Ecological studies of economic red algae. III. Growth and reproduction of natural and harvested populations of Gigartina stellata (Stackhouse) batters in New Hampshire

The growth in situ of populations of Gigartina stellata (Stackhouse) Batters has been recorded at three New Hampshire locations and correlated with seasonal and spatial variations of surface salinity, temperature, and nutrients. Annual growth began between February and May, depending upon the location and its hydrographic regime. Gigartina populations reached maximum biomass and size in August and September, and a general decrease in both occurred during the reproductive period from October to February. The period of fastest growth coincided with increasing summer temperatures, while maximum carpospore release occurred during the period of decreasing (coldest) temperatures. The largest plants and maximum biomass of Gigartina were found on the open coast (at a semi-exposed site) while smaller plants and reduced biomass were evident within the Great Bay Estuary System. Salinity is a dominant factor influencing both the local distribution and growth of the plant. Gigartina is primarily restricted to the littoral zone, and its maximum length and biomass occur between +0.45 and +1.0 m above M.L.W.     

The demography of fronds of Chondrus crispus Stackhouse

A demographic analysis was done of an intertidal stand of Chondrus crispus Stackhouse at Pubnico Point, Nova Scotia. The distribution of gametophytic and tetrasporophytic fronds was assessed. Fronds were placed in one of four size-classes. Size-specific measurements for fronds are presented. Gametophytic fronds dominated the stand. Fronds in Size-Class 2 exhibited high survivorship. Survivorship fell sharply when fronds entered Size-Class 3 and Size-Class 4. Fronds became fertile and highly branched when in the third and fourth size-classes. It is suggested that these two factors contribute to the higher mortality rates of such individuals.Monthly spore release rates·m^?2 for gametophytes and tetrasporophytes were measured. The annual release rate of carpospores exceeded the annual release rate of tetraspores by a ratio of 4.7:1. Rates did not fall below 10⁶ spores released·m^?2·month^?1 for either phase.     

In situ measurements of hydrodynamic forces imposed on Chondrus crispus Stackhouse

Among the hydrodynamic forces experienced by intertidal organisms, drag and the impingement force are thought to have the greatest effect on macroalgae. These forces are modified by biotic factors such as algal morphology, reconfiguration, and the presence of a canopy. However, much of what is known about the hydrodynamics of macroalgae has been garnered from low-velocity laboratory flume studies. Few field studies have measured drag and none have directly measured the effects of the canopy on force. To examine in situ hydrodynamic forces imposed on the turf forming macroalga Chondrus crispus, compact digital force sensors were developed that measure and record the 3-dimensional force imposed on a macroalga without disturbing the surrounding canopy. Sensors were positioned within natural Chondrus beds and the effects of the canopy, algal morphology, and sea state on in situ hydrodynamic force were examined. Additionally, the predictions of a new model for drag on flexible macroalgae were tested by simultaneously measuring force and water velocity. Digital force recordings indicated that Chondrus only experience drag; lift and impingement force were negligible in all combinations of factors. Canopies significantly reduced drag by 15-65%. Morphology and size also influenced drag, such that lower forces were imposed on small planar algae than large arborescent individuals. Further, planar algae experienced low drag in all combinations of sea and canopy state, indicating that these individuals may not be as susceptible to wave disturbance as arborescent individuals. Overall, these data indicate that the ability for Chondrus to grow large, arborescent individuals is dependent on the drag reducing properties of the canopy, while more hydrodynamically harsh habitats may be accessible to planar morphologies. Additionally, these data suggest that drag models for canopy forming macroalgae must incorporate the effects of the canopy to predict drag accurately in situ.     

Growth regulators from the seaweed Chondrus crispus L. (Lyn.)

The level of auxin-like compounds extracted from the seaweed Chondrus crispus L. (Lyn.) is reported. Their presence before and after alkaline hydrolysis is studied to confirm that the bound auxin-like substances are present in algae in greater concentration than the free form.     

Wavelength-dependent induction of UV-absorbing mycosporine-like amino acids in the red alga Chondrus crispus under natural solar radiation

Polychromatic response spectra for the induction of UV absorbing mycosporine-like amino acids (MAAs) were calculated after exposing small thalli of the red alga Chondrus crispus under various cut-off filters to natural solar radiation on the North Sea island Helgoland, Germany. The laboratory-grown specimens typically contain only traces of palythine and synthesise five different MAAs rapidly and in high concentrations after being transplanted into shallow water. The resulting qualitative and quantitative patterns of MAA induction differed markedly with respect to spectral distribution. Furthermore, the wavebands effective for MAA induction vary within the MAA. UV-B radiation had a negative effect on the accumulation of the major MAAs shinorine (λ_max=334 nm) and palythine (λ_max=320 nm), while short wavelength UV-A exhibits the highest quantum efficiency on their synthesis. In contrast, the synthesis of asterina-330 (λ_max=330 nm), palythinol (λ_max=332 nm) and palythene (λ_max=360 nm) was mainly induced by UV-B radiation. Whether the synthesis of shinorine and palythine is induced by a photoreceptor with an absorption maximum in the short wavelength UV-A and whether a second photoreceptor absorbing UV-B radiation is responsible for the induction of asterina-330, palythinol and palythene remains to be studied.Our results show that C. crispus has a high capacity to adapt flexibly the qualitative and quantitative MAA concentration to the prevailing spectral distribution of irradiance. On one hand, this is regarded as an important aspect with respect to the acclimation of algae to increasing UV-B irradiance in the context of ongoing depletion of stratospheric ozone. On the other hand, the experiment demonstrates that UV-A irradiance is more important for the induction of the major MAAs shinorine and palythine than UV-B.     

Inter-simple sequence repeat (ISSR) analysis of genetic variation of Chondrus crispus populations from North Atlantic

ISSR analysis was used to investigate genetic variations of 184 haploid and diploid samples from nine North Atlantic Chondrus crispus Stackhouse populations and one outgroup Yellow Sea Chondrus ocellatus Holmes population. Twenty-two of 50 primers were selected and 163 loci were scored for genetic diversity analysis. Genetic diversity varied among populations, percentage of polymorphic bands (PPB) ranged from 27.0 to 55.8%, H (Nei's genetic diversity) ranged from 0.11 to 0.20 and I (Shannon's information index) ranged from 0.16 to 0.30. Estimators PPB, H and I had similar values in intra-population genetic diversity, regardless of calculation methods. Analysis of molecular variance (AMOVA) apportioned inter-population and intra-population variations for C. crispus, showing more genetic variance (56.5%) occurred in intra-population, and 43.5% variation among nine populations. The Mantel test suggested that genetic differentiation between nine C. crispus populations was closely related with geographic distances (R = 0.78, P = 0.002). Results suggest that, on larger distance scale (ca. >1000 km), ISSR analysis is useful for determining genetic differentiations of C. crispus populations including morphologically inseparable haploid and diploid individuals.     

Red algae found on European salt-marshes. II. Catenella caespitosa (Rhabdoniaceae)

A survey is given of the taxonomy, life history and geography of Catenella caespitosa (With.) L. Irv. basedon study of living specimens, culture experiments, study of herbarium material and literature. This species is conspecific with Catenella repens (Lightf.) Batt. and Catenella opuntia (Good. & Woodw.) Grev. and several other taxa. Fragmentation is the most common mode of propagation of C. caespitosa. However, reproduction by tetraspores and by carpospores is also observed. The species is found in the tropics as well a in temperate climates.     

Experimental studies on the marine algae Iridaea and Gigartina

Field and laboratory studies of the economically important marine algae Iridaea cordata (Turner) Bory and Gigartina exasperata Harvey & Bailey (Rhodophyta: Gigartinales) have been made in order to establish information essential to the aquaculture of these species. The development of a simple and successful method for experimental transplantation of these species which permits periodic removal, measurement, and photography of the transplanted algae is described and its feasibility for successfully transplanting these algae to habitats in which they do not occur naturally, demonstrated. The growth rates of such transplanted algae and the maximum size which they attain are comparable to those of plants in the population from which the transplanted algae were taken.In field experiments, the optimum depth for maximum growth rates was 3–5 m below M.L.L.W. Laboratory experiments using a crossed-gradients (light and temperature) incubator indicated that optimum growth rates are obtained at temperatures of 10–14 °C and light intensities of 8.9–16.0 × 10³ erg/cm² sec (cool-white fluorescent); such light intensities are substantially higher than the optimum for many of the red algae that have been grown in laboratory culture. These results support the biological feasibility of cultivating these algae in the inland waters of the Pacific Northwest.     

Photosynthesis and respiration in a fast growing strain of Gigartina exasperata (Harvey and Bailey)

Photosynthesis and respiration rates of blades from a selected, fast growing strain of the marine red alga. Gigartina exasperata Harvey and Bailey, a carrageenan producer, were measured with an oxygen electrode and compared with rates similarly obtained from wild material of the same species. The measurements, expressed as μl O₂ · mg chl a^?1, min^?1. were made over a light intensity range from 5 to 800 μE · m^?2 · sec^?1 and a temperature range of 6 to 16°C. The photosynthesis light intensity data are best described by hyperbolic functions.     

Physiology and cryosensitivity of coral endosymbiotic algae (Symbiodinium)

Coral throughout the world are under threat. To save coral via cryopreservation methods, the Symbiodinium algae that live within many coral cells must also be considered. Coral juvenile must often take up these important cells from their surrounding water and when adult coral bleach, they lose their endosymbiotic algae and will die if they are not regained. The focus of this paper was to understand some of the cryo-physiology of the endosymbiotic algae, Symbiodinium, living within three species of Hawaiian coral, Fungia scutaria, Porites compressa and Pocillopora damicornis in Kaneohe Bay, Hawaii. Although cryopreservation of algae is common, the successful cryopreservation of these important coral endosymbionts is not common, and these species are often maintained in live serial cultures within stock centers worldwide. Freshly-extracted Symbiodinium were exposed to cryobiologically appropriate physiological stresses and their viability assessed with a Pulse Amplitude Fluorometer. Stresses included sensitivity to chilling temperatures, osmotic stress, and toxic effects of various concentrations and types of cryoprotectants (i.e., dimethyl sulfoxide, propylene glycol, glycerol and methanol). To determine the water and cryoprotectant permeabilities of Symbiodinium, uptake of radio-labeled glycerol and heavy water (D₂O) were measured. The three different Symbiodinium subtypes studied demonstrated remarkable similarities in their morphology, sensitivity to cryoprotectants and permeability characteristics; however, they differed greatly in their sensitivity to hypo- and hyposmotic challenges and sensitivity to chilling, suggesting that standard slow freezing cryopreservation may not work well for all Symbiodinium. An appendix describes our H₂O:D₂O water exchange experiments and compares the diffusionally determined permeability with the two parameter model osmotic permeability.     

Comparative studies of mucopolysaccharides from marine animals. II. Illex illecebrosvs (Lesueur) and various crustaceans

Various mucopolysaccharides (MPS) were extracted from the tentacles of the squid, Illex illecebrosus (Leseuer) and the viscera of crustaceans comprising the blue crab, Callinectes sapidus Rathbun, the green crab, Carcinus maenas (Linné), the red crab, Geryon quinquedens Smith, the rock crab, Cancer irroratus Say, the lobster (body and head), Homarus americana Milne Edwards, and the shrimp (head), Pandalus borealis (Kröyer). The MPS were analyzed for uronic acid, hexosamine, N-sulfate, protein, neutral sugar, and anticoagulant activity. Chemical analysis of the two fractions extracted from the squid tentacles, suggests that fraction F1 is similar to chondroitin sulfate and F2 is heparin-like. In the crustaceans, the MPS extracted appear to resemble chondroitin sulfate and heparin. The blood anticoagulant activity of the MPS from the red crab was ≈66 IU/mg, whereas those obtained from the other species ranged from 7 to 30 IU/mg. Based on these data and the chemical analysis, it appears that the MPS from the red crab is heparin-like, while the MPS from the other species are more like chondroitin sulfate.     

Inducible defence and its modulation by environmental stress in the red alga Chondrus yendoi (Yamada and Mikami in Mikami, 1965) from Honshu Island, Japan

Among the numerous anti-herbivore defences developed by macroalgae, chemical and morphological traits are best documented and understood. Plant defence theory suggests that these resistances, which can either be constitutive or inducible, are associated with metabolic costs. They should therefore be impaired under conditions of energy limitation, but evidence for this prediction is scarce. In two subsequent experiments, we tested whether a reduction of light availability is changing feeding rates of the two mesoherbivores Idotea ochotensis and Lacuna smithii on the red alga Chondrus yendoi. Algal individuals were kept in outdoor mesocosm facilities for 10 days, during which we manipulated the amount of incoming sunlight at 6 levels (0%-99% reduction, i.e. 2000-20 μmol s^− 1 m^− 2). Orthogonal to this, we established the presence or absence of one of the herbivores to test whether C. yendoi can generate a defence. Algal palatability was investigated afterwards in no-choice feeding assays using naïve grazer individuals. The consumption of algal tissue in L. smithii increased with decreasing light, while this was not the case for I. ochotensis. However, we found a defence induced as a reaction to herbivory only by the highly mobile isopod but not when the slowly moving snail grazed on C. yendoi. Isopod total consumption rates in our experiments were 40 times higher than those of the gastropods. We therefore suggest that C. yendoi exhibits grazer-specific reactions to herbivory, depending on the mobility and voracity of the consumers. Interestingly, only for one of the grazers, i.e. the snail, short-term light reduction influenced the palatability of algal tissue. We discuss different but not mutually exclusive models that could explain this pattern. In conclusion, we view this three-species system as an illustrative example for specificity in grazer-algal interactions and their modification by environmental stress.     

Micropropagation and bioreactor studies of the medicinally important plant Lessertia (Sutherlandia) frutescens L.

This study describes a protocol for rapid and efficient in vitro propagation of Lessertia frutescens (cancer bush), a medicinally important plant species native to southern Africa. Single node explants were grown in various culture regimes of MS medium containing 30 g/l sucrose supplemented with various concentrations of cytokinins and auxins and solidified with 8 g/l agar. These were (a) 2.22, 4.44, 13.32 and 22.19 µM BA; 2.32, 4.65, 13.95 and 23.23 µM K and 0.45, 2.27, 4.54 and 13.62 µM TDZ (b) a combination of 2.22 µM BA with 0.57, 2.85, 5.71 and 11.42 µM IAA, 0.49, 2.46, 4.9 and 9.8 µM IBA or 0.54, 2.69, 5.37 and 10.74 µM NAA and (c) different media types viz. MS, SH basal salt medium and WPM at 1, ½ and ¼ salt strength which were each supplemented with 2.22 µM BA and 0.54 µM NAA. Single node explants were also grown in MS liquid medium supplemented with 2.22 µM BA and 0.54 µM NAA in temporary and continuous immersion bioreactors. Maximum number of shoots (12.9) per single node explant was obtained in the temporary immersion bioreactor but 50% of these shoots showed symptoms of hyperhydricity. In solid culture the best shoot multiplication response (10 shoots) was obtained in full strength MS. Roots were induced using shoot tips cultured in ½ MS solid medium supplemented with various concentrations of IBA or NAA. The highest rooting percentage (78%) was achieved in 19.6 µM IBA. Rooted plantlets were cultured in a mixture of perlite and vermiculite (1:1; v/v) and successfully acclimatized in a growth chamber with an 85% survival rate.     

Establishment of vascular endothelial cell lines from the aortas of wild Japanese serows (Capricornis crispus)

Endothelial cell lines were established from the aortas of wild Japanese serows (Capricornis crispus) by transfection of a simian virus 40 (SV40) large T antigen gene. The cloned cell lines, designed SeET (Japanese serow endothelial-SV40T) cells, express SV40T antigen and retain cobblestone-like morphology. Although von Willbrand Factor (vWF) is expressed in the cells, the expression rate and the quantity are lower than in serow primary endothelial cells. The SeET cells exhibit positive uptake of acetylated low density lipoprotein and dose-dependent cell proliferation upon exposure to vascular endothelial growth factor. These results suggest that these SeET cells have preserved endothelial phenotypes and able to function with decreased expression of vWF. The SeET cell line will be a valuable tool for in vitro studies on the physiological properties of endothelial cells and for the propagation of viruses and parasites of Japanese serows.     

NMR and homology modeling studies of copper(II)-halocyanin from Natronobacterium pharaonis bacteria

Halocyanin from the haloalkaliphilic archaean Natronobacterium pharaonis is a peripheral membrane type 1 blue copper protein with a single polypeptide chain of 163 amino acid residues. Halocyanin participates as putative electron carrier protein associated to an electron acceptor role for a terminal oxidase and has the lowest redox potential value reported to date for a BCP. NMR studies and homology modeling calculations were performed to evaluate the electronic properties of Cu(II)-halocyanin from Natronobacterium pharaonis. The copper coordination site properties of Cu(II)-halocyanin are discussed. The ¹H NMR spectra, isotropic chemical shifts and relaxation times for halocyanin are compared with those of other BCPs such as azurin, amicyanin, plastocyanin and stellacyanin. The wild-type Cu(II)-halocyanin presents almost the same ¹H NMR spectra in comparison with Cu(II)-plastocyanin as expected from a similar coordination symmetry. However, minor differences were found. In order to get some insight on these differences, a computational model for Cu(II)-halocyanin from N. pharaonis was built. Model is based on sequential homology of halocyanin with two different families of proteins: plastocyanins and pseudoazurins. Homology modeling was performed using two different structural templates and copper ion was added for further refinement of the coordination site. Proposed structure was in good agreement with NMR experimental information and is the first three-dimensional model reported to date of an halocyanin. Small differences were found in the copper coordination site with respect to other BCP with known structure. This work is also an interesting example of expertise-driven homology modeling across different protein families.     

Falsocalcifolium nov. gen. and revision of the tribe Calcifolieae (Mississippian, incertae sedis algae)

A new genus, Falsocalcifolium is described. The type species is F. punctatum (Maslov) emend. The genus is placed in the tribe Calcifolieae Shuysky emend. of the family Calcifoliaceae Termier, Termier and Vachard nomen translat. Consequently, the generic make-up of the tribe is emended, and is now considered to be composed of Frustulata Saltovskaya emend., Falsocalcifolium nov. gen., and Calcifolium Shvetsov and Birina emend. Evolution within the tribe is shown by the progressive development of more petaloid morphologies and changes in the number and relationships of the internal structures called filaments (interpreted here as the emplacements containing endosymbiotic cyanobacteria). This evolution is also related to palaeoecological factors. The representatives of the tribe are considered excellent biostratigraphic markers in several western Tethyan regions (e.g. Great Britain, Ireland, southern France, southern Spain), where they are widely represented, but their geographical distribution in other basins seems to be incomplete, and thus their use in biostratigraphic correlation is at the moment limited.     

Interaction between benthic algae (Lyngbya bouillonii, Dictyota dichotoma) and scleractinian coral Porites lutea in direct contact

Competition between the massive scleractinian coral Porites lutea and two benthic algal species, thin-filamentous blue-green Lyngbya bouillonii (Cyanophyta) commonly observed growing as mats and fleshy brown Dictyota dichotoma (Phaeophyta), was investigated. Experiments were designed to expose coral fragments to different treatments to test the role of abrasion, shading and allelopathy by Lyngbya and Dictyota on coral growth and physiology in direct physical contact. The growth rates of coral fragments were significantly lower in both the algal/coral and the net control (only plastic net touched the corals) treatments than in the manipulation control (contact with algae and plastic net was prevented), demonstrating the importance of abrasion in Porites-Lyngbya and Porites-Dictyota interactions. Furthermore, coral fragments exposed to Lyngbya grew significantly slowly than net controls, but this effect was not statistically significant for P. lutea maintained in contact with Dictyota. Light levels were reduced equally in the algal/coral and shading mimic (plastic net touched the corals shaded with neutral-density filters) treatments. However, there were no significant differences in the growth rates between the shading mimic and the net control treatments, suggesting that shading had no measurable effect on coral growth. Thus, the growth of P. lutea in contact with Dictyota was reduced by abrasion whereas in direct contact with L. bouillonii, abrasion was supplemented by additional factors unique to Lyngbya in mediating coral-algal competition. Physical contact with L. bouillonii induced a significant reduction in photochemical efficiency (F_v / F_m) of PSII and chlorophyll concentration of in hospite zooxanthellae of P. lutea fragments, as well as a decrease of the symbiotic dinoflagellate density. Analysis of the growth rate and F_v / F_m of the investigated algae revealed a significant reduction in both parameters for D. dichotoma but not for L. bouillonii when in direct contact with P. lutea fragments. Thus, the competitive inhibition by the coral P. lutea and the brown alga D. dichotoma was mutual, while L. bouillonii acted as a one-sided inhibitor for scleractinian corals inducing bleaching and severe damage of live coral tissue. The fact that mats-forming blue-green alga L. bouillonii acts as a poison for scleractinian corals and is able to kill live coral tissue is reported for the first time. Allelochemical mechanism of the effect on live coral tissue by this alga is suggested. Possible mechanisms of competitive interactions for substrate between the coral polyps of scleractinians and algal species investigated are discussed.     

Synthesis and SAR studies of novel benzodiazepinedione-based inhibitors of Clostridium difficile (C. difficile) toxin B (TcdB)

Synthesis and structure-activity relationships (SAR) of a novel series of benzodiazepinedione-based inhibitors of Clostridium difficile toxin B (TcdB) are described. Compounds demonstrating low nanomolar affinity for TcdB, and which possess improved stability in mouse plasma vs. earlier compounds from this series, have been identified. Optimized compound 11d demonstrates a good pharmacokinetic (PK) profile in mouse and hamster and is efficacious in a hamster survival model of Clostridium difficile infection.     

Hybridization studies of Haemonchus contortus (rudolphi, 1803) and H. placei (place, 1893) (Nematoda: Trichostrongylidae)

Hybridization experiments between Haemonchus contortus from sheep and H. placei from cattle indicate that the H. placei vulvar morph type and the inability of the eggs to hatch and develop at 11°C were inherited as dominant traits in the hybrids. The size of the third stage larvae was similar to H. placei in hybrids from the mating of male H. placei × female H. contortus while larvae from the reciprocal mating were intermediate in size. Hybrids produced by the mating of thiabendazole resistant female H. contortus × non-resistant male H. placei were also resistant. The F₁ males of the mating between male H. contortus and female H. placei were sterile. Male sterility did not occur in the reciprocal cross until the F₂ generation. Female hybrids from these generations had a low level of fertility when backcrossed to males of either parent species. Cytological studies of the hybrid males indicated that sterility was due to several kinds of meiotic disturbance and that spermatogenesis stopped during metaphase I. The chromosomes in eggs of unfertilized females did not undergo meiosis and polar body formation; instead they increased in number by a process of endomitosis. Unfertilized eggs in the faeces were characterized by uneven cytoplasmic division and abnormal shape. It is proposed that hybrid sterility can lead to the local eradication of one species of Haemonchus by the other. Furthermore, the outcome of which species is eradicated can be influenced by such variables as initial population sizes, climate, and the local ratio of sheep to cattle.     

Heavy metal complexation in polluted molluscs II. Oysters (Ostrea edulis and Crassostrea gigas)

Cadmium, zinc and copper, accumulated from polluted habitats by the oysters Ostrea edulis and Crassostrea gigas, were studied. Three distinct low molecular weight (less than 3000 daltons) zinc complexes were separated from highly contaminated C. gigas, one of which may be a complex with the amino acid taurine. Only one of these zinc complexes was present in less contaminated specimens of O. edulis. On gel permeation chromatography of oyster extracts, copper was eluted together with amino acids (principally taurine) and the betaine homarine. No evidence of metallothionein type proteins was found.