Changes in free amino acids in the hemolymph of giant freshwater prawn Macrobrachium rosenbergii exposed to varying salinities: relationship to osmoregulatory ability

Changes in free amino acids (FAA) in the hemolymph of the giant freshwater prawn, Macrobrachium rosenbergii, were examined in individuals exposed to varying salinities for up to 1 week. In freshwater and under conditions of low salinity, total FAA concentrations were maintained between approximately 0.85 and 1 mM and did not exhibit changes in response to salinity exposure. Under high salinities, total FAA concentrations increased dramatically, reaching up to 2.1 mM depending on treatment. Examination of individual amino acid concentrations revealed that these increases were based on specific changes in glycine, arginine, alanine, proline and lysine. Among these, alanine showed the greatest increases, resulting in levels six-fold higher under high salinity than in freshwater and under low salinity. The other amino acid species showed increases of 2.5-fold compared to original values. These five FAAs in freshwater and under low salinity together occupied approximately 45% of total FAA contents and under high salinity comprised more than 70% of total FAA contents. These results suggest that specific hemolymph FAAs are involved in mediating response to salinity exposure in freshwater prawns.     

Quantification of lectin in freshwater prawn (Macrobrachium rosenbergii ) hemolymph by ELISA

An enzyme-linked immunoabsorbent assay was developed to quantify the lectin present in the hemolymph of the freshwater prawn Macrobrachium rosenbergii. This method involves the use of murine monoclonal IgG1 with kappa light chain (designated as 3G1) antibodies raised against the purified lectin, the assay that we developed recognized as little as 30 ng/ml of lectin, and was used to measure the lectin concentration in animals at different maturation stages. The highest concentration of lectin was identified in the hemolymph from post-larval prawns and the lowest in molt stage adult animals. The hemagglutination activity of the lectin was four-fold higher in adult than in juvenile specimens, although in all cases N-acetylated sugar residues, such as N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, and N-acetyl-D-neuraminic acid were inhibitors of the lectin activity, suggesting that lectin plays a role in the transport of N-acetylated sugar in juvenile prawns. Our results indicate that lectin concentration and hemagglutinating activity could be influenced by developmental conditions of the freshwater prawn.     

Adaptive shifts in osmoregulatory strategy and the invasion of freshwater by brachyuran crabs: evidence from Dilocarcinus pagei (Trichodactylidae)

To evaluate putative adaptive changes underpinning the invasion of freshwater by the Brachyura, this investigation examines anisosmotic extra and isosmotic intracellular osmoregulatory capabilities in Dilocarcinus pagei, a neotropical, hololimnetic crab, including its embryonic and juvenile phases. All ontogenetic stages show a remarkable ability to survive a high salinity medium (25 per thousand, 750 mOsm/kg H2O, 350 mm Na+, 400 mM Cl-). Adults hyper-regulate hemolymph osmolality up to isosmoticity at 744 mOsm kg/H2O (24 per thousand), [Na+] and [Cl-] becoming isoionic at 449 (22 per thousand) and 256 mM (16 per thousand), respectively. Hemolymph (420+/-39 mOsm/kg H2O) and urine (384+/-44 mOsm/kg H2O) are isosmotic in adults held in freshwater, and after 5-days exposure to 25 per thousand (787+/-9 mOsm/kg H2O and 777+/-43 mOs/kg H2O, respectively); D. pagei does not produce dilute urine. Total free amino acid (FAA) concentrations in embryos (14.9+/-1.2), juveniles (32.8+/-0.1) and adult muscle (10.9+/-2.1 mmol/kg wet weight) in freshwater are 30-fold less than in brackish/marine Crustacea, suggesting that FAA constitute a useful parameter to evaluate adaptation to freshwater. On acclimation to 25 per thousand, total FAA increase by approximately 100% in embryos and in adult muscle and nerve tissue and hemolymph, owing to large increases in proline, arginine and/or alanine. However, effective FAA contribution to intracellular osmolality increases only in embryos, from 3 to 4.5%. These findings suggest that gill-based, anisosmotic extracellular regulation has supplanted isosmotic intracellular regulatory mechanisms during the conquest of freshwater by the Brachyura, and indicate that D. pagei may be an old, well-adapted inhabitant of this biotope.     

Intra- and extracellular osmotic regulation in the hololimnetic Caridea and Anomura: a phylogenetic perspective on the conquest of fresh water by the decapod Crustacea

We investigate extra- and intracellular osmoregulatory capability in two species of hololimnetic Caridea and Anomura: Macrobrachium brasiliense, a palaemonid shrimp, and Aegla franca, an aeglid anomuran, both restricted to continental waters. We also appraise the sharing of physiological characteristics by the hololimnetic Decapoda, and their origins and role in the conquest of fresh water. Both species survive salinity exposure well. While overall hyperosmoregulatory capability is weak in A. franca and moderate in M. brasiliense, both species strongly hyporegulate hemolymph [Cl⁻] but not osmolality. Muscle total free amino acids (FAA) increase slowly but markedly in response to the rapid rise in hemolymph osmolality consequent to hyperosmotic challenge: 3.5-fold in A. franca and 1.9-fold in M. brasiliense. Glycine, taurine, arginine, alanine and proline constitute ≈85% of muscle FAA pools in fresh water; taurine, arginine, alanine each contribute ≈22% in A. franca, while glycine predominates (70%) in M. brasiliense. These FAA also show the greatest increases on salinity challenge. Muscle FAA titers correlate strongly (R = 0.82) with hemolymph osmolalities across the main decapod sub/infraorders, revealing that marine species with high hemolymph osmolalities achieve isosmoticity of the intra- and extracellular fluids partly through elevated intracellular FAA concentrations; freshwater species show low hemolymph osmolalities and exhibit reduced intracellular FAA titers, consistent with isosmoticity at a far lower external osmolality. Given the decapod phylogeny adopted here and their multiple, independent invasions of fresh water, particularly by the Caridea and Anomura, our findings suggest that homoplastic strategies underlie osmotic and ionic homeostasis in the extant freshwater Decapoda.     

Free amino acid pools as effectors of osmostic adjustment in different tissues of the freshwater shrimp macrobrachium olfersii (crustacea, decapoda) during long-term salinity acclimation

To examine osmotic regulation during long-term acclimation to a hyperosmotic medium, hemolymph osmolality, [Na⁺] and total protein, tissue hydration, and free amino acid (FAA) pools in abdominal muscle, gills, central nervous tissue and hemolymph were quantified in the diadromous freshwater (FW) shrimp, Macrobrachium olfersii, during direct exposure to 21‰S seawater over a 20-day period. Hemolymph osmolality and [Na⁺] reach stable maxima within 24 h while total protein is unchanged. Muscle and nerve tissues rapidly lose water while gills hydrate; all tissues attain maximum hydration (+5%) by 5 days, declining to FW values except for gills. Total FAA are highest in muscle, reach a maximum by 2 days (+64%), declining to FW values. Gill FAA increase by 110% after 24 h, diminishing to FW values. Nerve FAA increase 187% within 24 h, and remain elevated. Hemolymph FAA decrease (-75%) after 24 h, stabilizing well below the FW concentration. During acclimation, muscle glycine (+247%), gill taurine (+253%) and proline (+150%), and nerve proline (+426%), glycine (+415%) and alanine (+139%) increase, while hemolymph leucine (-70%) decreases. Total FAA pools contribute 10-20% to intracellular (22-70 mmol/kg) and 0.5-2.4% to hemolymph (3-7 mOsm/kg) osmolalities during direct acclimation from FW. These data emphasize the modest participation of FAA pools in intracellular osmotic regulation during physiological adaptation by M. olfersii to osmotic challenge, accentuating the role of anisosmotic extracellular regulation, suggesting that, during the invasion of freshwater by the Crustacea, dependence on intracellular adjustment employing FAA as osmotic effectors, has become progressively reduced.     

Sialylation is modulated through maturation in hemocytes from Macrobrachium rosenbergii

In this work we identified in adult and juvenile freshwater prawn, Macrobrachium rosenbergii, three major type of circulating hemocytes: fusiform; rounded; and large ovoid hemocytes. Rounded and large hemocytes represent the first defense line, since this type of cells exerts phagocytic activity as well as lectin synthesis. Considering that glycosylation plays important roles in cell communication and as a target for pathogenic microorganisms, in this report was also described the main glycosidic modifications that occur in the large and rounded hemocytes from the freshwater prawn during maturation as determined with lectins. Neu5Acalpha2,6Gal, was identified homogeneously distributed in the membrane in 90% of hemocytes from juvenile organisms. Maturation of the freshwater prawn induced a decrease or complete loss of Neu5Acalpha2,6Gal residues that were replaced with Neu5Acalpha2,3 molecules in practically all hemocytes from adult organisms. This change was paralleled by a diminution in 9-O-acetyl-neuraminic acid (Neu5,9Ac(2)) expression. T and Tn antigens (Galbetal,3 GalNAcalpha1-0-Ser/Thr or GalNAcalpha1-0-Ser/Thr, respectively), as well as N-glycosidically linked glycans, seem to be highly conserved throughout maturation. Our results show that sialylation of freshwater prawn hemocytes is modulated throughout the maturation process.     

Site of hemolymph lectin production and its activation in vitro by 20-hydroxyecdysone

To identify the tissues which produce hemolymph lectin in larvae of Bombyx mori, ovary, testis, fat body, and hemocytes from 5th-instar larvae were cultured in vitro and the culture medium was partially purified and assayed for hemagglutinating activity. Among the tissues tested, hemocytes appeared to be a major source of the hemolymph lectins. Ovary produced lectins to about one-tenth of the amount observed for the hemocytes, whereas testis and fat body were not productive. To study the hormonal control of hemolymph lectin production by hemocytes, hemocytes from 4th-instar larvae were cultured in vitro. Hemagglutinating activity in the hemolymph of 4th-instar larvae was immunostainable with the monoclonal antibody raised against 350,000 dalton lectin found in the 5th-instar hemolymph, but their molecular sizes were larger than the 5th-instar hemolymph lectins. When 20-hydroxyecdysone was added into the medium, production of the lectin by the hemocytes was remarkably enhanced, depending upon the hormone concentration.     

Free amino acid pools as effectors of osmostic adjustment in different tissues of the freshwater shrimp macrobrachium olfersii (crustacea,decapoda) during long-term salinity acclimation

To examine osmotic regulation during long-term acclimation to a hyperosmotic medium, hemolymph osmolality, [Na⁺] and total protein, tissue hydration, and free amino acid (FAA) pools in abdominal muscle, gills, central nervous tissue and hemolymph were quantified in the diadromous freshwater (FW) shrimp, Macrobrachium olfersii, during direct exposure to 21‰S seawater over a 20-day period. Hemolymph osmolality and [Na⁺] reach stable maxima within 24?h while total protein is unchanged. Muscle and nerve tissues rapidly lose water while gills hydrate; all tissues attain maximum hydration (+5%) by 5 days, declining to FW values except for gills. Total FAA are highest in muscle, reach a maximum by 2 days (+64%), declining to FW values. Gill FAA increase by 110% after 24?h, diminishing to FW values. Nerve FAA increase 187% within 24?h, and remain elevated. Hemolymph FAA decrease (?75%) after 24?h, stabilizing well below the FW concentration. During acclimation, muscle glycine (+247%), gill taurine (+253%) and proline (+150%), and nerve proline (+426%), glycine (+415%) and alanine (+139%) increase, while hemolymph leucine (?70%) decreases. Total FAA pools contribute 10–20% to intracellular (22–70?mmol/kg) and 0.5–2.4% to hemolymph (3–7?mOsm/kg) osmolalities during direct acclimation from FW. These data emphasize the modest participation of FAA pools in intracellular osmotic regulation during physiological adaptation by M. olfersii to osmotic challenge, accentuating the role of anisosmotic extracellular regulation, suggesting that, during the invasion of freshwater by the Crustacea, dependence on intracellular adjustment employing FAA as osmotic effectors, has become progressively reduced.     

Hemocytes of Leiobunum limbatum and two other species of harvestmen (Arachnida,Opiliones): Morphological classification and functional aspects

The hemocytes of Leiobunum limbatum, Mitopus morio, and Opilio ravennae number from about 8,000 (juveniles) to 41,000 (pregnant females) per microliter of hemolymph. Five different types of hemocytes occur in all three species and both sexes. According to their ultrastructural appearance and their similarities to other arthropod hemocytes these five types are designated as prohemocyte, plasmatocyte, granulocyte, coagulocyte, and spherulocyte. From the ultrastructural point of view the prohemocytes are interpreted as stem cells for plasmatocytes which on their part differentiate into granulocytes. Transitional stages which would indicate the origin of coagulocytes and spherulocytes could not be found. Granulocytes and spherulocytes are interpreted as being storage cells; coagulocytes burst when hemolymph is transferred to a microscopic slide. Plasmatocytes are involved in the removal of dead cells or cell fragments. Plasmatocytes are demonstrated as being able to phagocytize and digest bacteria.     

A novel glycosylation phenotype expressed by Lec23, a Chinese hamster ovary mutant deficient in alpha-glucosidase I.

Lec23 Chinese hamster ovary (CHO) cells have been shown to possess a unique lectin resistance phenotype and genotype compared with previously isolated CHO glycosylation mutants (Stanley, P., Sallustio, S., Krag, S. S., and Dunn, B. (1990) Somatic Cell Mol. Genet. 16, 211-223). In this paper, a biochemical basis for the lec23 mutation is identified. The carbohydrates associated with the G glycoprotein of vesicular stomatitis virus (VSV) grown in Lec23 cells (Lec23/VSV) were found to possess predominantly oligomannosyl carbohydrates that bound strongly to concanavalin A-Sepharose, eluted 3 sugar eq beyond a Man9GlcNAc marker oligosaccharide on ion suppression high pressure liquid chromatography, and were susceptible to digestion with jack bean alpha-mannosidase. Monosaccharide analyses revealed that the oligomannosyl carbohydrates contained glucose, indicating a defect in alpha-glucosidase activity. This was confirmed by further structural characterization of the Lec23/VSV oligomannosyl carbohydrates using purified rat mammary gland alpha-glucosidase I, jack bean alpha-mannosidase, and 1H NMR spectroscopy at 500 MHz. [3H]Glucose-labeled Glc3Man9GlcNAc was prepared from CHO/VSV labeled with [3H]galactose in the presence of the processing inhibitors castanospermine and deoxymannojirimycin. Subsequently, [3H]Glc2Man9GlcNAc was prepared by purified alpha-glucosidase I digestion of [3H]Glc3Man9GlcNAc. When these oligosaccharides were used as alpha-glucosidase substrates it was revealed that Lec23 cells are specifically defective in alpha-glucosidase I, a deficiency not previously identified among mammalian cell glycosylation mutants.     

Dynamics of vitellogenin synthesis in juvenile giant freshwater prawn Macrobrachium rosenbergii

The dynamics of vitellogenin (Vg) mRNA expression and patterns of Vg and vitellin distribution in the hepatopancreas and ovary of juvenile Macrobrachium rosenbergii were examined using real-time RT-PCR and immunohistochemical methods. Eyestalk ablation was seen to induce rapid development of the gonads and Vg synthesis in females. In the female hepatopancreas, Vg mRNA expression was observed several days following ablation, after which levels increased gradually with increasing gonadosomatic index (GSI). Vitellin accumulation in the oocytes also increased with increasing Vg mRNA synthesis; expression was however negligible in the ovary. Hemolymph Vg levels in females ranged from 0.04 to 2.2 mg/ml. SDS PAGE/Western blotting analysis of hemolymph samples revealed that juvenile Vg was composed of 199 and 90 kDa subunits; the 102 kDa subunit present in adult female Vg (Okuno et al., 2002. J Exp Zool 292:417-429) could not be detected at any stage of vitellogenesis in juveniles. Vg was not detectable in non-ablated juveniles. The results of this study confirmed that the mode of involvement of eyestalk factors in regulating vitellogenesis is intrinsic to both juveniles and adults, and that a basic pattern of Vg synthesis and processing is conserved. However, the fact that juveniles are not able to produce the same Vg levels observed in adult females, and do not reach high GSI levels culminating in spawning suggests that other factors and physiological conditions specific to adult females are necessary to demonstrate full reproductive ability.     

Identification of lectin isoforms in juvenile freshwater prawns Macrobrachium rosenbergii (DeMan, 1879)

From the serum of juvenile freshwater prawns, we isolated by affinity chromatography on glutaraldehyde-fixed rat erythrocytes stroma, immobilized in Sephadex G-25, a sialic acid specific lectin of 9.6[emsp4 ]kDa per subunit. Comparative analysis against adult organisms purified lectin, by chromatofocusing, showed that the lectin from juvenile specimens is composed by four main isoforms with a pl of 4.2, 4.6, 5.1, and 5.6, whereas the lectin from adults is eluted at pH 4.2. The amino acid composition of the lectin obtained from adult and juvenile stages suggest identity, but the compositions are not identical since a higher content of carbohydrates was found in the lectin from younger organisms. The freshwater prawn lectin showed specificity toward N-acetylated amino sugar residues such as GlcNAc, GalNAc, Neu5Ac and Neu5,9Ac; but in juvenile organisms the lectin showed three times less hemagglutinating activity than the lectin from adults. Both lectins agglutinated rat, rabbit and chicken erythrocytes, indicating that Neu5,9Ac in specific O-glycosydically linked glycans seems to be relevant for the interaction of M. rosenbergii lectins with their specific cellular receptor. Our results suggest that the physicochemical characteristics of the lectin from the freshwater prawn are regulated through maturation.     

Chemical characterization of the lectin from the freshwater prawn Macrobrachium rosenbergii (De Man) by MALDI-TOF

The serum of the freshwater prawn contains a sialic acid specific lectin (MrL) that agglutinates erythrocytes from rat and rabbit, as well as some Gram negative and positive bacterial strains. In this work, we performed the chemical characterization of the MrL purified by affinity chromatography on stroma from rat erythrocytes and by ion exchange chromatography. In its active form, MRL is a dimeric glycoprotein with 9.5 kDa per subunit. The amino acid sequence of the lectin was deduced from peptides obtained after trypsin treatment by matrix-assisted laser desorption ionization mass spectrometry-time of flight analysis (MALDI-TOF). The predicted amino acid sequence of the lectin showed 54% homology with the hyperglycemic hormone from Macrobrachium rosenbergii. It also showed homology with the variable region of the human immunoglobulin kappa (22%) and lambda (27%) light chains. The lectin is a glycoprotein with 11% (w/w) carbohydrate content and is constituted by Gal, Man, GlcNAc, GalNAc and NeuAc in a molar ratio of 4:3:2:1:0.6. The primary structure of the carbohydrate chains of the lectin from the freshwater prawn was determined by affinity chromatography of MrL-glycopeptides on Con A and LCA lectin columns, which indicated that the main carbohydrate chains conforming the lectin are N-glycosidically linked. Man3 GlcNAc2.1 oligosaccharides were the most abundant structures with 57%) followed by Gal1.3 Man3 GlcNAc2.8 with 24%. Our results suggest that the freshwater prawn possess a lectin in the hemolymph plasma, related to those from the immunoglobulin superfamily.     

Structural determination of the N-glycans of a lepidopteran arylphorin reveals the presence of a monoglucosylated oligosaccharide in the storage protein

The structures of the oligosaccharides attached to arylphorin from Chinese oak silkworm, Antheraea pernyi, have been determined. Arylphorin, a storage protein present in fifth larval hemolymph, contained 4.8% (w/w) of carbohydrate that was composed of Fuc:GlcNAc:Glc:Man=0.2:4.0:1.4:13.6 moles per mole protein. Four moles of GlcNAc in oligomannose-type oligosaccharides strongly suggest that the protein contains two N-glycosylation sites. Normal-phase HPLC and mass spectrometry oligosaccharide profiles confirmed that arylphorin contained mainly oligomannose-type glycans as well as truncated mannose-type structures with or without fucosylation. Interestingly, the most abundant oligosaccharide was monoglucosylated Man9-GlcNAc2, which was characterized by normal-phase HPLC, mass spectrometry, Aspergillus saitoi alpha-mannosidase digestion, and 1H 600 MHz NMR spectrometry. This glycan structure is not normally present in secreted mammalian glycoproteins; however, it has been identified in avian species. The Glc1Man9GlcNAc2 structure was present only in arylphorin, whereas other hemolymph proteins contained only oligomannose and truncated oligosaccharides. The oligosaccharide was also detected in the arylphorin of another silkworm, Bombyx mori, suggesting a specific function for the Glc1Man9GlcNAc2 glycan. There were no processed glucosylated oligosaccharides such as Glc1Man5-8GlcNAc2. Furthermore, Glc1Man9GlcNAc2 was not released from arylophorin by PNGase F under nondenaturing conditions, suggesting that the N-glycosidic linkage to Asn is protected by the protein. Glc1Man9GlcNAc2 may play a role in the folding of arylphorin or in the assembly of hexamers.     

Substrate specificity analysis of endoplasmic reticulum glucosidase II using synthetic high mannose-type glycans

Glucosidase II (Glc'ase II) is a glycan-processing enzyme that trims two alpha1,3-linked Glc residues in succession from the glycoprotein oligosaccharide Glc2Man9GlcNAc2 to give Glc1Man9GlcNAc2 and Man9GlcNAc2 in the endoplasmic reticulum (ER). Monoglucosylated glycans, such as Glc1-Man9GlcNAc2, generated by this process play a key role in glycoprotein quality control in the ER, because they are primary ligands for the lectin chaperones calnexin (CNX) and calreticulin (CRT). A precise analysis of the substrate specificity of Glc'ase II is expected to further our understanding of the molecular basis to glycoprotein quality control, because Glc'ase II potentially competes with CNX/CRT for the same glycans, Glc1Man7-9GlcNAc2. In this study, a quantitative analysis of the specificity of Glc'ase II using a series of structurally defined synthetic glycans was carried out. In the presence of CRT, Glc'ase II-mediated trimming from Glc2Man9GlcNAc2 stopped at Glc1Man9GlcNAc2, supporting the notion that the glycan structure delivered to the CNX/CRT cycle is Glc1Man9GlcNAc2. Unexpectedly, our experiments showed that Glc1Man8(B)GlcNAc2 had nearly the same reactivity as Glc1Man9GlcNAc2, which was markedly greater than that of its positional isomer Glc1Man8(C)GlcNAc2. An analysis with glycoprotein-like probes revealed the stepwise formation of Glc1Man9GlcNAc2 and Man9GlcNAc2 from Glc2Man9GlcNAc2, even in the presence of CRT. It was also shown that Glc1Man8(B)GlcNAc2 had even greater reactivity than Glc1Man9GlcNAc2 at the glycoprotein level. Moreover, inhibitory activities by nonglucosylated glycans suggested that Glc'ase II recognized the C arm (Manalpha1, 2Manalpha1, 6Man-) of high mannose-type glycans.     

Free amino acid composition in juveniles of Mytilus galloprovincialis: spatial variability after Prestige oil spill

Composition of free amino acids (FAA) in juveniles of Mytilus galloprovincialis was analysed along a large geographical coastline area in Galicia (NW Spain). Individuals were sampled in February 2003, three months after the Prestige oil spill. Pollution values at sampling time were reported as polycyclic aromatic hydrocarbons (PAHs) concentrations in soft tissues of individuals and varied between the highest amount observed in Carrumeiro mussels (502 ng/g dw) and the lowest in Pindo mussels (196 ng/g dw), both locations being close to each other in the centre of the geographical area under study. Pollution values in the other populations varied within the range of 241-347 ng/g dw. Total free amino acids (TFAA) were highest in Agui?o-Pindo-Carrumeiro juveniles at the centre of the Coastline area studied (420-462 micromol/g dw) as compared to the other populations at North and South of Galicia (312-347 micromol/g dw). TFAA results were based on the variability observed in protein free amino acids (PFAA micromol/g dw) among populations (214-249 micromol/g dw for Agui?o-Pindo-Carrumeiro mussels and 98-149 micromol/g dw for the other populations) whereas non-protein free amino acids (NPFAA) taurine and ornithine did not show any significant spatial pattern of variation. Glycine and alanine represented the most abundant PFAA (16-29% and 2.7-11.9% of TPFAA, respectively) and significant correlations between PFAA and both the protein content of soft tissues (r=-0.82) and the condition index of juveniles (r=0.86) were observed. No significant relationships were detected, however, between pollution values in soft tissues as PAHs and FAA profiles with the exception of alanine concentrations as percentage of TFAA (r=0.88; P<0.01). The latter seemed to be an "all or nothing" effect likely due to the influence of other abiotic factors at one of the sampling sites. Such relationship was found not significant when the outlier represented by Carrumeiro mussels was removed from the analysis. The most abundant free amino acid taurine (43.2-68.5%TFAA) followed an inverse variability of that of glycine and by extension of the group PFAA most likely as a compensatory decrease in mussel populations with low protein content (and high condition index). Accordingly, taurine:glycine (t:g) ratio varied between 1 and 2 in most mussel populations but increased up to 3.2-4.2 in Miranda and Bueu mussels at both ends of the geographical interval studied with a corresponding PAHs concentrations of 261 and 304 ng/g dw, respectively. These mussel populations with the highest t:g ratios were characterised by the lowest PFAA contents (below 40%) and condition index values (below 10%). Results of the present study established a significant link between energetic status of growing juveniles and FAA concentrations in environments with different pollution degrees. Variability of the free amino acids profiles in soft tissues were related to endogenous factors of juveniles (protein content, condition index) whereas no relationship with contamination values could be observed. The utility of t:g ratio as general condition factor for M. galloprovincialis is also corroborated for in situ growing juveniles.     

Surface carbohydrates of Eudiplozoon nipponicum pre- and post-fusion

The development of the monogenean Diplozoon (Nordmann, 1832) (Diplozoidae) necessitates fusion of two larval stages (diporpae) into one double organism. How diporpae find, distinguish and contact each other is unclear, nor is the nature of the stimuli responsible for the dedifferentiation of cells and the formation of new tissues at the site of somatic fusion. Previous studies have implied a role for carbohydrates and glycoproteins in the interactions between helminth parasites and their hosts. Hypothetically, glycoconjugates may also be involved in the establishment of parasite-parasite associations. Changes in the surface saccharide residues during the development of Eudiplozoon nipponicum, a gill ectoparasite of carp (Cyprinus carpio) are described. Flat-fixed specimens and sections of diporpae, juveniles (just-fused) and adult worms were examined following exposure to a panel of 12 FITC-conjugated lectins. All developmental stages exhibited a specific surface binding pattern with ten lectins, indicating that Man/Glc, GlcNAc, Gal and GalNAc are probably present on their surfaces. No reaction was observed with Fuc-specific lectins (UEA-I and LTA). There is evidence that parasite development is accompanied by both qualitative and quantitative changes in the saccharide pattern distribution. The diporpa sucker reacted with nine lectins, excluding BS-II. A very strong binding of PNA, LCA and ConA (Gal and Man/Glc-specific lectins) was observed with the papilla glands of juvenile worms. The role of glandular secretions in this unique fusion process is discussed.     

Agglutinin-mediated phagocytosis-associated generation of superoxide anion and nitric oxide by the hemocytes of the giant freshwater prawn Macrobrachium rosenbergii

Hemocyte mediated phagocytosis is one of the vital components of innate defence mechanisms in crustaceans and this phagocytic process is aided by serum agglutinins. However, literature on agglutinin mediated opsono-phagocytosis is unclear in the case of Macrobrachium rosenbergii hemocytes. Further, very few studies in the case of superoxide anion generation and none with regard to nitric oxide generation during phagocytosis exist among crustaceans. We investigated the occurrence of agglutinins in the serum and the role of serum agglutinins in mediating phagocytosis by the hemocytes. We show that the prawn serum possesses agglutinins that function as opsonins during phagocytosis of HB RBC by the hemocytes. Hemagglutination-inhibition assays revealed the specificity of serum agglutinins for N-acetylated hexoses, namely GalNAc, GlcNAc and ManNAc, with a higher affinity for ManNAc. In addition, ManNAc was able to inhibit the phagocytic response (by about 60%) of the hemocytes against serum pretreated HB RBC, wherein the serum was previously treated with ManNAc. We next investigated the ability of the hemocytes to generate superoxide anion and nitric oxide during HB RBC phagocytosis and results show generation of both these free radicals. In addition, there was an enhancement in generation (75% increase) of these free radicals during agglutinin mediated opsonophagocytosis, when compared to buffer treated targets and interestingly this enhanced generation was inhibited by ManNAc (27% for superoxide anion and 36% for nitric oxide), an inhibitory sugar for phagocytosis. Inhibition of phagocytosis induced superoxide anion generation by DPI (53%), sodium azide (56%) and tropolone (61%), reveals the possible involvement of NADPH-oxidases, peroxidases and probably phenoloxidases, respectively, in the generation of superoxide anion. Similarly, decrease in nitric oxide generation in the presence of l-NIO (47%) during phagocytosis lends support to the role of nitric oxide generation during cellular immune processes. These findings thus suggest a role for superoxide anion and nitric oxide in the innate defense mechanism, namely phagocytosis, in Macrobrachium rosenbergii.     

Helix pomatia lectin and annexin V,two molecular probes for insect microparticles: possible involvement in hemolymph coagulation

Insect hemolymph coagulation involves a complex reaction with contributions from hemocytes and soluble factors. Here we present evidence for the presence of microparticles in the coagulation reaction. These particles are formed by hemocytes in a calcium-dependent process. Both the particles and the remaining cells are labelled by annexin V indicating the presence of phosphatidylserine on the outer membrane. Microparticles are enriched in hemomucin, a surface protein of Drosophila hemocytes that is specifically recognised by a snail (Helix pomatia) lectin. Hemomucin is shown to bind to lipophorin, a multifunctional hemolymph molecule previously implied in coagulation. Our findings suggest similarities at a biochemical and cellular level between vertebrate blood and insect hemolymph coagulation.     

Nitrogen and carbohydrate pools of two rhizome types of Phragmites australis (Cav.) Trin. ex Steudel

Carbohydrate and total nitrogen contents as well as free amino acid (FAA) concentrations were determined in distinct types of rhizomes of two genetically homogeneous stands of reed (Phragmites australis (Cav.) Trin. ex Steudel) differing in morphology, productivity and nutrient supply in order to evaluate the storage capacity of vertical rhizomes and expansion rhizomes. The expansion rhizomes possess significantly higher amounts of FAA and of total nitrogen but similar carbohydrate concentrations in comparison to the vertical rhizomes. However, no significant differences were found for total nitrogen, FAA and total carbohydrates between both investigated stands indicating a comparable storage capacity of rhizomes independent of nutrient availability. Only the composition of the FAA pool varied in the alanine/asparagine ratio probably influenced by the oxygen supply of the rhizome/root system.