They are young, and they are many: dating freshwater lineages in unicellular dinophytes

Dinophytes are one of few protist groups that have an extensive fossil record and are therefore appropriate for time estimations. However, insufficient sequence data and strong rate heterogeneity have been hindering to put dinophyte evolution into a time frame until now. Marine‐to‐freshwater transitions within this group are considered geologically old and evolutionarily exceptional due to strong physiological constraints that prevent such processes. Phylogenies based on concatenated rRNA sequences (including 19 new GenBank entries) of two major dinophyte lineages, Gymnodiniaceae and Peridiniales, were carried out using an uncorrelated molecular clock and five calibration points based on fossils. Contrarily to previous assumptions, marine‐to‐freshwater transitions are more frequent in dinophytes (i.e. five marine‐freshwater transitions in Gymnodiniaceae, up to ten but seven strongly supported transitions in Peridiniales), and none of them occurred as early as 140 MYA. Furthermore, most marine‐to‐freshwater transitions, and the followed diversification, took place after the Cretaceous–Paleogene boundary. Not older than 40 MYA, the youngest transitions within Gymnodiniaceae and Peridiniales occurred under the influence of the Eocene climate shift. Our evolutionary scenario indicates a gradual diversification of dinophytes without noticeable impact of catastrophic events, and their freshwater lineages have originated several times independently at different points in time

Characterization of acetylcholinesterase inhibition and energy allocation in Daphnia magna exposed to carbaryl

The inhibition of acetylcholinesterase (AChE) activity and energy allocation in the freshwater organism Daphnia magna exposed to carbaryl and potential recovery from the effects was examined. The binding of carbaryl-AChE was characterized through in vitro assays. To evaluate the recovery from inhibition and the alteration in energy budget, in vivo exposure and recovery regime tests were conducted. In comparison to diazoxon, the active metabolite of the insecticide diazinon, the stability of enzyme-carbaryl complex was fifteen times lower and the reactivity toward the active site was two times lower, resulting in approximately 30 times lower overall inhibition rate than for diazoxon. The in vitro reactivation rate constant of the inhibited enzyme and the in vivo recovery rate constant of AChE activity were 1.9h-1 and 0.12h-1 for carbaryl, respectively, which are much higher than the corresponding rate constants for diazoxon. The lower AChE inhibition and greater reactivation/recovery rates are in accordance with the lower toxicity of carbaryl compared to diazinon. Carbaryl exposure also altered the profile of the energy reserve: the decrease in lipid and glycogen and the increase in protein content resulted in the reduction of the total energy budget by about 45mJ/gww. This corresponds to 26 percent of the available energy, which might allocate for external stressors. The mechanistic model of AChE inhibition is helpful to get an insight into (eco-)toxicological effects of AChE inhibitors on freshwater crustaceans under environmentally realistic conditions

Spatial fragmentation in the distribution of diatom endosymbionts from the taxonomically clarified dinophyte Kryptoperidinium triquetrum (= Kryptoperidinium foliaceum, Peridiniales)

Abstract Among the photosynthetically active dinophytes, the Kryptoperidiniaceae are unique in having a diatom as endosymbiont instead of the widely present peridinin chloroplast. Phylogenetically, it is unresolved at present how the endosymbionts are inherited, and the taxonomic identities of two iconic dinophyte names, Kryptoperidinium foliaceum and Kryptoperidinium triquetrum, are also unclear. Multiple strains were newly established from the type locality in the German Baltic Sea off Wismar and inspected using microscopy as well as molecular sequence diagnostics of both host and endosymbiont. All strains were bi-nucleate, shared the same plate formula (i.e., po, X, 4′, 2a, 7′′, 5c, 7s, 5′′′, 2′′′′) and exhibited a narrow and characteristically L-shaped precingular plate 7′′. Within the molecular phylogeny of Bacillariaceae, endosymbionts were scattered over the tree in a highly polyphyletic pattern, even if they were gained from different strains of a single species, namely K. triquetrum. Notably, endosymbionts from the Baltic Sea show molecular sequences distinct from the Atlantic and the Mediterranean Sea, which is the first report of such a spatial fragmentation in a planktonic species of dinophytes. The two names K. foliaceum and K. triquetrum are taxonomically clarified by epitypification, with K. triquetrum having priority over its synonym K. foliaceum. Our study underlines the need of stable taxonomy for central questions in evolutionary biology

Taxonomic clarification of the dinophyte <i>Rhabdosphaera erinaceus</i> Kamptner, ≡ <i>Scrippsiella erinaceus</i> comb. nov. (Thoracosphaeraceae, Peridiniales)

<div><p>The <i>Scrippsiella trochoidea</i> species complex (Thoracosphaeraceae, Peridiniales) consists of a cryptic diversity and multiple species, for which established scientific names are not available at present. Previously, the name <i>Scrippsiella trochoidea</i> has been taxonomically clarified, leaving a reliable determination of morphologically similar, but only distantly related species impossible. We isolated and cultivated <i>Scrippsiella erinaceus</i> comb. nov. (strains GeoM*533 and GeoM*534) from material collected near the type locality off Rovinj, Republic of Croatia (Adriatic Sea). We barcoded the species of the Thoracosphaeraceae using rRNA sequences (including 22 new sequences) and investigated the morphology of the strains using light and electron microscopy. The <i>Scrippsiella trochoidea</i> species complex was composed of three primary clades, and the Adriatic strains were reliably assigned to one of them being distinct from the true <i>Scrippsiella trochoidea</i>. We designate an epitype for the basionym <i>Rhabdosphaera erinaceus</i> prepared from the strain GeoM*534. The unambiguous links between a scientific species name, its protologue, genetic characterization and spatial distribution have particular importance for character-poor, unicellular organisms such as the Dinophyceae. </p></div

Mechanistic Toxicodynamic Model for Receptor-Mediated Toxicity of Diazoxon, the Active Metabolite of Diazinon, in Daphnia magna

The organothiophosphate diazinon inhibits the target site acetylcholinesterase only after activation to its metabolite diazoxon. Commonly, the toxicity of xenobiotics toward aquatic organisms is expressed as a function of the external concentration and the resulting effect on the individual level after fixed exposure times. This approach does not account for the time dependency of internal processes such as uptake, metabolism, and interaction of the toxicant with the target site. Here, we develop a mechanistic toxicodynamic model for Daphnia magna and diazoxon, which accounts for the inhibition of the internal target site acetylcholinesterase and its link to the observable effect, immobilization, and mortality. The model was parametrized by experiments performed in vitro with the active metabolite diazoxon on enzyme extracts and in vivo with the parent compound diazinon. The mechanism of acetylcholinesterase inhibition was shown to occur irreversibly in two steps via formation of a reversible enzyme inhibitor complex. The corresponding kinetic parameters revealed a very high sensitivity of acetylcholinesterase from D. magna toward diazoxon, which corresponds well with the high toxicity of diazinon toward this species. Recovery of enzyme activity but no recovery from immobilization was observed after in vivo exposure to diazinon. The toxicodynamic model combining all in vitro and in vivo parameters was successfully applied to describe the time course of immobilization in dependence of acetylcholinesterase activity during exposure to diazinon. The threshold value for enzyme activity below which immobilization set in amounted to 40% of the control activity. Furthermore, the model enabled the prediction of the time-dependent diazoxon concentration directly present at the target site

Assessing dinophyte biodiversity in Bavarian lakes (Germany) by 18S v4 amplicon sequencing

Seventeen surface plankton tow samples were collected from piers at thirteen localities in Upper Bavaria (Germany) in April 2017 using a plankton net (mesh size 20µm). The localities included eleven lakes (one lake was sampled at two sites) and one subsidiary river, to cover standing and flowing bodies of water as well. Four sites had been sampled twice. Environmental DNA was extracted using the Genomic DNA from Soil kit (Machery-Nagel; Düren, Germany) following the manufacturer's protocol. The small subunit (SSU or 18S) of the ribosomal RNA (rRNA) operon V4 region (~410 bp) was the amplification target. Due to PCR biases or PCR errors that may artificially increase diversity, each PCR reaction was performed in triplicates. Forward and reverse primers were those used by Xiao et al. (2017) (DOI: 10.1007/s12010-016-2358-3). DNA amplification (PCR) for subsequent Illumina amplicon sequencing (Illumina; San Diego, USA-CA) was carried out using 5ng/µl template DNA, 1 µM of each primer and 2x KAPA Hifi HotStart Ready Mix (Roche; Penzberg, Germany). Resulting PCR products were visualised in 1% agarose gels and were purified using AMPure XP Beads (Beckman Coulter; Brea, USA-CA). Dual indices and Illumina sequence adapters were attached by means of an Index PCR using the Nextera XT Index Kit (Illumina), and final PCR products were again purified using AMPure XP Beads. The library was validated using an Agilent 2100 Bioanalyzer Software and a DNA 1000 Chip (Agilent Technologies; Santa Clara, USA-CA) to verify the size of the resulting fragments. The final DNA libraries were equimolarly pooled and run in a MiSeq System (Illumina) after combining the denatured PhiX control library (15%) and the denatured amplicon library. Some 6.5 million 2 x 300 bp paired-end reads were produced and demultiplexed into seventeen samples from thirteen sites. Using Trimmomatic (v0.38), 3'-ends of the reads were trimmed based on read quality information. PEAR (v0.9.10) with default settings was used to merge the paired-end reads. Sequences, which could not be merged, were discarded. Primer-matching sequence segments were truncated from the amplicons by cutadapt (v1.9) and amplicons were only kept in the sequence pool, if both, the segment of the forward and of the reverse primer could be found. Remaining sequences were filtered for further quality features by vsearch (v2.3.0). Sequences were discarded, if they were outside a 50 bp radius above or below the median length of the primer-truncated amplicon (~387 bp), if they carry any ambiguity or if the expected number of miscalled bases of a sequence (sum of all base error probabilities of a sequence) was above 1. Chimera were predicted also by vsearch utilising the UCHIME algorithm with default settings in de-novo mode for each sample separately and removed from the sample files. About 4 million sequences passed all filtering steps and were used as input for the OTU-clustering, which was done by the tool Swarm (v2.1.8) with default settings. The most abundant amplicon of each OTU-cluster was used as an OTU representative. These sequences were annotated by the RDP classifier implemented in mothur (v1.38.1) using the Ref_NR99 version of release 128 of the SILVA SSU sequence set using a reference with a confidence cutoff of 90. The annotation of each representative sequence was used as annotation of the OTU cluster as well and added to the corresponding line of the OTU table