Mussels flexing their muscles: a new method for quantifying bivalve behaviour

We employed a novel technique to quantify how blue mussels Mytilus edulis react to predation risk in their environment by quantifying mussel gape using a Hall sensor attached to one shell valve reacting to a magnet attached to the other. Change in gape angle per second (CHIGA) versus gape angle plots resulted in a distribution with a boundary, which defined the maximum CHIGA of a mussel at all gape angles. CHIGA boundary plots for all individual mussels were similar in form. However, the CHIGA boundary increased in extent with mussel length (maximum CHIGA for mussel valve closures for mussels 2.98 and 79.6 mm long were −1.5 and −11°s⁻¹, respectively), showing that larger mussels opened and closed most rapidly. Mussel extract added to the seawater, a factor believed to signal predation, caused mussels to close significantly faster than otherwise (P < 0.001). This approach for assessing how mussels react to their environment indicates that mussel response to predation is graded and complex and may well indicate animal-based assessments of the trade-off between effective feeding and the likelihood of predation.     

A new method for obtaining barnacle cement in the liquid state for polymerization studies

A new method for collecting barnacle cement in the liquid state is described. This permits studies on the transformation of cement from liquid to solid states not heretofore possible. Preliminary analyses showed that polymerization, i.e., the in vitro change from a clear liquid to an opaque solid mass is not affected by temperatures ranging from-10° to 45°C, by diluting with distilled water up to two-fold, or by treating with selective enzymes; some chemical inhibitors induce the formation of a white precipitate, possibly indicating denaturation of the protein. It is concluded that the liquid cement contains all the necessary components for self-assembly, progressively changing from a clear liquid to an opaque rubbery insoluble mass. The polymerization process is time-dependent and requires no exogenous catalyst. A mechanism that may be involved in the adhesion process of the barnacle is suggested.     

A new method for estimating phytoplankton growth rates and carbon biomass

A new method is described for the determination of phytoplankton growth rates and carbon biomass. This procedure is easy to apply and utilizes the labeling of chlorophyll a (chl a) with ¹⁴C. Pure chl a is isolated using two-way thin-layer chromatography, and the specific activity of chl a carbon is determined. Data from laboratory cultures indicate that the specific activity of chl a carbon becomes nearly equal to that of total phytoplankton carbon in incubations lasting 6 to 12 h and can be used to calculate phytoplankton growth rates and carbon biomass. Application of the method to the phytoplankton community in an eutrophic estuary in Hawaii indicates that the cells are growing with a doubling time of about 2 d and that about 85% of the particulate carbon consists of phytoplankton carbon.     

Coral weight increment in situ. A new method to determine coral growth

An underwater weighing apparatus is described. This consits basically of a float, the buoyancy of which is neutralized by a known amount of lead weights. The standard error of the method is ±0.1g. The entire weighing procedure takes place on the reef using SCUBA diving gear. The advantages are that the animals are not harmed by experimental treatment, and that the pure calcium-carbonate increment is registrated. Some results obtained by this method show growth of Montastrea annularis and Madracis mirabilis.     

Digestive enzymes in marine invertebrates from hydrothermal vents and other reducing environments

The present study demonstrates the potential hydrolytic activities in the symbiont-containing tissues of the vent invertebrates Riftia pachyptila, Bathymodiolus thermophilus (collected in 1991 at the East Pacific Rise) and the shallow-water bivalve Lucinoma aequizonata (collected in 1991 from the Santa Barbara Basin). Activities of phosphatases, esterases, -glucuronidase and leucineaminopeptidase were comparable to those of digestive tract tissues of other marine invertebrates. A lack in most glycosidases as well as in trypsin and chymotrypsin was observed. Activities of lysozyme and chitobiase were rather high. In all vent invertebrates with symbionts and in L. aequizonata, the symbiont-containing tissues and the symbiont-free tissues had similar levels of enzymatic activities, indicating that polymeric nutrients could be hydrolysed after release from the symbionts and cellular uptake. The high activities of -fucosidase in all vent invertebrates as well as in the shallow-water bivalve L. aequizonata could point to the existence of a yet undescribed substrate available to hydrolysation. The ectosymbionts-carrying polychaete Alvinella pompejana (collected in 1991 at the East Pacific Rise, EPR) shows high lysozyme activities in its gut, consistent with the proposed food source of bacteria. For the vent crab Bythogrea thermydron (also collected in 1991 at the EPR) hydrolytic activities were highest in the gut, dominated by esterase and peptidase activities which support their proposed carnivorous food source. A snail and a limpet collected from R. pachyptila tubes showed high levels of chitobiase suggesting a food source of grazed bacteria or ingested R. pachyptila tube.     

A novel hydrothermal method to synthesize brookite titanium dioxide nanosquares for efficient pollutant degradation

Environmental Chemistry Letters - Photocatalysis is a cost-effective method to degrade and remove pollutants, using semiconductor catalysts such as titanium dioxide. The brookite form of titanium...     

A new method for spatially moving correlation analysis in geomedicine

A method of correlation analysis within a spatially moving window was applied to two sets of epidemiological/geochemical data in Norway, (1) mortality/disability rates of multiple sclerosis (MS) versus atmospheric fallout of Mg and concentrations of Se in overbank sediment, and (2) incidence rates of malignant melanoma of the skin (MM) versus concentrations of Ca and K in overbank sediment. It appears that n = 17 observation sites within the moving window is a practical compromise between noise in the data at small values of n and a spatial resolution good enough to detect trends in the distribution patterns of the correlation coefficient. For MS versus Mg, MS versus Se and MM versus Ca the correlation coefficients are generally negative and depict systematic distribution patterns with anomalous clusters of sites with good correlation. For MS versus Se the correlation coefficients also form an additional cluster of positive coefficients. Tests with permutated data show that more than 70% of the negative correlation coefficients for MS versus Mg and for MM versus Ca are both significantly different from zero at p<0.05, while less than 15% of those for MS versus Se are significant at the same level. For MM versus K the correlation coefficients are randomly distributed and not significantly different from zero. The described correlations may be effects of confounders and do not per se indicate any causal relationships. However, further research based on these results may well lead to the identification of possible aetiological factors.     

A new bathymodioline mussel symbiosis at the Juan de Fuca hydrothermal vents

Until recently, the only major hydrothermal vent biogeographic province not known to include bathymodioline mussels was the spreading centers of the northeast Pacific, but deep-sea dives using DSV Alvin on the Endeavor segment of the Juan de Fuca Ridge (47°56N 129°06W; ∼2,200 m depth) in August 1999 yielded the only recorded bathymodioline mytilids from these northeastern Pacific vents. One specimen in good condition was evaluated for its relatedness to other deep-sea bathymodioline mussels and for the occurrence of chemoautotrophic and/or methanotrophic symbionts in the gills. Phylogenetic analyses of the host cytochrome oxidase I gene show this mussel shares evolutionary alliances with hydrothermal vent and cold seep mussels from the genus Bathymodiolus, and is distinct from other known species of deep-sea bathymodiolines, suggesting this mussel is a newly discovered species. Ultrastructural analyses of gill tissue revealed the presence of coccoid bacteria that lacked the intracellular membranes observed in methanotrophic symbionts. The bacteria may be extracellular but poor condition of the fixed tissue complicated conclusions regarding symbiont location. A single gamma-proteobacterial 16S rRNA sequence was amplified from gill tissue and directly sequenced from gill tissue. This sequence clusters with other mussel chemoautotrophic symbiont 16S rRNA sequences, which suggests a chemoautotrophic, rather than methanotrophic, symbiosis in this mussel. Stable carbon (δ¹³C = −26.6%) and nitrogen (δ¹⁵N = +5.19%) isotope ratios were also consistent with those reported for other chemoautotroph-mussel symbioses. Despite the apparent rarity of these mussels at the Juan de Fuca vent sites, this finding extends the range of the bathymodioline mussels to all hydrothermal vent biogeographic provinces studied to date.     

A high-resolution reverse-phase liquid chromatography method for the analysis of mycosporine-like amino acids (MAAs) in marine organisms

Mycosporine-like amino acids (MAAs) are a group of about 20 structurally related water-soluble compounds, widely distributed among freshwater and marine organisms. To provide a better assessment of the diversity and concentration of MAAs in aquatic environments a high-performance liquid chromatography (HPLC) method of analysis based on reverse-phase C₁₈ column and trifluoroacetic acid and an ammonium-containing mobile phase was developed. The improvements with respect to previous methods and the extraction and clean-up procedures are described here. With this method the clean-up recovery of MAAs of high polarity (shinorine), medium polarity (palythinol), and low polarity (palythene) is greater than 99% (±1%). The method is selective enough to resolve in a single run most of the characterized MAAs found in marine organisms, including the critical and highly polar compounds shinorine, mycosporine-2-glycine, and palythine-serine, the medium polarity pair palythenic acid and shinorine methyl ester (M-333), and the low polarity isomeric pair usujirene and palythene. A chromatogram of a mixture of over 20 MAAs such as might be found in complex samples of marine organisms is given. Good precision was obtained in the separations. The relative standard deviation for retention times was below 1% and the mean relative standard deviation for integrated area estimations was below 2%. A mean column recovery of standards was 99% (±1%) whereas limits of detection (signal-to-noise, S/N=2) for different MAAs varied between 0.08 and 0.47 pmol injected. The applicability of the method was tested using extracts of three microalgae cultures, three natural phytoplankton populations, two scleractinian corals, and one species of sea anemone. Results reveal the occurrence of several unknown MAAs not previously reported in the literature. The selectivity of the method toward some recently discovered MAAs makes it especially suitable not only for studying new field samples, but also for re-examining the MAA composition of previously studied organisms.Communicated by O. Kinne, Oldendorf/Luhe     

K-select analysis: a new method to analyse habitat selection in radio-tracking studies

Two kinds of wildlife habitat studies can be distinguished in the literature: hindcasting and forecasting studies. Hindcasting studies aim to emphasize among a large set of habitat variables those that are of interest for the focus species, whereas forecasting studies are intended to predict habitat selection according to a small number of habitat variables for a given area. We provide here a new analytical tool which relies on the concept of ecological niche, the K-select analysis, for hindcasting studies of habitat selection by animals using radio-tracking data. Each habitat variable defines one dimension in the ecological space. For each animal, the difference between the vector of average available habitat conditions and the vector of average used conditions defines the marginality vector. Its size is proportional to the importance of habitat selection, and its direction indicates which variables are selected. By performing a non-centered principal component analysis of the table containing the coordinates of the marginality vectors of each animal (row) on the habitat variables (column), the K-select analysis returns a linear combination of habitat variables for which the average marginality is greatest. It is a synthesis of variables which contributes the most to the habitat selection. As with principal component analysis, the biological significance of the factorial axes is deduced from the loading of variables. An example is provided: habitat selection by wild boar is studied in a Mediterranean habitat using the K-select analysis. The numerous advantages of the analysis (a large number of variables that can be included, individual variability in habitat selection taken into account, a lack of too strict underlying hypotheses) make it a powerful approach in radio-tracking studies designed to identify habitat variables that are selected by animals.     

Intron-targeted PCR: a new approach to survey neutral DNA polymorphism in bivalve populations

PCR (polymerase chain reaction) amplification of non-coding introns in phylogenetically widespread genes, using DNA primers based on the conserved exon sequences, provides a widely applicable strategy for finding DNA polymorphisms in eukaryotic genomes. Polymorphisms in introns provide a new source of potentially neurtral genetic markers for use in population biology. Here we use this approach to design PCR primers for an intron of calmodulin genes. We show that there are at least two calmodulin genes in mussels of theMytilus edulis species complex, and using gene- and species-specific primers we resolve two alleles at a calmodulin intron locus. Population surveys using PcR of adult mussel DNA reveal that genotype frequencies at most sites surveyed in England, Scotland and Italy, conform to Hardy-Weinberg equilibrium, suggesting that this is a novel neutral genetic marker. The data also provide preliminary evidence for restricted gene flow between mussel populations on the west and northeast coasts of Britain, and for local effects around the Thames estuary.     

A non-negative and high-resolution finite volume method for the depth-integrated solute transport equation using an unstructured triangular mesh

This paper proposes a new high-resolution finite volume method for solving the two-dimensional (2D) solute transport equation using an unstructured mesh. A new simple r-factor algorithm is introduced into the Total Variation Diminishing flux limiter to achieve a more efficient yet accurate high-resolution scheme for solving the advection term. To avoid the physically-meaningless negative solutions resulted from using the Green–Gauss theorem, a nonlinear two-point flux approximation scheme is adopted to deal with the anisotropic diffusion term. The developed method can be readily coupled with a two-dimensional finite-volume-based flow models under unstructured triangular mesh. By integrating with the ELCIRC flow model, the proposed method was verified using three idealized benchmark cases (i.e., advection of a circle-shaped solute field, advection in a cyclogenesis flow field and transport of a initially square-shaped solute plume), and further applied to simulate the non-reactive solute transport process driven by irregular tides in the Deep Bay, eastern Pearl River Estuary of China. These cases are also simulated by models using other existing methods, including different r-factor for advection term and the Green–Gauss theorem for diffusion term. The comparison between the results from the new method and those from other existing methods demonstrated the new method could describe advection induced concentration shock and discontinuities, and anisotropic diffusion at high resolution without providing spurious oscillations and negative values.     

A new method for detecting pollution effects on marine macrobenthic communities

A method is described by which the pollution status of a marine macrobenthic community may be assessed without reference to a temporal or spatial series of control samples. Theoretical considerations suggest that the distribution of numbers of individuals among species should behave differently from the distribution of biomass among species when influenced by pollution-induced disturbance. Combined k-dominance plots for species biomass and numbers take three possible forms representing unpolluted, moderately polluted and grossly polluted conditions, one curve acting as an internal control against which the other can be compared. Field data from unpolluted communities and from a well documented temporal pollution gradient support the model, but further empirical testing is required.     

A simple and precise method for fitting a von Bertalanffy growth curve

The parameter K of the von Bertalanffy equation, as developed by Beverton and Holt (1957), is first estimated by the relation $$\log _e \left( {dL_t /dt} \right) = A - Kt$$ where dLt/dt denotes growth increments per a unit of age, t denotes age, and A is a constant. The K estimate is used to evaluate L∞; $$L_\infty = \left( {e^K \sum\limits_2^n {L_t - \sum\limits_1^{n - 1} {L_t } } } \right)/\left( {n - 1} \right)\left( {e^K - 1} \right)$$ The L∞ estimate is used to estimate t _o, and to obtain a better estimate for K; $$\log _e \left( {1 - L_t /L_\infty } \right) = - Kt + Kt_0 $$ The K estimate may be used to obtain another estimate for L∞. Solved examples show that a single iteration is sufficient to obtain fitted equations which are, on the average, as precise as equations fitted by the least squares method shown by Tomlinson and Abramson (1961). This new method can be used, with a slight modification, for the second equation given above, if growth data have unequal age intervals. The variance of K, t _o and log _e L∞ can be estimated by applying the simple methods used in the case of straight-line relationships.     

Cytoenzymatic investigation of intracellular digestion in the symbiont-bearing hydrothermal bivalve <Emphasis Type="Italic">Bathymodiolus azoricus</Emphasis>

Invertebrates harbouring endosymbiotic chemoautotrophic bacteria are widely distributed in a variety of reducing marine habitats, including deep-sea hydrothermal vents. Bathymodiolids are dominants of the biomass at geochemically distinct vent sites of the Mid Atlantic Ridge (MAR) and thus are good candidates to study biological processes in response to site-specific conditions. To satisfy their nutritional requirements, these organisms depend to varying extent on two types of chemoautotrophic symbionts and on filterfeeding. The quantitative relationships of the nutritional modes are poorly understood. Using enzyme cytochemistry, electron microscopy and X-ray microanalysis, the structural and functional aspects of the cellular equipment necessary for lysosomal digestion was studied. We provide evidence for the following: (1) the basis of intracellular digestion of symbionts in Bathymodiolus azoricus from two geochemically distinct vent sites was not mainly in the large lysosomal bodies as previously thought (based on the membranous content resembling bacteria); (2) senescent bacteria are autolysed, possibly by bacterial acid phosphatase, that is more likely a cell cycling of the symbionts rather than an active lysosomal digestion by the host; (3) the consistent absence of hydrolases may indicate the improper use of the name “lysosome” for large vesicles at the base of the gill bacteriocytes (4) nutrient transfer in B. azoricus, therefore, may more likely be accomplished through leaking of metabolites from the symbiont to the host, not excluding lysosomal resorption of dead bacteria as an auxiliary strategy for organic molecule transfer; (5) evidence is provided for microvillar transfer of substances from the seawater that may indicate filter-feeding, in non-symbiotic ciliated gill cells of mussels from Lucky Strike; (6) two types of lysosomal vesicles can be distinguished in digestive cells based on their enzymatic content and their elemental composition.     

A new derivatization method for a determination of MX and related hydroxyfuranones in water

An extremely potent mutagen, 3‐chloro‐4(dichloromethyl)‐5‐hydroxy‐2(5/f)‐furanone (MX) is commonly present in chlorinated drinking water. Due to its high mutagenic activity and according to WHO guidelines its concentration should be controlled in drinking waters. Determination of MX is difficult due to ppt levels at which the compound usually exists in drinking waters. Derivatization with 2‐propanol is presented as a method which significantly lowers GC/MS detection level of MX. Suitability of 2‐propylation for derivatization of other hydroxyfuranones is also shown.     

A model for measuring leaf growth rate of vegetative shoot in the seagrass, Zostera marina

A new model for determining leaf growth in vegetative shoots of the seagrass Zostera marina (eelgrass) is described. This model requires the weights of individual mature and immature whole leaves and leaf plastochrone interval (P_L) as parameters, differing from the conventional leaf marking technique (CLM) that requires cutting and separation between new and old tissue of leaves. The techniques required for the model are the same as for the plastochrone method, but the parameters differ between both methods in use of the weight of individual immature leaves. In a mesocosm study, eelgrass growth was examined, and parameters for the new model and plastochrone method (the weights of individual mature and immature leaves and P_L) were measured. Leaf growth rate was measured using the CLM and determined by the new method and the plastochrone method. The results were then compared between the CLM, the new model, and the plastochrone method. The results obtained with the new model were similar to those obtained with the CLM. However, the results of the plastochrone method differed from those of the CLM, while the weight of immature leaves varied seasonally. The new model was also used to determine leaf growth in a natural eelgrass bed in Mikawa Bay, Japan, and revealed the growth rates in all shoots and those of different ages. This method would be advantageous as an accurate means of direct measurement in fieldwork, and should therefore be a useful tool for monitoring seagrass growth.     

A new method to determine in situ growth rates of decapod shrimp: a case study with brown shrimp Crangon crangon

Shrimps are economically and ecologically very important, yet a lack of ageing techniques and hence unknown growth rates often impairs analytical assessments and management. A new method for the determination of in situ growth rates of shrimps is presented, based on dry weight condition. Since this index oscillates from low values directly after moult to highest values prior to moult in constantly feeding shrimp, the lowest observed pre-moult condition followed by a moult was introduced as a reference value to separate growing and starving individuals in field data. Experiments with Crangon crangon confirmed that (1) post-moult condition varies in a narrow physiologically optimal range, regardless of recent growth increments, and (2) dry weight condition prior to moult is closely related to the subsequent length increment. The method was applied to estimate growth increments from in situ dry weight condition data of C. crangon. The new method can easily be applied to other related species, since the required data can be obtained from very simple short-term experiments.     

A new model for the CBL growth based on the turbulent kinetic energy equation

Starting from the evolution equation for the turbulent energy density spectrum (EDS), we develop a new model for the growth of the Convective boundary layer (CBL). We apply dimensional analysis to parameterize the unknown inertial transport and convective source term in the dynamic equation for the three-dimensional (3-D) spectrum and solve the 3-D EDS equation. The one-dimensional vertical spectrum is derived from the 3-D spectrum, employing a weight function. This allows us to select the magnitude of the vertical spectral component for the construction of the growing 3-D EDS. Furthermore, we employ the vertical component of the energy spectrum to calculate the eddy diffusivity (required in dispersion models). Currently there are no available experimental data to directly verify our EDS model.