Heterologous expression,refolding and functional characterization of two antifreeze proteins from Fragilariopsis cylindrus (Bacillariophyceae)

Antifreeze proteins (AFPs) provide protection for organisms subjected to the presence of ice crystals. The psychrophilic diatom Fragilariopsis cylindrus which is frequently found in polar sea ice carries a multitude of AFP isoforms. In this study we report the heterologous expression of two antifreeze protein isoforms from F. cylindrus in Escherichia coli. Refolding from inclusion bodies produced proteins functionally active with respect to crystal deformation, recrystallization inhibition and thermal hysteresis. We observed a reduction of activity in the presence of the pelB leader peptide in comparison with the GS-linked SUMO-tag. Activity was positively correlated to protein concentration and buffer salinity. Thermal hysteresis and crystal deformation habit suggest the affiliation of the proteins to the hyperactive group of AFPs. One isoform, carrying a signal peptide for secretion, produced a thermal hysteresis up to 1.53 °C ± 0.53 °C and ice crystals of hexagonal bipyramidal shape. The second isoform, which has a long preceding N-terminal sequence of unknown function, produced thermal hysteresis of up to 2.34 °C ± 0.25 °C. Ice crystals grew in form of a hexagonal column in presence of this protein. The different sequences preceding the ice binding domain point to distinct localizations of the proteins inside or outside the cell. We thus propose that AFPs have different functions in vivo, also reflected in their specific TH capability.     

Characterization of an antifreeze protein from the polar diatom Fragilariopsis cylindrus and its relevance in sea ice

Antifreeze proteins (AFPs), characterized by their ability to separate the melting and growth temperatures of ice and to inhibit ice recrystallization, play an important role in cold adaptation of several polar and cold-tolerant organisms. Recently, a multigene family of AFP genes was found in the diatom Fragilariopsis cylindrus, a dominant species within polar sea ice assemblages. This study presents the AFP from F. cylindrus set in a molecular and crystallographic frame. Differential protein expression after exposure of the diatoms to environmentally relevant conditions underlined the importance of certain AFP isoforms in response to cold. Analyses of the recombinant AFP showed freezing point depression comparable to the activity of other moderate AFPs and further enhanced by salt (up to 0.9 °C in low salinity buffer, 2.5 °C at high salinity). However, unlike other moderate AFPs, its fastest growth direction is perpendicular to the c-axis. The protein also caused strong inhibition of recrystallization at concentrations of 1.2 and 0.12 μM at low and high salinity, respectively. Observations of crystal habit modifications and pitting activity suggested binding of AFPs to multiple faces of the ice crystals. Further analyses showed striations caused by AFPs, interpreted as inclusion in the ice. We suggest that the influence on ice microstructure is the main characteristic of these AFPs in sea ice.     

Antifreeze proteins in polar sea ice diatoms: diversity and gene expression in the genus Fragilariopsis

Fragilariopsis is a dominating psychrophilic diatom genus in polar sea ice. The two species Fragilariopsis cylindrus and Fragilariopsis curta are able to grow and divide below freezing temperature of sea water and above average sea water salinity. Here we show that antifreeze proteins (AFPs), involved in cold adaptation in several psychrophilic organisms, are widespread in the two polar species. The presence of AFP genes (afps) as a multigene family indicated the importance of this group of genes for the genus Fragilariopsis, possibly contributing to its success in sea ice. Protein phylogeny showed the potential mobility of afps, which appear to have crossed kingdom and domain borders, occurring in Bacteria, diatoms, crustaceans and fungi. Our results revealed a broad distribution of AFPs not only in polar organisms but also in taxa apparently not related to cold environments, suggesting that these proteins may be multifunctional. The relevance of AFPs to Fragilariopsis was also shown by gene expression analysis. Under stress conditions typical for sea ice, with subzero temperatures and high salinities, F. cylindrus and F. curta strongly expressed selected afps. An E/G point mutation in the Fragilariopsis AFPs may play a role in gene expression activity and protein function.     

Heterologous expression, refolding and functional characterization of two antifreeze proteins from Fragilariopsis cylindrus (Bacillariophyceae)

Antifreeze proteins (AFPs) provide protection for organisms subjected to the presence of ice crystals. The psychrophilic diatom Fragilariopsis cylindrus which is frequently found in polar sea ice carries a multitude of AFP isoforms. In this study we report the heterologous expression of two antifreeze protein isoforms from F. cylindrus in Escherichia coli. Refolding from inclusion bodies produced proteins functionally active with respect to crystal deformation, recrystallization inhibition and thermal hysteresis. We observed a reduction of activity in the presence of the pelB leader peptide in comparison with the GS-linked SUMO-tag. Activity was positively correlated to protein concentration and buffer salinity. Thermal hysteresis and crystal deformation habit suggest the affiliation of the proteins to the hyperactive group of AFPs. One isoform, carrying a signal peptide for secretion, produced a thermal hysteresis up to 1.53 °C ± 0.53 °C and ice crystals of hexagonal bipyramidal shape. The second isoform, which has a long preceding N-terminal sequence of unknown function, produced thermal hysteresis of up to 2.34 °C ± 0.25 °C. Ice crystals grew in form of a hexagonal column in presence of this protein. The different sequences preceding the ice binding domain point to distinct localizations of the proteins inside or outside the cell. We thus propose that AFPs have different functions in vivo, also reflected in their specific TH capability.     

Antifreeze activity enhancement by site directed mutagenesis on an antifreeze protein from the beetle Rhagium mordax

The ice binding motifs of insect antifreeze proteins (AFPs) mainly consist of repetitive TxT motifs aligned on a flat face of the protein. However, these motifs often contain non-threonines that disrupt the TxT pattern. We substituted two such disruptive amino acids located in the ice binding face of an AFP from Rhagium mordax with threonine. Furthermore, a mutant with an extra ice facing TxT motif was constructed. These mutants showed enhanced antifreeze activity compared to the wild type at low concentrations. However, extrapolating the data indicates that the wild type will become the most active at concentrations above 270 μmol.     

Environmental and climatic changes in the central Mediterranean Sea (Siculo–Tunisian Strait) during the last 30 ka based on dinoflagellate cyst and planktonic foraminifera assemblages

A high resolution micropalaeontological study of the core MD 04-2797 CQ recovered in the Sicilian–Tunisian Strait provides insights into the paleoclimatic history of the Mediterranean Sea at the transition between the western and eastern basin over the last 30 ka. Using the analysis of dinoflagellate cyst and planktonic foraminiferal assemblages, we reconstruct the paleoenvironmental changes that took place in this region. High abundances of cold temperate dinocyst species (Nematosphaeropsis labyrinthus, Spiniferites elongatus, Bitectatodinium tepikiense) and the polar planktonic foraminifera Neogloboquadrina pachyderma (left coiling) reveal three major cooling events synchronous with North Atlantic Henrich events 1 and 2 (H1 and H2) and the European and North Atlantic Younger Dryas event. During the Holocene, the presence of warm dinocyst species (Spiniferites mirabilis and Impagidinium aculeatum) and planktonic foraminifera (Globorotalia inflata and Globigerinoides ruber), reflects a significant increase of sea surface temperatures in the western Mediterranean basin, but a full warming was not recorded until 1500 years after the onset of the Holocene. Moreover, our results show that the Holocene was interrupted by at least four brief cooling events at ~ 9.2 ka, ~ 8 ka, ~ 7 ka and ~ 2.2 ka cal. BP, which may be correlated to climatic events recorded in Greenland ice cores and in the Atlantic Ocean.     

Perturbation of bacterial ice nucleation activity by a grass antifreeze protein

Certain plant-associating bacteria produce ice nucleation proteins (INPs) which allow the crystallization of water at high subzero temperatures. Many of these microbes are considered plant pathogens since the formed ice can damage tissues, allowing access to nutrients. Intriguingly, certain plants that host these bacteria synthesize antifreeze proteins (AFPs). Once freezing has occurred, plant AFPs likely function to inhibit the growth of large damaging ice crystals. However, we postulated that such AFPs might also serve as defensive mechanisms against bacterial-mediated ice nucleation. Recombinant AFP derived from the perennial ryegrass Lolium perenne (LpAFP) was combined with INP preparations originating from the grass epiphyte, Pseudomonas syringae. The presence of INPs had no effect on AFP activity, including thermal hysteresis and ice recrystallization inhibition. Strikingly, the ice nucleation point of the INP was depressed up to 1.9 °C in the presence of LpAFP, but a recombinant fish AFP did not lower the INP-imposed freezing point. Assays with mutant LpAFPs and the visualization of bacterially-displayed fluorescent plant AFP suggest that INP and LpAFP can interact. Thus, we postulate that in addition to controlling ice growth, plant AFPs may also function as a defensive strategy against the damaging effects of ice-nucleating bacteria.     

Effect of salinity and seawater calcite saturation state on Mg and Sr incorporation in cultured planktonic foraminifera

Trace elements incorporated in planktonic foraminiferal test carbonate are commonly used as paleoproxies. For instance, Mg/Ca ratios are frequently used for reconstructing sea surface temperature and, together with the foraminiferal stable oxygen isotope ratios, are also used as paleosalinity proxy. Foraminiferal Sr/Ca ratios constitute another example of the application of trace elements in paleostudies since they may reflect the Sr/Ca values of seawater. However, over the past few decades it has been proven that the incorporation of trace elements in foraminiferal calcite is controlled by more than one environmental parameter. To quantify the effect of salinity on Mg and Sr incorporation planktonic foraminifera Globigerinoides sacculifer (sensu stricto) were grown in the laboratory under different environmental conditions. Laboratory experiments allowed us to separate a direct salinity effect from a possible independent impact through differences in the calcite saturation state of the seawater (Ω). Although the temperature effect is more important than the salinity effect, a change of 4 salinity units is equivalent to a 1 °C bias on Mg/Ca-based temperatures. This effect of salinity on Mg incorporation is minor. However, when using Mg/Ca-based temperatures in combination with foraminiferal δ¹⁸O to calculate salinity, it cannot be neglected. The present study shows salinity as the overriding control on Mg incorporation within the range of Ω studied (Ω between 5.25 and 6.50; [CO₃²⁻] between 218 and 270 μmol/kg) at a constant temperature of 26 °C. In contrast, Ω appears to be the main control on foraminiferal Sr incorporation (0.10 mmol/mol per 100 µmol/kg rise in [CO₃²⁻]), whereas salinity has a non significant influence on Sr/Ca.     

Effects of salt,alkali and salt–alkali mixed stresses on seed germination of the halophyte Salsola ferganica (Chenopodiaceae)

Salsola ferganica L. (Chenopodianceae) is an annual halophytic species. Experiments were carried out in laboratory to determine the effects of temperature, perianths and various types of salinity on seed germination and germination recovery. Seeds were germinated at 6 levels of temperature with perianths, plus perianths and removed perianths in complete darkness for 9 days. The germination responses of the seeds without perianths at 25 °C were determined over a wide range of NaCl, NaHCO₃ or NaCl–NaHCO₃ mixed stress for 13 days. Perianths seriously affected germination as a barrier for seed germination and the optimal temperature was at 25 °C. Highest germination percentage was obtained under control and seed germination was progressively inhibited with the increase of salinity concentration. The negative effect of NaHCO₃ at the same concentration on germination was stronger than that of NaCl and NaCl–NaHCO₃ mixed. When substrate salinity was removed, seeds exposed to a high NaCl concentration (400–800 mM), NaHCO₃ (50–200 mM) and NaCl–NaHCO₃ mixed (100–400 mM) germinated well. Final germination of Salsola ferganica seeds was significantly affected by types of salt at the low salinity (?200 mM) and with increased salinity it was influenced mainly by salinity concentration for various proportion of salt–alkali mixed stress.     

Effects of salt stress on volatile compounds,total phenolic content and antioxidant activities of Salvia mirzayanii

Salvia mirzayanii is a medicinal and aromatic plant belonging to the Lamiaceae family, which is an endemic plant in Iran. In this study, the effects of different salt concentrations on total phenolic content, antioxidant activities and volatile components of the aerial parts of S. mirzayanii were studied. The results showed that total phenolic content increased with the increase in salt concentration. The increase was more pronounced under moderate salinity (3.8 mg GAE g^− 1 DW at 6.8 dS m^− 1 NaCl). Plants grown at 6.8 dS m^− 1 NaCl displayed the highest DPPH˚ scavenging activity with the lowest IC₅₀ value (2.13 mg ml^− 1) compared to the control. The volatile components were identified and analyzed by HS (headspace)-GC–MS using the Combi PAL System technique. The main components of control plants were α-terpinyl acetate, 1,8-cineole and bicyclogermacrene. The proportions of these main compounds were differently affected by salinity stress. The results showed that the synthesis of both total phenolic and some important volatile components was induced by moderate salinity.     

Caulerpa taxifolia responses to hyposalinity stress

An investigation into salinity responses of Caulerpa taxifolia was undertaken in a series of laboratory trials to evaluate the use of hyposalinity stress as an eradication strategy. The effect of instantaneous (or shock) exposure to reduced salinity (10 ppt) on the effective quantum yield (EQY) of C. taxifolia for different incubation periods (15, 30, 45, 60, 90, 180, 360, 720 and 1440 min) indicated that 180 min or more at this salinity was required to kill the alga. Average EQY declined by 88.2 ± 4.6% (mean ± S.E.) of the pre-treatment level for the 180 min treatment but were as much as 96.6 ± 2.5% lower than pre-treatment EQY for the 1440 min exposure. Exposure for 90 min or less resulted in an intermediate response, whereas lesser exposures (60, 30 and 15 min) had no lasting effect on C. taxifolia health. The effect of gradual changes in salinity, as might be anticipated in an eradication scenario, on EQY of C. taxifolia was investigated through the dilution of seawater (35 ppt) over different time scales (5 h, 2, 4, 7 and 27 days). In all trials >5 h, the response to hyposalinity was the same regardless of the rate of change in salinity with all treatments resulting in a marked loss in EQY below ∼15 ppt. Declines in the average ratio of EQY after to EQY before for 4, 7 and 27 days treatments (85.2 ± 8.2%, 78.8 ± 9.05% and 77.3 ± 18.2% of pre-treatment levels, respectively), were significantly larger than the 5 h treatment (2.6 ± 4.4% of pre-treatment levels). The 2 days salinity reduction (48.5 ± 17.1%) resulted in an intermediate response. In the 5 h treatment, the exposure to salinities below 15 ppt was less than 3 h, which given the result of the preceding trial explains the lack of substantial EQY response as the minimum exposure period required to kill the alga at 10 ppt is ≥180 min. There is thus no evidence that C. taxifolia is capable of acclimation to gradual reductions in salinity. Consequently, hyposalinity is an effective means of killing the algae and may prove highly effective for populations in relatively small, contained water bodies.     

Interactive effects of salinity and water depth on the growth of Melaleuca ericifolia Sm. (Swamp paperbark) seedlings

Melaleuca ericifolia Sm. (Swamp paperbark) is a common tree species in freshwater and brackish wetlands in southern and eastern Australia. The survival of this species in many wetlands is now threatened by increased salinity and inappropriate water regimes. We examined the response of 5-month-old M. ericifolia seedlings to three water depths (exposed, waterlogged and submerged) at three salinities (2, 49 and 60 dS m⁻¹). Increasing water depth at the lowest salinity did not affect survival, but strongly inhibited seedling growth. Total biomass, leaf area and maximum root length were highest in exposed plants, intermediate in waterlogged plants and lowest in submerged plants. Although completely submerged plants survived for 10 weeks at the lowest salinity, they demonstrated negative growth rates and were unable to extend their shoots above the water surface. At the higher salinities, M. ericifolia seedlings were intolerant of waterlogging and submergence: all plants died after 9 weeks at 60 dS m⁻¹. Soil salinities increased over time, and by Week 10, exceeded external water column salinities in both the exposed and waterlogged treatments. In exposed sediment, ∼90% of plants survived for 10 weeks at 60 dS m⁻¹ even though soil salinities reached ∼76 dS m⁻¹. No mortality occurred in the exposed plants at 49 dS m⁻¹, and small but positive relative growth rates were recorded at Week 10. We conclude that at low salinities M. ericifolia seedlings are highly tolerant of sediment waterlogging, but are unlikely to tolerate prolonged submergence. However, at the higher salinities, M. ericifolia seedlings are intolerant of waterlogging and submergence and died rapidly after 5 weeks exposure to this combination of environmental stressors. This research demonstrates that salinity may restrict the range of water regimes tolerated by aquatic plants.     

Enhancing the activity and thermostability of thermostable β-glucosidase from a marine Aspergillus niger at high salinity

To evaluate the effect of salinity on the catalyzing ability of β-glucosidase in the marine fungus Aspergillus niger, the thermodynamic parameters of the β-glucosidase were investigated at different salinities. At the optimum salinity of 6% NaCl (w/v) solution, the optimum temperature and pH of the β-glucosidase activity was 66 °C and 5.0, respectively. Under these conditions, the β-glucosidase activity increased 1.46 fold. The half-life of denaturation in 6% NaCl (w/v) solution was approximately twice as long as that in NaCl free solution. The Gibb's free energy for denaturation, ΔG, was 2 kJ/mol higher in 6% NaCl (w/v) solution than in NaCl free solution. The melting point (68.51 °C) in 6% NaCl (w/v) solution was 1.71 °C higher than that (66.80 °C) in NaCl free solution. Similarly, the activity and thermostability of the pure β-glucosidase increased remarkably at high salinity. The thermostable β-glucosidase, of which the activity and the thermostability are remarkably enhanced at high salinity, is valuable for industrial hydrolyzation of cellulose in high salinity environments.     

Vitrification as a method for genome resource banking oocytes from the endangered Tasmanian devil (Sarcophilus harrisii)

Populations of Australia’s largest terrestrial marsupial carnivore, the Tasmanian devil (Sarcophilus harrisii), are rapidly declining in the wild due to Tasmanian Devil Facial Tumour Disease (TDFTD). One tool which can reduce the loss of genetic diversity is genome resource banking. This study examines the application of an oocyte vitrification protocol, initially developed in a model marsupial carnivore, to the endangered Tasmanian devil. Ovarian tissue was transported to the laboratory on ice from Tasmania which took up to 48 h. Individual granulosa oocyte complexes (GOC) were isolated enzymatically and the viability of oocytes from primary GOC was assessed immediately following isolation or after exposure to cold shock, vitrification and thawing media without exposure to liquid nitrogen or the full vitrification and thawing process. There was no decline in oocyte viability following cold shock or exposure to the vitrification and thawing media. Following the full vitrification and thawing process there was a decline in oocyte viability (χ² = 20.0, P < 0.001) but approximately 70% of oocytes remained viable. This study provides further evidence that oocyte vitrification is a promising strategy for genome resource banking in carnivorous marsupials and suggests that it should be considered in conservation plans for the survival of the iconic Tasmanian devil.     

Distribution of large Emiliania huxleyi in the Central and Northeast Atlantic as a tracer of surface ocean dynamics during the last 25,000 years

The coccolithophore species Emiliania huxleyi is characterized by a wide range of sizes, which can be easily distinguished in the light microscope. In this study we have quantified the abundance of large (coccoliths > 4 µm in maximum length) E. huxleyi specimens during the last 25 kyr in sedimentary records from eleven cores and drill sites in the NE Atlantic and W Mediterranean Sea, to prove its usefulness in the reconstruction of water mass dynamics and biostratigraphic potential.During the Last Glacial Maximum this large form, a cold-water indicator, was common in the NE Atlantic and Mediterranean, and its regional variation in abundance indicates a displacement of the climatic zones southwards in agreement with the development of ice sheets and sea ice in the Northern Hemisphere during this period. On the other hand, the gradient between northern and southern surface water masses in the Subtropical Gyre appears to have been more pronounced than at present, while the Portugal and Canary Currents were more intense. In the western Mediterranean basin temperatures were cooler than in the adjacent Atlantic, provoking a quasi-endemism of these specimens until the end of Heinrich Event 1. This may have been due to a restriction in the communication between the Atlantic and Mediterranean through the Strait of Gibraltar, the arrival of cold surface water and the amplification of cooling after the development of ice sheets in the Northern Hemisphere.During the deglaciation, large E. huxleyi specimens decreased in abundance at medium and low latitudes, but were still numerous close to the Subarctic region during the Holocene. In transitional waters this decrease to present day abundances occurred after Termination Ib.The abrupt change in abundance of this large E. huxleyi form is proposed as a new biostratigraphic event to characterize the Holocene in mid- to low-latitude water masses in the North Atlantic, although this horizon seems to be diachronous by 5 kyr from tropical to subarctic regions, in agreement with the gradual onset of warm conditions.     

Succinic acid production from sucrose and molasses by metabolically engineered E. coli using a cell surface display system

To achieve sucrose-metabolizing capability, different sucrose utilization operons have been introduced into E. coli that cannot utilize sucrose. However, these engineered strains still suffer from low growth rates and low sucrose uptake rates. In this study, cell surface display system was adopted in engineered E. coli AFP111 for succinic acid production from sucrose and molasses directly. Invertase (CscA) from E. coli W was successfully anchored to outer membrane by fusion with OmpC anchoring motif, and the displayed CscA showed high extracellular activity. Compared with the sucrose permease system, the cell surface display system consumed less ATP during sucrose metabolism. When less ATP was consumed by AFP111/pTrcC-cscA, the succinic acid productivity from sucrose was 23% higher than that by AFP111/pCR2.1-cscBKA that having the sucrose permease system. As a result, 41 g L⁻¹ and 36.3 g L⁻¹ succinic acid were produced by AFP111/pTrcC-cscA from sucrose and sugarcane molasses respectively at 34 h in 3-L fermentor during dual-phase fermentation. In addition, 79 g L⁻¹ succinic acid was accumulated with recovered AFP111/pTrcC-cscA cells at the end of dual-phase fermentation in 3-L fermentor, and the overall yield was 1.19 mol mol⁻¹ hexose.     

Tarbellastraea (Scleractinia): A new stable isotope archive for Late Miocene paleoenvironments in the Mediterranean

Geochemical proxy records of sea surface temperature (SST) or sea surface salinity (SSS) variability on intra- and interannual time-scales in corals from geological periods older than Pleistocene are extremely rare due to pervasive diagenetic alteration of coralline aragonite. Very recently, however, stable isotope data (δ¹⁸O, δ¹³C) from specimens of Porites of Late Miocene age (10 Ma) have been shown to preserve original environmental signatures. In this paper we describe new finds of the zooxanthellate corals Porites and Tarbellastraea in exceptional aragonite preservation from the island of Crete in sediments of Tortonian (～ 9 Ma) and Early Messinian (～ 7 Ma) age. Systematic, comparative stable isotope analysis of massive Tarbellastraea and Porites sampled from the same beds and localities reveal identical stable isotope fractionation patterns in both genera. Therefore, extinct Tarbellastraea represents an additional environmental archive fully compatible and mutually exchangeable with Porites. Provided that seasonal variations in δ¹⁸O reflect SST changes only, seasonal SST contrasts of 7.3 °C for the Tortonian and 4.8 °C for the Early Messinian are inferred, implying warmer summer and cooler winter SSTs during the Tortonian than during the Messinian. However, reduced δ¹⁸O seasonality (1.1‰ in the Tortonian and 0.7‰ in the Messinian) and slightly less negative mean δ¹⁸O in Messinian corals (? 2.4‰) compared to Tortonian specimens (? 2.7‰) may not necessarily indicate a long-term fall in SSTs, but changes in surface water δ¹⁸O, i.e. global ice build-up or enhanced evaporation during summer or increased precipitation/river discharge during winter and changes in insolation. On the other hand, coral communities of Tortonian and Messinian age in central Crete are identical, and compatible annual extension rates indicate similar average SSTs during the two investigated time periods. In addition, lithological and paleobotanical data from Central Crete document a change from humid to dry climatic conditions during the Late Miocene. Therefore, a likely explanation for the observed shift in coral mean δ¹⁸O and reduced δ¹⁸O seasonality from the Tortonian to the Early Messinian is a change in ambient seawater δ¹⁸O caused by a change in the hydrological balance towards high evaporation/high salinity during summer.     

Structural characteristics of a novel antifreeze protein from the longhorn beetle Rhagium inquisitor

Antifreeze proteins (AFPs) are characterized by their capacity to inhibit the growth of ice and are produced by a variety of polar fish, terrestrial arthropods and other organisms inhabiting cold environments. This capacity reflects their role as stabilizers of supercooled body fluids. The longhorn beetle Rhagium inquisitor is known to express AFPs in its body fluids. In this work we report on the primary structure and structural characteristics of a 12.8 kDa AFP from this beetle (RiAFP). It has a high capacity to evoke antifreeze activity as compared to other known insect AFPs and it is structurally unique in several aspects. In contrast to the high content of disulfide bond-formation observed in other coleopteran AFPs, RiAFP contains only a single such bond. Six internal repeat segments of a thirteen residue repeat pattern is irregularly spaced apart throughout its sequence. The central part of these repeat segments is preserved as TxTxTxT, which is effectively an expansion of the TxT ice-binding motif found in the AFPs of several known insect AFPs.     

A new record of Myxobolus brachysporus and M. israelensis in the tilapia (Oreochromis niloticus) collected from the Nile River,Egypt

The present study was carried out as part of an ongoing general survey for myxosporean parasites infecting tilapias in the River Nile, Egypt. In the present study, 77 Nile tilapia (Oreochromis niloticus) were collected from boat landing sites at Beni-Suef governorate, Egypt and examined for the myxosporean infection. The infection was encountered as a huge number of free spores in the kidney and the spleen. The infection showed a prevalence of 51.9% (40/77) for Myxobolus brachysporus while it was 25.9% (20/77) for Myxobolus israelensis. Mature spores of M. brachysporus were ellipsoidal and measured 8.6 × 13.2 μm. The polar capsules were subcircular with 5–6 filament turns and measured 4.7 × 3.6 μm. Spores of M. israelensis were ellipsoidal in the frontal view and fusiform in the lateral view. Spore measurements were 13.4 μm long and 8.7 μm wide. The polar capsules were elongated with 6–7 filament coils and measured 8.6 × 3.1 μm. The findings presented here proved that tilapia fishes in the Nile River are still suffering from infections with Myxobolus species. Therefore, further studies should be carried out to survey the Myxobolus infection among tilapias under culture conditions to clarify the pathological impacts of this parasite in tilapias aquaculture.     

Effect of environmental and nutritional factors on growth and azaspiracid production of the dinoflagellate Azadinium spinosum

Azadinium spinosum, a small dinoflagellate isolated from the North Sea, is a producer of azaspiracids (AZAs), a group of biotoxins associated with human illness following ingestion of contaminated shellfish. Using batch and continuous cultures of A. spinosum, the present study investigated the effects of different environmental and nutritional factors (salinity, temperature, photon flux density, aeration, culture media, nitrogen sources, phosphate source, and N/P ratios) on growth, maximum cell concentration, and AZA cell quota.Azadinium spinosum grew in a wide range of conditions; from 10 ̊C to 26 ̊C and salinities from 30 to 40, under irradiances ranging from 50 μmol m⁻² s⁻¹ to 250 μmol m⁻² s⁻¹, with or without aeration. Growth and maximum cell concentration were highest at a salinity of 35, at temperatures between 18 ̊C and 22 ̊C, and with aeration. Concerning AZA cell quota, the most significant effect was observed at low temperature; the AZA cell quota was more than 20 times higher at 10 ̊C (220 fg cell⁻¹) than at temperatures between 18 ̊C and 26 ̊C. A. spinosum grew on all media tested with only slight differences in growth rate and AZA cell quota. In continuous culture, lowering the concentration of nutrients (0.5 strength of a modified K-medium) in the inflow improved AZA cell quota whereas higher concentration (doubling the normal strength of K-medium) improved maximal cell concentration. A. spinosum grew on different sources of nitrogen tested (nitrate, urea, ammonium) with almost no effect on toxin cell quota and growth, except that adding ammonium caused a decrease in growth.These first experiments on Azadinium spinosum increased our knowledge on factors affecting its growth and toxin production; furthermore, these results allowed and improved particularly A. spinosum production in pilot scale photobioreactors for AZA isolation.