Antioxidant Responses in Copepods Are Driven Primarily by Food Intake, Not by Toxin-Producing Cyanobacteria in the Diet

The association between oxidative processes and physiological responses has received much attention in ecotoxicity assessment. In the Baltic Sea, bloom-forming cyanobacterium Nodularia spumigena is a significant producer of various bioactive compounds, and both positive and adverse effects on grazers feeding in cyanobacteria blooms are reported. To elucidate the effect mechanisms and species sensitivity to the cyanobacteria-dominating diet, we exposed two Baltic copepods, Acartia bifilosa and Eurytemora affinis, to a diet consisting of toxin-producing cyanobacteria N. spumigena and a high-quality food Rhodomonas salina at 0–300 μg C L−1; the control food was R. salina provided as a monodiet at the same food levels. The subcellular responses to food type and availability were assayed using a suite of biomarkers – antioxidant enzymes [superoxide dismutases (SOD), catalase (CAT), and glutathione S-transferases (GST)] and acetylcholinesterase (AChE). In parallel, we measured feeding activity using gut content (GC) assayed by real-time PCR analysis that quantified amounts of the prey DNA in copepod stomachs. As growth and reproduction endpoints, individual RNA content (a proxy for protein synthesis capacity), egg production rate (EPR), and egg viability (EV%) were used. In both toxic and nontoxic foods, copepod GC, RNA content, and EPR increased with food availability. Antioxidant enzyme activities increased with food availability regardless of the diet type. Moreover, CAT (both copepods), SOD, and GST (A. bifilosa) were upregulated in the copepods receiving cyanobacteria; the response was detectable when adjusted for the feeding and/or growth responses. By contrast, the diet effects were not significant when food concentration was used as a co-variable. A bimodal response in AChE was observed in A. bifilosa feeding on cyanobacteria, with up to 52% increase at the lower levels (5–25 μg C L−1) and 32% inhibition at the highest food concentrations. These findings contribute to the refinement of biomarker use for assessing environmental stress and mechanistic understanding of cyanobacteria effects in grazers. They also suggest that antioxidant and AChE responses to feeding activity and diet should be accounted for when using biomarker profiles in field-collected animals in the Baltic Sea and, perhaps other systems, where toxic cyanobacteria are common.

Single-cell quantification of a broad RNA spectrum reveals unique noncoding patterns associated with cell types and states

The ability to interrogate total RNA content of single cells would enable better mapping of the transcriptional logic behind emerging cell types and states. However, current single-cell RNA-sequencing (RNA-seq) methods are unable to simultaneously monitor all forms of RNA transcripts at the single-cell level, and thus deliver only a partial snapshot of the cellular RNAome. Here we describe Smart-seq-total, a method capable of assaying a broad spectrum of coding and noncoding RNA from a single cell. Smart-seq-total does not require splitting the RNA content of a cell and allows the incorporation of unique molecular identifiers into short and long RNA molecules for absolute quantification. It outperforms current poly(A)-independent total RNA-seq protocols by capturing transcripts of a broad size range, thus enabling simultaneous analysis of protein-coding, long-noncoding, microRNA, and other noncoding RNA transcripts from single cells. We used Smart-seq-total to analyze the total RNAome of human primary fibroblasts, HEK293T, and MCF7 cells, as well as that of induced murine embryonic stem cells differentiated into embryoid bodies. By analyzing the coexpression patterns of both noncoding RNA and mRNA from the same cell, we were able to discover new roles of noncoding RNA throughout essential processes, such as cell cycle and lineage commitment during embryonic development. Moreover, we show that independent classes of short-noncoding RNA can be used to determine cell-type identity.

Identification of the stress granule transcriptome via RNA-editing in single cells and in vivo

Stress granules are phase separated assemblies formed around mRNAs whose identities remain elusive. The techniques available to identify the RNA content of stress granules rely on their physical purification, and are therefore not suitable for single cells and tissues displaying cell heterogeneity. Here, we adapted TRIBE (Target of RNA-binding proteins Identified by Editing) to detect stress granule RNAs by fusing a stress granule RNA-binding protein (FMR1) to the catalytic domain of an RNA-editing enzyme (ADAR). RNAs colocalized with this fusion are edited, producing mutations that are detectable by sequencing. We first optimized the expression of this fusion protein so that RNA editing preferentially occurs in stress granules. We then show that this purification-free method can reliably identify stress granule RNAs in bulk and single S2 cells, and in Drosophila tissues, such as 398 neuronal stress granule mRNAs encoding ATP binding, cell cycle and transcription factors. This new method opens the possibility to identify the RNA content of stress granules as well other RNA based assemblies in single cells derived from tissues.

Short term fluctuating temperature alleviates Daphnia stoichiometric constraints

In this study, we analysed how short term temperature fluctuation interacts with nutrient limitation in the vertical migrating Daphnia commutata. We hypothesize that short term (daily) temperature fluctuation will alleviate nutrient limitation. We carried out experiments analysing growth rates, phosphorus and RNA content of D. commutate grown under four different temperature regimes and two P-limited conditions. Our experiments showed that individuals grown under fluctuating temperature grew more than at the mean temperature. We estimated the expected sizes for the 15 °C treatment based on the Q10 and for the fluctuating temperature treatment. These expected sizes for both treatments resulted well below the observed ones. The P and RNA content of individuals grown at 10 °C were significantly higher than those at 20 °C, and when individuals grown at 10 °C were translocated to 20 °C they exerted an increased growth rate. Our results suggest that, under a regime of diel vertical migration, the temperature alternation would allow migrating organisms to alleviate the effect of severe nutrient limitation maintaining population growth. Under a scenario of global warming, where epilimnetic temperatures will increase, lake temperature will interact with nutrient limitation for consumers, but, organisms may be able to face these changes if they can still regularly move from a cold hypolimnion to a warmer epilimnion.

Study of Condition Indices in Goby, Parachaeturichthys Ocellatus (Day, 1873) from the Creeks of Mumbai

Condition indices study like RNA content, DNA content, RNA: DNA, RNA: protein, RNA: lipid was carried out in goby, Parachaeturichthys ocellatus from the creeks of Mumbai to assess its nutritional status in different months. The study was carried out from June 2010 to September 2011.The range of RNA content in male was 72-185.6 µg/100 mg while in female was 82-145.46 µg/100 mg. RNA content was high during spawning months. The DNA content showed slight variations with range of 22.56- 39.31 µg/100 mg in males and 25.20-32.52 µg/100 mg in females. The range of ratio of RNA: DNA in males was 2.08-5.13 with an average of 3.74 while in female was 2.92-5.07 with an average of 3.99. The ratio above 2 indicates good condition. The RNA: protein showed an average of 0.0015 in males and 0.0017 in females while the average of RNA: lipid was 0.0176 in males and 0.0127 in females. RNA: protein and RNA: lipid showed the lowest values in post reproductive stages while it increased with the onset of reproductive cycles. The condition indices study showed that P. ocellatus was in good condition throughout the year and the creeks of Mumbai were suitable habitat for feeding and reproduction.