A Screening Tool for the Direct Analysis of Marine and Freshwater Phycotoxins in Organic SPATT Extracts from the Chesapeake Bay

Many detection methods for phycotoxins, bioactive compounds produced by harmful algae, focus on one compound or a class of related compounds. Multiple harmful algal species often co-occur in the environment, however, emphasizing the need to analyze for the presence of multiple groups of marine and freshwater phycotoxins in environmental samples, e.g., extracts from solid phase adsorption toxin tracking (SPATT). Two methods were developed to screen for 13 phycotoxins (microcystin-RR, -LR, -YR, azaspiracid-1, -2, karlotoxin 3, goniodomin A, brevetoxin-2, yessotoxin, pectenotoxin-2, dinophysistoxin-1, -2, and okadaic acid) in organic SPATT extracts using ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) equipped with a trapping dimension (trap) and at-column dilution (ACD). The performance of each compound under 36 combinations of chromatographic conditions was characterized, and two final methods, acidic and basic, were selected based on peak shapes, signal intensities, resolution, and the separation in time of positive and negative MS ionization modes. Injection volumes of up to 1 mL were possible through trap/ACD technology, resulting in limits of detection between 0.001 and 0.05 µg/L across the analytes. Benefits highlighted in this study, beyond the improved detection limits and co-detection of multiple toxin groups, include the ability to inject samples of 100% organic solvent, ensuring analyte stability and streamlining workflow through the elimination of laborious sample preparation steps

Effects of dietary taurine level on visual function in European sea bass (Dicentrarchus labrax)

Dietary insufficiencies have been well documented to decrease growth rates and survival (and therefore overall production) in fish aquaculture. By contrast, the effects of dietary insufficiencies on the sensory biology of cultured fish remains largely unstudied. Diets based solely on plant protein sources could have advantages over fish-based diets because of the cost and ecological effects of the latter, but plant proteins lack the amino acid taurine. Adequate levels of taurine are, however, necessary for the development of a fully functional visual system in mammals. As part of ongoing studies to determine the suitability of plant-based diets, we investigated the effects of normal and reduced taurine dietary levels on retinal anatomy and function in European sea bass (Dicentrarchus labrax). We could not demonstrate any effects of dietary taurine level on retinal anatomy, nor the functional properties of luminous sensitivity and temporal resolution (measured as flicker fusion frequency). We did, however, find an effect on spectral sensitivity. The peak of spectral sensitivity of individuals fed a 5% taurine diet was rightward shifted (i.e., towards longer wavelengths) relative to that of fish fed a 0% or 1.5% taurine diet. This difference in in spectral sensitivity was due to a relatively lower level of middle wavelength pigment (maximum absorbance .500 nm) in fish fed a 5% taurine diet. Changes in spectral sensitivity resulting from diets containing different taurine levels are unlikely to be detrimental to fish destined for market, but could be in fishes that are being reared for stock enhancement programs

De novo assembly and characterisation of transcriptomes from Amphidinium species.

Amphidinium Claparede et Lachmann is one of the most abundant and diverse dinoflagellate genera found in marine benthic, sand dwelling and pelagic habitats. Species of Amphidinium have been widely studied for their potential to produce natural products, in particular, long chain and linear polyketide and macrolide compounds. More than 40 compounds produced by different species of Amphidinium have been isolated. These compounds with varying bioactivities are being investigated for their efficacy as antimicrobial and/or therapeutic agents. Some compounds produced by Amphidinium species have bioactivities against fish gill cells, and may lead to fish kills. Structural elucidation of many toxic compounds produced by dinoflagellates suggest that they may be based on a polyether ladder backbone, and that polyketide synthase (PKS) enzymes may have a role in their biosynthesis. The aim of our study was to screen Amphidinium species for the production of polyketide compounds and test their toxicity. We conducted a transcriptomic sequencing study to determine the presence of PKS genes (both mono-functionally expressed catalytic domains and genes encoding multimodular PKS enzyme complexes) in Amphidinium. The ketosynthase domain encoding transcripts were found to form distinct clades in the phylogenetic analysis, in comparison to similar genes from other organisms. We also show the presence of genes encoding six key enzymes essential for fatty acid production in Amphidinium. The results presented here are a step forward towards recognising the genes encoding critical steps in toxin biosynthesis and designing tools to monitor for and mitigate human illnesses due to harmful algal blooms

Spatiotemporal distribution of phycotoxins and their co-occurrence within nearshore waters

Harmful algal blooms (HABs), varying in intensity and causative species, have historically occurred throughout the Chesapeake Bay, U.S.; however, phycotoxin data are sparse. The spatiotemporal distribution of phycotoxins was investigated using solid-phase adsorption toxin tracking (SPATT) across 12 shallow, nearshore sites within the lower Chesapeake Bay and Virginia\u27s coastal bays over one year (2017-2018). Eight toxins, azaspiracid-1 (AZA1), azaspiracid-2 (AZA2), microcystin-LR (MC-LR), domoic acid (DA), okadaic acid (OA), dinophysistoxin-1 (DTX1), pectenotoxin-2 (PTX2), and goniodomin A (GDA) were detected in SPATT extracts. Temporally, phycotoxins were always present in the region, with at least one phycotoxin group (i.e., consisting of OA and DTX1) detected at every time point. Co-occurrence of phycotoxins was also common; two or more toxin groups were observed in 76% of the samples analyzed. Toxin maximums: 0.03 ng AZA2/g resin/day, 0.25 ng DA/g resin/day, 15 ng DTX1/g resin/day, 61 ng OA/g resin/day, 72 ng PTX2/g resin/day, and 102,050 ng GDA/g resin/day were seasonal, with peaks occurring in summer and fall. Spatially, the southern tributary and coastal bay regions harbored the highest amount of total phycotoxins on SPATT over the year, and the former contained the greatest diversity of phycotoxins. The novel detection of AZAs in the region, before a causative species has been identified, supports the use of SPATT as an explorative tool in respect to emerging threats. The lack of karlotoxin in SPATT extracts, but detection of Karlodinium veneficum by microscopy, however, emphasizes that this tool should be considered complementary to, but not a replacement for, more traditional HAB management and monitoring methods

From Stop to Start: Tandem Gene Arrangement, Copy Number and Trans-Splicing Sites in the Dinoflagellate Amphidinium carterae

Dinoflagellate genomes present unique challenges including large size, modified DNA bases, lack of nucleosomes, and condensed chromosomes. EST sequencing has shown that many genes are found as many slightly different variants implying that many copies are present in the genome. As a preliminary survey of the genome our goal was to obtain genomic sequences for 47 genes from the dinoflagellate Amphidinium carterae. A PCR approach was used to avoid problems with large insert libraries. One primer set was oriented inward to amplify the genomic complement of the cDNA and a second primer set would amplify outward between tandem repeats of the same gene. Each gene was also tested for a spliced leader using cDNA as template. Almost all (14/15) of the highly expressed genes (i.e. those with high representation in the cDNA pool) were shown to be in tandem arrays with short intergenic spacers, and most were trans-spliced. Only two moderately expressed genes were found in tandem arrays. A polyadenylation signal was found in genomic copies containing the sequence AAAAG/C at the exact polyadenylation site and was conserved between species. Four genes were found to have a high intron density (>5 introns) while most either lacked introns, or had only one to three. Actin was selected for deeper sequencing of both genomic and cDNA copies. Two clusters of actin copies were found, separated from each other by many non-coding features such as intron size and sequence. One intron-rich gene was selected for genomic walking using inverse PCR, and was not shown to be in a tandem repeat. The first glimpse of dinoflagellate genome indicates two general categories of genes in dinoflagellates, a highly expressed tandem repeat class and an intron rich less expressed class. This combination of features appears to be unique among eukaryotes

Conventionally assessed voluntary activation does not represent relative voluntary torque production

The ability to voluntarily activate a muscle is commonly assessed by some variant of the twitch interpolation technique (ITT), which assumes that the stimulated force increment decreases linearly as voluntary force increases. In the present study, subjects (n = 7) with exceptional ability for maximal voluntary activation (VA) of the knee extensors were used to study the relationship between superimposed and voluntary torque. This includes very high contraction intensities (90–100%VA), which are difficult to consistently obtain in regular healthy subjects (VA of ∼90%). Subjects were tested at 30, 60, and 90° knee angles on two experimental days. At each angle, isometric knee extensions were performed with supramaximal superimposed nerve stimulation (triplet: three pulses at 300 Hz). Surface EMG signals were obtained from rectus femoris, vastus lateralis, and medialis muscles. Maximal VA was similar and very high across knee angles: 97 ± 2.3% (mean ± SD). At high contraction intensities, the increase in voluntary torque was far greater than would be expected based on the decrement of superimposed torque. When voluntary torque increased from 79.6 ± 6.1 to 100%MVC, superimposed torque decreased from 8.5 ± 2.6 to 2.8 ± 2.3% of resting triplet. Therefore, an increase in VA of 5.7% (from 91.5 ± 2.6 to 97 ± 2.3%) coincided with a much larger increase in voluntary torque (20.4 ± 6.1%MVC) and EMG (33.9 ± 6.6%max). Moreover, a conventionally assessed VA of 91.5 ± 2.6% represented a voluntary torque of only 79.6 ± 6.1%MVC. In conclusion, when maximal VA is calculated to be ∼90% (as in regular healthy subjects), this probably represents a considerable overestimation of the subjects’ ability to maximally drive their quadriceps muscles