Particle size influences decay rates of environmental DNA in aquatic systems

Environmental DNA (eDNA) analysis is a powerful tool for remote detection of target organisms. However, obtaining quantitative and longitudinal information from eDNA data is challenging, requiring a deep understanding of eDNA ecology. Notably, if the various size components of eDNA decay at different rates, and we can separate them within a sample, their changing proportions could be used to obtain longitudinal dynamics information on targets. To test this possibility, we conducted an aquatic mesocosm experiment in which we separated fish-derived eDNA components using sequential filtration to evaluate the decay rate and changing proportion of various eDNA particle sizes over time. We then fit four alternative mathematical decay models to the data, building towards a predictive framework to interpret eDNA data from various particle sizes. We found that medium-sized particles (1–10 μm) decayed more slowly than other size classes (i.e., <1 and > 10 μm), and thus made up an increasing proportion of eDNA particles over time. We also observed distinct eDNA particle size distribution (PSD) between our Common carp and Rainbow trout samples, suggesting that target-specific assays are required to determine starting eDNA PSDs. Additionally, we found evidence that different sizes of eDNA particles do not decay independently, with particle size conversion replenishing smaller particles over time. Nonetheless, a parsimonious mathematical model where particle sizes decay independently best explained the data. Given these results, we suggest a framework to discern target distance and abundance with eDNA data by applying sequential filtration, which theoretically has both metabarcoding and single-target applications.     

AUXOSPORULATION IN CYCLOTELLA OCELLATA (BACILLARIOPHYCEAE) UNDER NATURAL AND EXPERIMENTAL CONDITIONS1

Growth and sexual reproduction in a population of Cyclotella ocellata Pantocseck were studied during one annual cycle in a reservoir and in short-term enclosure experiments performed in situ involving different nutrient conditions and concentrations of zooplankton species. Three phases of auxosporulation in this diatom were distinguishable morphologically: 1) preauxospore, from the beginning of zygote formation until the valves were longitudinally separated, 2) primary auxospore, when the zygote grew too large to fit inside the valves and before it reached its full size, and 3) mature auxospore, characterized by a well-developed, markedly scalloped edge. Under experimental and natural conditions, sexual reproduction was associated with changes in cell size. In the natural system, the auxospore appeared to act as a resting structure during conditions adverse for population growth. A threshold population of small cells appeared to be necessary for sexual reproduction in the natural system, whereas auxosporulation was associated with phosphorus fertilization in the enclosures. In both environments only cells smaller than 9.5 μm in diameter were capable of auxospore formation. Our results suggest that, once having reached the critical cell size, the factors that trigger sexual reproduction may depend on ambient environmental conditions.     

Morphology, ontogeny and histochemistry of secretory trichomes of Geranium robertianum (Geraniaceae)

Geranium robertianum bears three types of glandular uniseriate trichomes which originate from a single protodermal cell and develop through periclinal divisions. Type I trichomes are procumbent and have an oval apical cell, two stalk cells and a basal cell. Type II trichomes are erect and have a pear shaped apical cell, two stalk cells and a basal cell. Type III trichomes are much longer than the other two types and have an elongated apical cell, five long stalk cells and a basal cell. Type I and type II trichomes are common on leaves while III trichomes are more abundant on flower structures.
Type I and type II trichomes secrete terpenoids and phenols. Type III trichomes are characterized by the accumulation of anthocyanins in the apical cell and secrete flavonoids.     

Differential scanning calorimetric study of the thermal unfolding of myosin rod,light meromyosin,and subfragment 2

The thermal unfolding of myosin rod, light meromyosin (LMM), and myosin subfragment 2 (S-2) was studied by differential scanning calorimetry (DSC) over the pH range of 6.5–9.0 in 0.5M KCl and either 0.20M sodium phosphate or 0.15M sodium pyrophosphate. Two rod samples were examined: one was purified by Sephadex G-200 without prior denaturation (native rod), and the other was purified by a cycle of denaturation-renaturation followed by Sephacryl S-200 chromatography (renatured rod). There were clearly distinguishable differences in the calorimetric behavior of these two samples. At pH 7.0 in phosphate the DSC curves of native rod were deconvoluted into six endothermic two-state transitions with melting temperatures in the range of 46–67°C and a total enthalpy of 4346 kJ/mol. Under identical conditions the melting profile of LMM was resolved into five endothermic peaks with transition temperatures in the range of 45–66°C, and the thermal profile of long S-2 was resolved into two endotherms, 46 and 57°C. Transition 4 observed with native rod was present in the deconvoluted DSC curve for long S-2, but absent in the DSC curve for LMM. This transition was identified with the high-temperature transition detected with long S-2 and attributed to the melting of the coiled-coil α-helical segment of subfragment 2 (short S-2). The low-temperature transition of long S-2 was attributed to the unfolding of the hinge region. The smallest transition temperatures observed for all three fragments were 45–46°C. It is suggested that the most unstable domain in rod (domain 1) responsible for the 46°C transition includes both the hinge region, which is the C-terminal segment of long S-2, and a short N-terminal segment of LMM. This domain, accounting for 21% of the rod structure, contains the S-2/LMM junction, and upon proteolytic cleavage yields the C-terminal and N-terminal ends of long S-2 and LMM, respectively. Over the pH range of 6.5–7.5, the observed specific heat of denaturation of rod was approximately equal to the sum of the specific heats of LMM and S-2. This finding provides an additional argument for the existence of independent domains in myosin rod.     

Protein stabilization by compatible solutes. Effect of diglycerol phosphate on the dynamics of Desulfovibrio gigas rubredoxin studied by NMR.

Heteronuclear NMR relaxation measurements and hydrogen exchange data have been used to characterize protein dynamics in the presence or absence of stabilizing solutes from hyperthermophiles. Rubredoxin from Desulfovibrio gigas was selected as a model protein and the effect of diglycerol phosphate on its dynamic behaviour was studied. The presence of 100 mM diglycerol phosphate induces a fourfold increase in the half-life for thermal denaturation of D. gigas rubredoxin. A model-free analysis of the protein backbone relaxation parameters shows an average increase of generalized order parameters of 0.015 reflecting a small overall reduction in mobility of fast-scale motions. Hydrogen exchange data acquired over a temperature span of 20 degrees C yielded thermodynamic parameters for the structural opening reactions that allow for the exchange. This shows that the closed form of the protein is stabilized by an additional 1.6 kJ x mol(-1) in the presence of the solute. The results seem to indicate that the stabilizing effect is due mainly to a reduction in mobility of the slower, larger-scale motions within the protein structure with an associated increase in the enthalpy of interactions.     

Physical mapping of rDNA genes in the ground beetle Carabus and related genera (Carabidae: Coleoptera)

The major ribosomal DNA (rDNA) loci were localized on meiotic and mitotic chromosomes and in interphase nuclei of 18 ground-beetle species belonging to three tribes of the supertribe Carabitae by fluorescence in situ hybridization (FISH), using a PCR-amplified 18S rDNA as a probe. Meiotic observations indicate that the 18S rDNA sequences are located on the largest autosomal bivalent in 12 species of Carabus , two species of Calosoma (both genera belonging to the tribe Carabini), and three sibling species of Ceroglossus chilensis (tribe Ceroglossini). The data suggest the occurrence of a conservative pattern in these three genera despite the chromosomal rearrangements that have led to karyotypes with higher chromosome numbers in Ceroglossus . A different result is found in Cychrus caraboides (tribe Cychrini), where ribosomal cistrons are located in two medium-sized autosomal pairs. Further species of Cychrini should be studied for corroborating the occurrence of molecular and karyotypical apomorphies in Cychrus with regard to the genera Carabus, Calosoma and Ceroglossus .     

Absence of plasma membrane H+-ATPase in plasmodesmata located in pit-fields of the young reactive pulvinus ofMimosa pudica L.

Summary Immunocytochemical techniques were employed to study the spatial distribution of the plasma membrane H⁺-ATPase within various cell types of the young reactive primary pulvinus ofMimosa pudica L. These cells were interconnected by large numbers of plasmodesmata, being concentrated within pit-fields. Although we could routinely detect evidence of the H⁺-ATPase along the plasma membrane, immunolabelling was rarely, if ever, observed along the plasma membranes of the plasmodesmata. This finding is discussed with respect to the likely specialized supramolecular structure of the plasmodesma.Abbreviations SEL size exclusion limit of plasmodesmata     

Control of Vascular Sap pH by the Vessel-Associated Cells in Woody Species (Physiological and Immunological Studies)

In Robinia wood, the vessel-associated cells form a continuous sleeve around the vessels. Variations in pH of the solution perfused through the vessels during the annual cycle and the opposing effects of carbonyl cyanide-m-chlorophenylhydrazone and fusicoccin on this pH value indicate that some living cells of the wood are involved in the control of vascular sap pH and that this control fluctuates with the seasons. The immunolocalization of the plasma membrane HT+-ATPase in Robinia wood was studied by the immunogold-silver-staining technique using an antibody raised against a conserved stretch of the cytoplasmic domain of the H+-ATPase. The immunostaining is much stronger in vessel-associated cells than in other living cell types (ray and axial parenchyma elements) of the secondary xylem. Our data show an efficient involvement of this cell type in the control of vascular sap pH.     

Effect of water-miscible aprotic solvents on kyotorphin synthesis catalyzed by immobilized α-chymotrypsin

Summary Immobilized -chymotrypsin was used as catalyst to synthesize a kyotorphin derivative (Bz-Tyr-Arg-OEt) in the presence of five water-miscible aprotic solvents (dimethylsulphoxide, dimethylformamide, acetonitrile, acetone and tetrahydrofurane) at 30 °C. By using a kinetically-controlled approach, the maximum synthetic activity was obtained when Arg-OEt was used as nucleophile donor at a concentration 1.5-times higher than the acyl-acceptor substrate (Bz-Tyr-OEt). The water-miscible aprotic solvents enhanced greatly the synthetic activity proportionally to their hidrophilicity properties adequately measured by the log P parameter. At the optimum solvent concentration for the enzymatic peptide synthesis, both the water activity (Aw) of the media and the water content of the immobilized derivative showed a saturation profile against the log P parameter. As a function of the solvent hydrophilicity, these water parameters were shown as key parameters for the increase in the synthetic activity of the enzyme by the presence of these solvents.