Two ACTH analogs exert differential effects on monocytes/macrophages function regulation in ayu (Plecoglossus altivelis).

Adrenocorticotropic hormone (ACTH), a bioactive peptide of the family of melanocortins, is generated from pro-opiomelanocortin (POMC). So far, the research on the specific functions of ACTH in the immune system of teleosts is limited. We determined two complementary DNA (cDNA) sequences of POMC in ayu (Plecoglossus altivelis), termed PaPOMC-A and PaPOMC-B. PaPOMCs transcripts occurred in all examined tissues, and their expression in immune tissues changed following experimental infection with Vibrio anguillarum. PaACTH-B, but not PaACTH-A, suppressed the phagocytosis of monocytes/macrophages (MO/MФ). Two isoforms of PaACTH increased the bactericidal capacity of MO/MФ. PaACTH-A increased anti-inflammatory cytokine expression, while PaACTH-B decreased pro-inflammatory cytokine expression in MO/MФ. Compared with PaACTH-B treatment, the PaACTH-A treatment improved survival rate and reduced the bacterial load in V. anguillarum-infected ayu through interleukin (IL)-10. Our results indicate that the two PaACTH isoforms exert different effects in the host defense against bacterial infection.

CXCR3.1 and CXCR3.2 Differentially Contribute to Macrophage Polarization in Teleost Fish.

The study of multiple copies of chemokine receptor genes in various teleosts has long appealed to investigators seeking to understand the evolution of the immune system. The CXCR CXCR3 gene has two isoforms, CXCR3.1 and CXCR3.2, which are both expressed in macrophages. The distinct roles of teleost CXCR3s have not been identified previously. In this article, we found that CXCR3.1 and CXCR3.2 differentially contributed to macrophage polarization in the teleosts: ayu (Plecoglossus altivelis), grass carp (Ctenopharyngodon idella), and spotted green pufferfish (Tetraodon nigroviridis). In ayu macrophages, the P. altivelis CXCR3.1 (PaCXCR3.1) gene was constitutively expressed, whereas the P. altivelis CXCR3.2 (PaCXCR3.2) gene was induced postinfection with Escherichia coli Upon E. coli infection, PaCXCR3.1+ and PaCXCR3.2+ macrophages showed an M1 and an M2 phenotype, respectively. CXCL9-11-like proteins mediated M1 and M2 polarization by interacting with the PaCXCR3.1 and PaCXCR3.2 proteins on macrophages, respectively. The transcription factors P. altivelis STAT1 and P. altivelis STAT3 were activated in PaCXCR3.1+ and PaCXCR3.2+ macrophages, respectively. Furthermore, the prognosis of septic ayu adoptively transferred with PaCXCR3.2+ macrophages was improved. Our data reveal a previously unknown mechanism for macrophage polarization, suggesting that redundant genes may regulate crucial functions in the teleost immune system.

A model system using confocal fluorescence microscopy for examining real-time intracellular sodium ion regulation.

The gills of euryhaline fish are the ultimate ionoregulatory tissue, achieving ion homeostasis despite rapid and significant changes in external salinity. Cellular handling of sodium is not only critical for salt and water balance but is also directly linked to other essential functions such as acid-base homeostasis and nitrogen excretion. However, although measurement of intracellular sodium ([Na(+)]i) is important for an understanding of gill transport function, it is challenging and subject to methodological artifacts. Using gill filaments from a model euryhaline fish, inanga (Galaxias maculatus), the suitability of the fluorescent dye CoroNa Green as a probe for measuring [Na(+)]i in intact ionocytes was confirmed via confocal microscopy. Cell viability was verified, optimal dye loading parameters were determined, and the dye-ion dissociation constant was measured. Application of the technique to freshwater- and 100% seawater-acclimated inanga showed salinity-dependent changes in branchial [Na(+)]i, whereas no significant differences in branchial [Na(+)]i were determined in 50% seawater-acclimated fish. This technique facilitates the examination of real-time changes in gill [Na(+)]i in response to environmental factors and may offer significant insight into key homeostatic functions associated with the fish gill and the principles of sodium ion transport in other tissues and organisms.

A transmembrane C-type lectin receptor mediates LECT2 effects on head kidney-derived monocytes/macrophages in a teleost, Plecoglossus altivelis.

Leukocyte cell-derived chemotaxin 2 (LECT2) is a multifunctional cytokine involved in many diseases in which immune dysfunction is present. Ayu LECT2 (PaLECT2), which interacts with a C-type lectin receptor (PaCLR), was shown to activate ayu head kidney-derived monocytes/macrophages (MO/MΦ) to improve the outcomes of fish upon bacterial infections. However, it is not known if PaCLR mediates PaLECT2 effects on ayu MO/MΦ. In this study, we determined the role of PaCLR in signal transduction of PaLECT2 on ayu MO/MΦ. We expressed the PaCLR ectodomain in Escherichia coli and produced a refolded recombinant protein (rPaCLR) that was then used to produce the anti-PaCLR IgG (anti-PaCLR) for neutralization. Addition of the refolded PaLECT2 mature peptide (rPaLECT2m) to ayu MO/MΦ cultures, increased cytokine expression, induced chemotaxis, and enhanced phagocytosis and bactericidal activity of these cells were observed. When we added anti-PaCLR to block the ectodomain of PaCLR, these effects were significantly inhibited. Based on our previous works and the data presented here, we conclude that PaCLR mediates the immunomodulatory effects of PaLECT2 on ayu MO/MΦ, thus defining a mechanism by which LECT2 protects fish against pathogens.

Physiological effects of salinity on Delta Smelt, Hypomesus transpacificus.

Abiotic factors like salinity are relevant to survival of pelagic fishes of the San Francisco Bay Estuary. We tested the effects of 4 parts per thousand (ppt) salinity increases on Delta Smelt (DS) in a laboratory experiment simulating salinity increases that might occur around the low-salinity zone (LSZ) (<6 ppt). Adult DS, fed 2% body mass per day, starting at 0.5 ppt [freshwater (FW)], were exposed to weekly step-increases of 4 ppt to a maximum of 10 ppt saltwater (SW) over 19 days, and compared to FW controls. DS (n = 12/treatment per sampling) were sampled at 24, 72, and 96 h (1, 3, and 4 days) post-salinity increase for analyses of hematocrit, plasma osmolality, muscle water content, gill chloride cell (CC) Na(+)/K(+)-ATPase (NKA) and apoptosis after being weighed and measured (n = 3 tanks per treatment). No apparent increase in length or weight occurred nor did a difference in survival. Following step-increases in SW, hematocrit increased over time. Other fish responses generally showed a pattern; specifically plasma osmolality became elevated at 1 day and diminished over 4 days in SW. Percent muscle water content (%) did not show significant changes. CCs showed increased NKA, cell size and apoptosis over time in SW, indicating that CCs turnover in DS. The cell renewal process takes days, at least over 19 days. In summary, DS are affected by salinities of the LSZ and ≤10 ppt, though they employ physiological strategies to acclimate.

A novel lipopolysaccharide-binding protein (LBP) gene from sweetfish Plecoglossus altivelis: molecular characterization and its role in the immune response of monocytes/macrophages.

Lipopolysaccharide-binding protein (LBP) belongs to the lipid transfer/LBP (LT-LBP) family, and plays a crucial role in the recognition of bacterial components that modulate cellular signals in phagocytic cells. Although several LBPs have been identified in teleosts, the effects of LBP homologs on teleost phagocytic cells are still obscure. Here, we report the cloning a novel full-length cDNA sequence of LBP-like protein (paLBP) gene from sweetfish, Plecoglossus altivelis. The paLBP cDNA encoded a 464 aa polypeptide, which was closest to that of rainbow smelt (Osmerus mordax). paLBP mRNA was detected mainly in the spleen, liver, and head kidney and levels dramatically increased in various tissues after Listonella anguillarum infection. In contrast to mammalian studies, paLBP mRNA could also be detected in sweetfish monocytes/macrophages. Recombinant paLBP showed LPS-binding activity and Western blot results revealed a significant increase of paLBP in the supernatant of sweetfish monocytes/macrophages challenged with L. anguillarum. Moreover, paLBP neutralization led to up-regulation of IL-1ß and TNF-α mRNA as well as respiratory burst activity in sweetfish monocytes/macrophages in response to L. anguillarum or LPS challenge. Therefore, these results suggest that paLBP is an inducible acute-phase protein mediating the immune response of sweetfish monocytes/macrophages upon bacterial challenge.

Physiological and biochemical strategies for withstanding emersion in two galaxiid fishes.

The galaxiid fishes of the Southern hemisphere display variable tolerance to aerial exposure. Brown mudfish (Neochanna apoda), for example, pseudoaestivate, inhabiting moist soil for months at a time, whereas inanga (Galaxias maculatus) emerse under unfavourable water conditions, but only for periods of a few hours. This study sought to identify the physiological and biochemical strategies that determine emersion tolerance in these species. Nitrogenous waste excretion was measured before and after an experimental emersion period (14 days for mudfish, 6 h for inanga). Both species showed significantly elevated ammonia "washout" upon return to water, but no increase in plasma or muscle ammonia. Post-emersion urea levels were elevated in plasma and muscle in both fish, however the extent of the accumulation did not indicate significant de novo urea production. This was supported by the lack of carbamoyl phosphate synthetase activity in tissues. Consequently, mudfish metabolism was examined to determine whether changes in parameters such as oxygen consumption, carbon dioxide excretion, and/or altered metabolic costs (represented by the key ionoregulatory enzyme Na(+), K(+)-ATPase; NKA) could explain emersion tolerance. Oxygen consumption rates, already very low in immersed mudfish, were largely maintained over the course of emersion. Carbon dioxide excretion decreased during emersion, and a small, but significant, decrease in NKA was noted. These data suggest that the extended emersion capacity of mudfish may result from a generally low metabolic rate that is maintained throughout aerial exposure via cutaneous gas exchange, and which limits the production of potentially toxic nitrogenous waste.

The importance of cutaneous gas exchange during aerial and aquatic respiration in galaxiids.

The Canterbury mudfish Neochanna burrowsius was found to be a pseudo-aestivating galaxiid with a low metabolic rate and significant cutaneous oxygen uptake (c. 43%) in both air and water. Another galaxiid, inanga Galaxias maculatus, had a higher metabolic rate in both media but the proportion of oxygen uptake met by cutaneous respiration rose significantly from 38 to 63% when the fish were exposed to air. Besides its important role in oxygen uptake, the skin of both species also contributed significantly to excretion of carbon dioxide in air, indicating the critical role of the integument as a respiratory tissue. In air, G. maculatus may increase cutaneous gas exchange to meet metabolic demands owing to the reduced utility of the gills, but as emersed G. maculatus were only able to maintain metabolic rates at c. 67% of that measured in water, this strategy probably only permits short-term survival. By contrast, the low and unchanging metabolic rate in water and air in N. burrowsius is a feature that may facilitate tolerance of long periods of emersion in the desiccating environments they inhabit.

Glycerol synthesis in freeze-resistant rainbow smelt: towards the characterization of a key enzyme glycerol-3-phosphatase.

Rainbow smelt (Osmerus mordax) synthesize high amounts of glycerol in winter as a cryoprotectant through the direct dephosphorylation of glycerol-3-phosphate by a phosphatase, glycerol-3-phosphatase (G3Pase). Such a protein is well described in a few species including fungi, bacteria and plants but never studied beyond tissue homogenates in any animal species. Purification, identification and characterization of this enzyme is thus crucial for a better comprehension of the biochemical adaptation in rainbow smelt in response to low temperature and more generally of the biochemical mechanisms involved in glycerol synthesis in animals. This work presents the first attempt to purify G3Pase from smelt liver, the main site of glycerol synthesis for the whole animal. A partial purification was performed, and some characteristics of the protein determined, including optimal pH, K(m) and cation requirements. Smelt G3Pase is most likely a low molecular weight, Mg⁺-dependent and cytosolic phosphatase.

Binding profiles and cytokine-inducing effects of fish rhamnose-binding lectins on Burkitt's lymphoma Raji cells.

Rhamnose-binding lectin (RBL) is one of the animal lectin categories which take part in the innate immune responses of fish. Osmerus lanceolatus lectin (OLL) from shishamo smelt eggs is an RBL composed of two tandem-repeated domains, both of which are considered to be a carbohydrate-recognition domain. SAL, catfish (Silurus asotus) egg RBL composed of three domains, binds to Burkitt's lymphoma Raji cells through globotriaosylceramide (Gb3) carbohydrate chain and to reduce cell size and growth by altering membrane composition without causing cell death. In this experiment, we tried to compare the binding effects of these two RBLs on Raji cells. Flow cytometric and fluorescence microscopic analyses revealed that OLL also directly bound to and shrunk Raji cells with ten times less reactivity than SAL but reduced cell growth with decreasing cell viability. Anti-Gb3 antibody completely blocked the binding of SAL to Raji cells but not that of OLL. In addition, the direct bindings of OLL and SAL to Raji cells were comparably inhibited by melibiose, but lactose was more effective inhibitor for the binding of OLL than that of SAL. These results suggest that OLL has slightly different cell-binding property compared with SAL and binds not only to Gb3 but also to the other carbohydrate receptor-bearing ß-galactoside chains. The quantitative RT-PCR analysis revealed that SAL induced the expression of TNF-α but not of IFN-γ, IL-1ß, and IL-10. Thus, SAL-induced cytostatic effect on Raji cells might be partially caused by TNF-α-mediated signaling pathway.

A complete biosynthetic pathway of the long-chain polyunsaturated fatty acids in an amphidromous fish, ayu sweetfish Plecoglossus altivelis (Stomiati; Osmeriformes).

The biosynthetic capability of the long-chain polyunsaturated fatty acids (LC-PUFA) in teleosts are highly diversified due to evolutionary events such as gene loss and subsequent neo- and/or sub-functionalisation of enzymes encoded by existing genes. In the present study, we have comprehensively characterised genes potentially involved in LC-PUFA biosynthesis, namely one front-end desaturase (fads2) and eight fatty acid elongases (elovl1a, elovl1b, elovl4a, elovl4b, elovl5, elovl7, elovl8a and elovl8b) from an amphidromous teleost, Ayu sweetfish, Plecoglossus altivelis. Functional analysis confirmed Fads2 with Δ6, Δ5 and Δ8 desaturase activities towards multiple PUFA substrates and several Elovl enzymes exhibited elongation capacities towards C18-20 or C18-22 PUFA substrates. Consequently, P. altivelis possesses a complete enzymatic capability to synthesise physiologically important LC-PUFA including arachidonic acid (ARA, 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) from their C18 precursors. Interestingly, the loss of elovl2 gene in P. altivelis was corroborated by genomic and phylogenetic analyses. However, this constraint would possibly be overcome by the function of alternative Elovl enzymes, such as Elovl1b, which has not hitherto been functionally characterised in teleosts. The present study contributes novel insights into LC-PUFA biosynthesis in the relatively understudied teleost group, Osmeriformes (Stomiati), thereby enhancing our understanding of the complement of LC-PUFA biosynthetic genes within teleosts.

Glycerol-3-phosphatase and not lipid recycling is the primary pathway in the accumulation of high concentrations of glycerol in rainbow smelt (Osmerus mordax).

Rainbow smelt is a small fish that accumulates glycerol in winter as a cryoprotectant when the animal is in seawater. Glycerol is synthesized in liver from different substrates that all lead to the formation of glycerol-3-phosphate (G3P). This study assesses whether glycerol is produced by a direct dephosphorylation of G3P by a phosphatase (G3Pase) or by a cycling through the glycerolipid pool followed by lipolysis. Foremost, concentrations of on-board glycerolipids and activity of G3Pase and of enzymes involved in lipid metabolism were measured in smelt liver over the glycerol cycle. Concentrations of on-board glycerolipids did not change over the cycle and were too low to significantly contribute directly to glycerol production but activities of enzymes involved in both potential pathways were up-regulated at the onset of glycerol accumulation. A second experiment conducted with isolated hepatic cells producing glycerol showed 1) that on-board glycerolipids were not sufficient to produce the glycerol released even though phospholipids could account for up to 17% of it, 2) that carbon cycling through the glycerolipid pool was not involved as glycerol was produced at similar rates following inhibition of this pathway, and 3) that G3Pase activity measured was sufficient to allow the synthesis of glycerol at the rate observed. These results are the first to clearly support G3Pase as the metabolic step leading to glycerol production in rainbow smelt and the first to provide strong support for a G3Pase in any animal species.

Molecular characterization of an IL-1ß gene from ayu, Plecoglossus altivelis.

IL-1ß plays a crucial role as a prototypical proinflammatory cytokine in immune responses and has been shown to affect macrophage functions. However, the effects of putative IL-1ß homologs on fish macrophages are still less known. Here, we cloned the full-length cDNA sequence of IL-1ß (aIL-1ß) gene from ayu, Plecoglossus altivelis. Phylogenetic analysis indicated that aIL-1ß was closest to that of Atlantic salmon (Salmo salar). Real-time quantitative PCR (RT-qPCR) revealed that aIL-1ß transcript was mainly expressed in spleen, head kidney and gill, and dramatically increased in various tissues after Listonella anguillarum infection. Subsequently, aIL-1ß was prokaryotic expressed and purified to prepare anti-aIL-1ß antibody. After L. anguillarum challenge, the aIL-1ß mRNA and protein levels were significantly up-regulated in ayu monocytes/macrophages. Moreover, aIL-1ß neutralization did not change phagocytic capability, but reduced bacterial killing capability in ayu head kidney-derived monocytes/macrophages. Therefore, aIL-1ß may play an important role in immune response of ayu, especially, contributing to bacterial killing of monocytes/macrophages.

Domain composition of rhamnose-binding lectin from shishamo smelt eggs and its carbohydrate-binding profiles.

Osmerus (Spirinchus) lanceolatus egg lectin (OLL) is a member of the rhamnose-binding lectin (RBL) family which is mainly found in aqueous beings. cDNA of OLL was cloned, and its genomic architecture was revealed. The deduced amino acid (aa) sequence indicated that OLL was composed of 213 aa including 95 aa of domain N and 97 aa of domain C. N and C showed 73 % sequence identity and contained both -ANYGR- and -DPC-KYL-peptide motifs which are conserved in most of the RBL carbohydrate recognition domains. The calculated molecular mass of mature OLL was 20,852, consistent with the result, and 20,677.716, from mass spectrometry. OLL was encoded by eight exons: exons 1 and 2 for a signal peptide; exons 3-5 and 6-8 for N- and C-domains, respectively. Surface plasmon resonance spectrometric analyses revealed that OLL showed comparable affinity for Galα- and ß-linkages, whereas Silurus asotus lectin (SAL), a catfish RBL, bound preferentially to α-linkages of neoglycoproteins. The Kd values of OLL and SAL against globotriaosylceramide (Gb3) were 1.69 × 10⁻5 M for and 2.81 × 10⁻6 M, respectively. Thus, the carbohydrate recognition property of OLL is slightly different from that of SAL. On the other hand, frontal affinity chromatography revealed that both OLL and SAL interacted with only glycolipid-type oligosaccharides such as Gb3 trisaccharides, not with N-linked oligosaccharides. The domain composition of these RBLs and an analytical environment such as the "cluster effect" of a ligand might influence the binding between RBL and sugar chains.

Transposon mutagenesis and genome sequencing identify two novel, tandem genes involved in the colony spreading of Flavobacterium collinsii, isolated from an ayu fish, Plecoglossus altivelis.

Bacteria of the family Flavobacteriaceae (flavobacteria) primarily comprise nonpathogenic bacteria that inhabit soil and water (both marine and freshwater). However, some bacterial species in the family, including Flavobacterium psychrophilum and Flavobacterium columnare, are known to be pathogenic to fish. Flavobacteria, including the abovementioned pathogenic bacteria, belong to the phylum Bacteroidota and possess two phylum-specific features, gliding motility and a protein secretion system, which are energized by a common motor complex. Herein, we focused on Flavobacterium collinsii (GiFuPREF103) isolated from a diseased fish (Plecoglossus altivelis). Genomic analysis of F. collinsii GiFuPREF103 revealed the presence of a type IX secretion system and additional genes associated with gliding motility and spreading. Using transposon mutagenesis, we isolated two mutants with altered colony morphology and colony spreading ability; these mutants had transposon insertions in pep25 and lbp26. The glycosylation material profiles revealed that these mutants lacked the high-molecular-weight glycosylated materials present in the wild-type strain. In addition, the wild-type strains exhibited fast cell population movement at the edge of the spreading colony, whereas reduced cell population behavior was observed in the pep25- and lbp26-mutant strains. In the aqueous environment, the surface layers of these mutant strains were more hydrophobic, and they formed biofilms with enhanced microcolony growth compared to those with the wild-type. In Flavobacterium johnsoniae, the Fjoh_0352 and Fjoh_0353 mutant strains were generated, which were based on the ortholog genes of pep25 and lbp26. In these F. johnsoniae mutants, as in F. collinsii GiFuPREF103, colonies with diminished spreading capacity were formed. Furthermore, cell population migration was observed at the edge of the colony in wild-type F. johnsoniae, whereas individual cells, and not cell populations, migrated in these mutant strains. The findings of the present study indicate that pep25 and lbp26 contribute to the colony spreading of F. collinsii.

Glycerol uptake is by passive diffusion in the heart but by facilitated transport in RBCs at high glycerol levels in cold acclimated rainbow smelt (Osmerus mordax).

Rainbow smelt (Osmerus mordax) is a small fish that accumulates glycerol at low winter seawater temperatures. In laboratory-held fish, glycerol concentration typically reaches 225 mM in plasma and in all cells. Glycerol uptake by the heart and red blood cells (RBCs) was assessed by tracking [(14)C(U)]glycerol into the acid-soluble pool. In fish acclimated to 9-10°C a decrease in perfusion/incubation temperature from 8 to 1°C resulted in a decrease in glycerol uptake with a Q(10) of 3.2 in heart and 2.4 in RBCs. Acclimation to ∼1.5°C did not result in an adaptive enhancement of glycerol uptake as rates were unchanged in heart and RBCs. Glycerol uptake at 1°C was by passive diffusion in heart as evidenced by a linear relationship between glycerol uptake and extracellular glycerol concentration and a lack of inhibition by phloretin. In contrast, in RBCs, glycerol uptake with respect to glycerol concentration showed two linear relationships with a transition point around 50 mM extracellular glycerol. The slope of the second phase was much steeper and eliminated with the inclusion of phloretin. In RBCs from Atlantic salmon (Salmo salar), a related species that does not accumulate glycerol, glycerol uptake showed only a single linear curve and was not inhibited by phloretin. The data imply a strong facilitated component to glycerol uptake in rainbow smelt RBCs at high glycerol concentrations. We propose this is related to cyclic changes in RBC glycerol content involving a loss of glycerol at the gill and a reaccumulation during passage through the liver.

Influence of acute cadmium exposure on the liver proteome of a teleost fish, ayu (Plecoglossus altivelis).

Cadmium (Cd) is a toxic heavy metal that causes the disruption of a variety of physiological processes. In this study, the effect of Cd on liver proteome of ayu, Plecoglossus altivelis, was investigated by two-dimensional gel electrophoresis (2-DE) and matrix assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS). Twenty-three altered protein spots were successfully identified. They were involved in oxidative stress response, metal metabolism, methylation, and so on. The mRNA expression of 60S acidic ribosomal protein P0, heat shock protein 70, apolipoprotein A-I, betaine-homocysteine S-methyltransferase, parahox cluster neighbor, and transferrin was subsequently determined by real-time PCR. The mRNA expression of these genes was consistent with proteomic results. These findings enrich our knowledge on the influence of Cd toxicity to teleost fish, and may be worthy of further investigation to develop biomarkers.

A novel oxyconforming response in the freshwater fish Galaxias maculatus.

How fish oxygen consumption is modulated by external PO(2) has long been a matter of interest, yet is an experimentally complicated question to answer. In this study closed and semi-closed respirometry were used to evaluate the oxygen consumption rate of the scaleless galaxiid fish, inanga (Galaxias maculatus) as a function of decreasing external PO(2). Both respirometry techniques showed that as environmental oxygen levels declined, oxygen consumption rates also decreased. At no point did inanga regulate oxygen consumption. This is strong evidence that inanga is an oxyconformer. Partitioned respirometry experiments showed that skin plays an important role in oxygen uptake in this fish species, and cutaneous oxygen uptake may have an important role in shaping the oxygen consumption response to hypoxia.

Cystidicola farionis, a Swim Bladder Parasite of European Smelt: Characterization of the Nematode Trehalose Strategy.

The molecular identification of Cystidicola farionis (a swim bladder nematode of European smelt from the Vistula Lagoon in Poland) was performed. Their prevalence level was determined, and changes in the trehalose synthesis pathway in larvae and adult nematodes were demonstrated. The trehalose level was almost four times higher in adult nematodes than in larvae. In contrast, the activity of both enzymes (trehalose 6-phosphate synthase, TPS and trehalose 6-phosphate phosphatase, TPP) involved in the synthesis of trehalose was higher in larvae than in adults under optimal conditions. The optimum pH for TPS isolated from larvae and adults was pH 7.0. The optimum pH for TPP from larvae and adults was pH 7.0 and pH 8.0, respectively. The optimal temperature was 20 °C, and Mg2+ ions were an activator for trehalose-synthetizing enzymes from both sources. Enzymes isolated from adult nematodes were less susceptible to divalent ion chelator and inorganic phosphate than larval enzymes. The dynamic transformation of trehalose in the nematode developing inside the swim bladder of the smelt appears to be an important metabolic pathway in the nematode survival strategy. These studies are aimed at a better understanding of the issue of the metabolic adaptation of parasites, which, in the future, may indirectly contribute to the elimination of the parasite from aquacultures, which will impact public health.

Glycerol loss to water exceeds glycerol catabolism via glycerol kinase in freeze-resistant rainbow smelt (Osmerus mordax).

Rainbow smelt accumulate high amounts of glycerol in winter. In smelt, there is a predictable profile of plasma glycerol levels that starts to increase in November (<5 µmol/ml), peaks in mid-February (>200 µmol/ml), and thereafter decreases to reach the initial levels in the beginning of May. The aim of this study was to investigate the respective role of the two main mechanisms that might be involved in glycerol clearance from mid-February: 1) breakdown of glycerol to glycerol-3-phosphate through the action of the glycerol kinase (GK) and 2) direct loss toward the environment. Over the entire glycerol cycle, loss to water represents a daily loss of ∼10% of the total glycerol content of fish. GK activities were very low in all tissues investigated and likely have a minor quantitative role in the glycerol cycle. These results suggest that glycerol levels are dictated by the rate of glycerol synthesis (accelerated and deactivated during the accumulation and decrease stages, respectively). Although not important in glycerol clearance, GK in liver might have an important metabolic function for other purposes, such as gluconeogenesis, as evidenced by the significant increase of activity at the end of the cycle.