and lobster (Homarus gammarus) were tested. Chromium was also tested with the prawn. Mortality was recorded after 48 h (for lobster) or 72 h (for other species) incubation at 18 degrees C, and the median lethal concentrations (LC(50) +/- 95% confidence intervals) per individual and per mass unit were calculated. The LC(50) values were, 74 microg Hg/L, 3,304 microg Cu/L, 1,686 microg Cd/L, 12,486 microg Cr/L for prawn; 72 microg Hg/L, 50 microg Cu/L, 158 microg Cd/L for spider crab; and 48 microg Hg/L, 46 microg Cu/L, 34 microg Cd/L for lobster. Therefore, larvae of spider crab and lobster were markedly more sensitive than prawn to heavy metals and thus more suitable to use in seawater quality bioassays. Of these two species, spider crab is recommended due to its abundance, easier maintenance, and higher fecundity. The mercury LC(50) values for different larval stages of P. serratus (zoea I, II, V, and VI) were obtained, and no ontogenetic change in sensitivity to the metal could be detected.

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Degradation of weathered oil by mixed marine bacteria and the toxicity of accumulated water-soluble material to two marine crustacea.

Artificially weathered crude oil was degraded by four diverse cultures of mixed marine bacteria under optimized conditions for 7 and 14 days. Loss in total weight of starting oil (30 g) ranged from 6.8-17.3% in biologically active incubations compared with only 0. 9-1.1% in sterile and nutrient-limited controls. In all incubations, both neutral and acidic water-soluble fractions (WSF) were accumulated. In biologically active systems, 50.9-249.0 mg neutral and 63.3-406.8 mg acidic WSF were accumulated whereas only 6.5-11.1 mg neutral and 1.7-2.2 mg acidic WSF were accumulated in control incubations. Analysis by gas chromatography demonstrated that accumulated WSF in biologically active systems contained compounds different from those washed from the starting crude oil. Exposure of grass shrimp (Palaemonetes pugio) embryos to neutral WSF from each of the biologically active cultures resulted in high embryo mortalities relative to sterile and nutrient-limited controls which exhibited >90% hatching success and larval survival. Toxicity of neutral WSF was also demonstrated on larvae of mysids (Mysidopsis bahia). In both cases, toxicity occurred only on exposure to neutral material accumulated by active, oil-degrading cultures and not with material washed from the weathered crude oil. These results imply that unique compounds were accumulated during degradation that may have been responsible for increased toxicity.

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The sublethal effects and accumulation of cadmium in the terrestrial isopod Porcellio laevis latr. (Crustacea, Isopoda).

This study investigated the sublethal effects of CdSO4 on the terrestrial isopod species Porcellio laevis and its capacity to accumulate and compartmentalize cadmium. The ability of P. laevis to discriminate between uncontaminated and CdSO4-contaminated leaves was also studied. It was shown that sublethal effects in terms of mass changes of exposed isopods occur at cadmium concentrations between 10 and 80 mg/kg. Moreover, P. laevis can be considered as a so-called macroconcentrator, accumulating and storing cadmium in its hepatopancreas tissue. However, the accumulation capacity is not proportional to the cadmium concentrations in the leaves presented as food, as shown by decreasing concentration factors with increasing cadmium concentrations in the diet. This is probably an effect of the isopod's ability to discriminate between different cadmium concentrations in the food, as shown by means of feed-choice experiments, in which P. laevis was able to avoid contaminated leaves.

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The photoactivation energy of the visual pigment in two spectrally different populations of Mysis relicta (Crustacea, Mysida).

We report the first study of the relation between the wavelength of maximum absorbance (lambdamax) and the photoactivation energy (Ea) in invertebrate visual pigments. Two populations of the opossum shrimp Mysis relicta were compared. The two have been separated for 9,000 years and have adapted to different spectral environments ("Sea" and "Lake") with porphyropsins peaking at lambdamax=529 nm and 554 nm, respectively. The estimation of Ea was based on measurement of temperature effects on the spectral sensitivity of the eye. In accordance with theory (Stiles in Transactions of the optical convention of the worshipful company of spectacle makers. Spectacle Makers' Co., London, 1948), relative sensitivity to long wavelengths increased with rising temperature. The estimates calculated from this effect are Ea,529=47.8+/-1.8 kcal/mol and Ea,554=41.5+/-0.7 kcal/mol (different at P<0.01). Thus the red-shift of lambdamax in the "Lake" population, correlating with the long-wavelength dominated light environment, is achieved by changes in the opsin that decrease the energy gap between the ground state and the first excited state of the chromophore. We propose that this will carry a cost in terms of increased thermal noise, and that evolutionary adaptation of the visual pigment to the light environment is directed towards maximizing the signal-to-noise ratio rather than the quantum catch.

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Visual pigment absorbance and spectral sensitivity of the Mysis relicta species group (Crustacea, Mysida) in different light environments.

Visual-pigment absorbance spectra and eye spectral sensitivities were examined in eight populations of opossum shrimp from different light environments. Four Finnish populations, two from the Baltic Sea and two from freshwater lakes, represent Mysis relicta, sensu stricto. The sibling species M. salemaai and M. diluviana are represented by, respectively, two Baltic Sea populations and two populations from freshwater lakes in Idaho, USA. In M. relicta, the visual pigments of the two lake populations were similar (lambda(max)=554.3+/-0.8 nm and 556.4+/-0.4 nm), but significantly red-shifted compared with the sea populations (at 529 and 535 nm) and with M. salemaai (at 521 and 525 nm). All these pigments had only A2 chromophore and the lake/sea difference indicates adaptive evolution of the opsin. In M. diluviana, lambda(max) varied in the range 505-529 nm and the shapes of spectra suggested varying A1/A2 chromophore proportions, with pure A1 in the 505 nm animals. Eye sensitivity spectra were flatter and peaked at longer wavelengths than the relevant visual-pigment templates, but declined with the same slope beyond ca. 700 nm. The deviations from visual-pigment spectra can be explained by ocular light filters based on three types of identified screening pigments.

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An antibody to recombinant crustacean hyperglycaemic hormone of Nephrops norvegicus cross-reacts with neuroendocrine organs of several taxa of malacostracan Crustacea.

The crustacean hyperglycaemic hormones (cHHs) are multifunctional neuropeptides that play a central role in the physiology of crustaceans. A partial cDNA coding for cHH of the Norway lobster, Nephrops norvegicus, was cloned; this cDNA was fused to glutathione- S-transferase (GST) to obtain a recombinant fusion protein that was used to raise a rabbit antiserum and to perform a biological assay. The specificity of the purified antibody was demonstrated by means of Western blotting. To validate the specificity of the purified antibody to the cHH of N. norvegicus and its cross-reactivity with other species, we performed standard immunocytochemistry of the eyestalk on: (1) paraffin sections of the decapod species N. norvegicus, Munida rugosa and Astacus leptodactylus and of the stomatopod Squilla mantis; (2) semithin resin sections of N. norvegicus and Palaemon elegans; (3) ultrathin sections of N. norvegicus sinus gland (transmission electron microscopy studies). The pattern of immunoreactivity shown by N. norvegicus eyestalk sections conforms to distribution, relative amount and ultrastructural features of cHH-containing neurons and nerve endings as reported in the previous literature. In all the crustacean species examined, the antibody marks precisely the X organ-sinus gland complex and unspecific staining is completely lacking. In addition, its specific cross-reaction by immunoprecipitation depletes shrimp eyestalk extract of hyperglycaemic activity in an in vivo bioassay. The results obtained show a cHH-specific molecular recognition despite the fact that the species tested belong to systematic groups increasingly remote in the phylogenetic tree. The antibody could be used for advancing our knowledge on cHH activity in a variety of crustacean species, e.g. for monitoring reproductive and stress conditions.

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Crustacean hyperglycaemic hormone in the nervous system of the primitive crustacean species Daphnia magna and Artemia salina (Crustacea: Branchiopoda).

Crustacean hyperglycaemic hormone-immunoreactive neuronal systems are detected in the central and peripheral nervous systems of two entomostracan crustaceans, Daphnia magna and Artemia salina, by immunocytochemistry using specific antisera against crustacean hyperglycaemic hormones of the decapod crustaceans Orconectes limosus and Carcinus maenas. In D. magna, four small putative interneurones are detected in the brain. In the thorax, ten bipolar peripheral neurones are stained by both antisera. They are obviously segmental homologues with centrally projecting axons that form interdigitating varicose fibres and terminals in putative neurohaemal areas next to the surface of the anterior part of the thoracic ganglia. Similar immunopositive neurones occur both in the central and peripheral nervous systems of A. salina. A total of five groups of neurones occur in the protocerebrum, the deutocerebrum and the mandibular ganglion. Some of the protocerebral neurones are bipolar and project to the dorsal frontal organ. A single pair of peripheral multipolar neurones in the maxillary segment projects centrally into the ventral nerve cord and innervates unidentified somatic muscles and tissues in the maxillary and the first appendage segments. None of the brain neurones in both species show similarities to decapod X-organ sinus gland neurosecretory neurones. Chromatography of brain extracts of D. magna combined with immunodot blotting revealed two strongly immunoreactive fractions at retention times close to that of the crustacean hyperglycaemic hormone of crayfish. Moreover, preabsorption controls suggest that the cross-reacting peptides of D. magna and A. salina are structurally closely related to those of decapods.

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A comparative immunocytochemical study of the hyperglycaemic, moult-inhibiting and vitellogenesis-inhibiting neurohormone family in three species of decapod crustacea.

Eyestalks of the palinuran species Jasus lalandii and Panulirus homarus, and the brachyuran species Carcinus maenas, were examined with antisera raised against purified crustacean hyperglycaemic hormone (cHH) of the astacidean species Homarus americanus and Procambarus bouvieri, as well as the brachyuran species Cancer pagurus. Other antisera used in this investigation were raised against purified moult-inhibiting hormone (MIH) of C. pagurus and vitellogenesis-inhibiting hormone (VIH) of H. americanus. Positive immunoreactions to all the antisera were localised in perikarya of the X-organ and the axon terminals in the sinus gland of all the crustaceans investigated. These results illustrate the existence of an immunological similarity, detectable at the immunocytochemical level, between the cHH/MIH/VIH neurohormones of the Astacidae, Palinura and Brachyura infraorders. Furthermore, results from consecutive tissue sections indicate that cHH, MIH and VIH are co-localised in a subpopulation of X-organ neurons.

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