Comparative analysis of fish environmental DNA reveals higher sensitivity achieved through targeted sequence-based metabarcoding.

Environmental DNA (eDNA)-based methods of species detection are enabling various applications in ecology and conservation including large-scale biomonitoring efforts. qPCR is widely used as the standard approach for species-specific detection, often targeting a fish species of interest from aquatic eDNA. However, DNA metabarcoding has the potential to displace qPCR in certain eDNA applications. In this study, we compare the sensitivity of the latest Illumina NovaSeq 6000 NGS platform to qPCR TaqMan assays by measuring limits of detection and by analysing eDNA from water samples collected from Churchill River and Lake Melville, NL, Canada. Species-specific, targeted next generation sequencing (NGS) assays had significantly higher sensitivity than qPCR, with limits of detection 14- to 29-fold lower. For example, when analysing eDNA, qPCR detected Gadus ogac (Greenland cod) in 21% of samples, but targeted NGS detected this species in 29% of samples. General NGS assays were as sensitive as qPCR, while simultaneously detecting 15 fish species from eDNA samples. With over 34,000 fish species on the planet, parallel and sensitive methods such as NGS will be required to support effective biomonitoring at both regional and global scales.

Harnessing the power of eDNA metabarcoding for the detection of deep-sea fishes.

The deep ocean is the largest biome on Earth and faces increasing anthropogenic pressures from climate change and commercial fisheries. Our ability to sustainably manage this expansive habitat is impeded by our poor understanding of its inhabitants and by the difficulties in surveying and monitoring these areas. Environmental DNA (eDNA) metabarcoding has great potential to improve our understanding of this region and to facilitate monitoring across a broad range of taxa. Here, we evaluate two eDNA sampling protocols and seven primer sets for elucidating fish diversity from deep sea water samples. We found that deep sea water samples (> 1400 m depth) had significantly lower DNA concentrations than surface or mid-depth samples necessitating a refined protocol with a larger sampling volume. We recovered significantly more DNA in large volume water samples (1.5 L) filtered at sea compared to small volume samples (250 mL) held for lab filtration. Furthermore, the number of unique sequences (exact sequence variants; ESVs) recovered per sample was higher in large volume samples. Since the number of ESVs recovered from large volume samples was less variable and consistently high, we recommend the larger volumes when sampling water from the deep ocean. We also identified three primer sets which detected the most fish taxa but recommend using multiple markers due the variability in detection probabilities and taxonomic resolution among fishes for each primer set. Overall, fish diversity results obtained from metabarcoding were comparable to conventional survey methods. While eDNA sampling and processing need be optimized for this unique environment, the results of this study demonstrate that eDNA metabarcoding can facilitate biodiversity surveys in the deep ocean, require less dedicated survey effort per unit identification, and are capable of simultaneously providing valuable information on other taxonomic groups.

An established Arabidopsis thaliana var. Landsberg erecta cell suspension culture accumulates chlorophyll and exhibits a stay-green phenotype in response to high external sucrose concentrations.

An established cell suspension culture of Arabidopsis thaliana var. Landsberg erecta was grown in liquid media containing 0-15%(w/v) sucrose. Exponential growth rates of about 0.40d-1 were maintained between 1.5-6%(w/v) sucrose, which decreased to about 0.30d-1 between 6 and 15%(w/v) sucrose. Despite the presence of external sucrose, cells maintained a stay-green phenotype at 0-15% (w/v) sucrose. Sucrose stimulated transcript levels of genes involved in the chlorophyll biosynthetic pathway (ChlH, ChlI2, DVR). Although most of the genes associated with photosystem II and photosystem I reaction centers and light harvesting complexes as well as genes associated with the cytochrome b6f and the ATP synthase complexes were downregulated or remained unaffected by high sucrose, immunoblotting indicated that protein levels of PsaA, Lhcb2 and Rubisco per gram fresh weight changed minimallyon a Chl basis as a function of external sucrose concentration. The green cell culture was photosynthetically competent based on light-dependent, CO2-saturated rates of O2 evolution as well as Fv/Fm and P700 oxidation. Similar to Arabidopsis WT seedlings, the suspension cells etiolated in the dark and but remained green in the light. However, the exponential growth rate of the cell suspension cultures in the dark (0.45±0.07d-1) was comparable to that in the light (0.42±0.02d-1). High external sucrose levels induced feedback inhibition of photosynthesis as indicated by the increase in excitation pressure measured as a function of external sucrose concentration. Regardless, the cell suspension culture still maintained a stay-green phenotype in the light at sucrose concentrations from 0 to 15%(w/v) due, in part, to a stimulation of photoprotection through nonphotochemical quenching. The stay-green, sugar-insensitive phenotype of the cell suspension contrasted with the sugar-dependent, non-green phenotype of Arabidopsis Landsberg erecta WT seedlings grown at comparable external sucrose concentrations. It appears that the commonly used Arabidopsis thaliana var. Landsberg erecta cell suspension culture has undergone significant genetic change since its original generation in 1993. We suggest that this genetic alteration has inhibited the sucrose sensing/signaling pathway coupled with a stimulation of chlorophyll an accumulation in the light with minimal effects on the composition and function of its photosynthetic apparatus. Therefore, caution must be exercised in the interpretation of physiological and biochemical data obtained from experimental use of this culture in any comparison with wild-type Arabidopsis seedlings.

The nitrogen costs of photosynthesis in a diatom under current and future pCO2.

With each cellular generation, oxygenic photoautotrophs must accumulate abundant protein complexes that mediate light capture, photosynthetic electron transport and carbon fixation. In addition to this net synthesis, oxygenic photoautotrophs must counter the light-dependent photoinactivation of Photosystem II (PSII), using metabolically expensive proteolysis, disassembly, resynthesis and re-assembly of protein subunits. We used growth rates, elemental analyses and protein quantitations to estimate the nitrogen (N) metabolism costs to both accumulate the photosynthetic system and to maintain PSII function in the diatom Thalassiosira pseudonana, growing at two pCO2 levels across a range of light levels. The photosynthetic system contains c. 15-25% of total cellular N. Under low growth light, N (re)cycling through PSII repair is only c. 1% of the cellular N assimilation rate. As growth light increases to inhibitory levels, N metabolite cycling through PSII repair increases to c. 14% of the cellular N assimilation rate. Cells growing under the assumed future 750 ppmv pCO2 show higher growth rates under optimal light, coinciding with a lowered N metabolic cost to maintain photosynthesis, but then suffer greater photoinhibition of growth under excess light, coincident with rising costs to maintain photosynthesis. We predict this quantitative trait response to light will vary across taxa.

ELEVATED CARBON DIOXIDE DIFFERENTIALLY ALTERS THE PHOTOPHYSIOLOGY OF THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE) AND EMILIANIA HUXLEYI (HAPTOPHYTA)(1).

Increasing anthropogenic carbon dioxide is causing changes to ocean chemistry, which will continue in a predictable manner. Dissolution of additional atmospheric carbon dioxide leads to increased concentrations of dissolved carbon dioxide and bicarbonate and decreased pH in ocean water. The concomitant effects on phytoplankton ecophysiology, leading potentially to changes in community structure, are now a focus of concern. Therefore, we grew the coccolithophore Emiliania huxleyi (Lohmann) W. W. Hay et H. Mohler and the diatom strains Thalassiosira pseudonana (Hust.) Hasle et Heimdal CCMP 1014 and T. pseudonana CCMP 1335 under low light in turbidostat photobioreactors bubbled with air containing 390 ppmv or 750 ppmv CO2 . Increased pCO2 led to increased growth rates in all three strains. In addition, protein levels of RUBISCO increased in the coastal strains of both species, showing a larger capacity for CO2 assimilation at 750 ppmv CO2 . With increased pCO2 , both T. pseudonana strains displayed an increased susceptibility to PSII photoinactivation and, to compensate, an augmented capacity for PSII repair. Consequently, the cost of maintaining PSII function for the diatoms increased at increased pCO2 . In E. huxleyi, PSII photoinactivation and the counter-acting repair, while both intrinsically larger than in T. pseudonana, did not change between the current and high-pCO2 treatments. The content of the photosynthetic electron transport intermediary cytochrome b6/f complex increased significantly in the diatoms under elevated pCO2 , suggesting changes in electron transport function.

Distinctive photosystem II photoinactivation and protein dynamics in marine diatoms.

Diatoms host chlorophyll a/c chloroplasts distinct from green chloroplasts. Diatoms now dominate the eukaryotic oceanic phytoplankton, in part through their exploitation of environments with variable light. We grew marine diatoms across a range of temperatures and then analyzed their PSII function and subunit turnover during an increase in light to mimic an upward mixing event. The small diatom Thalassiosira pseudonana initially responds to increased photoinactivation under blue or white light with rapid acceleration of the photosystem II (PSII) repair cycle. Increased red light provoked only modest PSII photoinactivation but triggered a rapid clearance of a subpool of PsbA. Furthermore, PsbD and PsbB content was greater than PsbA content, indicating a large pool of partly assembled PSII repair cycle intermediates lacking PsbA. The initial replacement rates for PsbD (D2) were, surprisingly, comparable to or higher than those for PsbA (D1), and even the supposedly stable PsbB (CP47) dropped rapidly upon the light shift, showing a novel aspect of rapid protein subunit turnover in the PSII repair cycle in small diatoms. Under sustained high light, T. pseudonana induces sustained nonphotochemical quenching, which correlates with stabilization of PSII function and the PsbA pool. The larger diatom Coscinodiscus radiatus showed generally similar responses but had a smaller allocation of PSII complexes relative to total protein content, with nearly equal stiochiometries of PsbA and PsbD subunits. Fast turnover of multiple PSII subunits, pools of PSII repair cycle intermediates, and photoprotective induction of nonphotochemical quenching are important interacting factors, particularly for small diatoms, to withstand and exploit high, fluctuating light.

Cell size trade-offs govern light exploitation strategies in marine phytoplankton.

Marine phytoplankton show complex community structures and biogeographic distributions, the net results of physiological and ecological trade-offs of species responses to fluctuating, heterogeneous environments. We analysed photosynthesis, responses to variable light and macromolecular allocations across a size panel of marine centric diatoms. The diatoms have strong capacities to withstand and exploit fluctuating light, when compared with picophytoplankton. Within marine diatoms, small species show larger effective cross-sections for photochemistry, and fast metabolic repair of photosystem II after photoinactivation. In contrast, large diatoms show lower susceptibility to photoinactivation, and therefore incur lower costs to endure short-term exposures to high light, especially under conditions that limit metabolic rates. This size scaling of key photophysiological parameters thus helps explain the relative competitive advantages of larger versus smaller species under different environmental regimes, with implications for the function of the biogenic carbon pump. These results provide a mechanistic framework to explain and predict shifts in marine phytoplankton community size structure with changes in surface irradiance and mixed layer depth.