De novo assembly of a transcriptome for Calanus finmarchicus (Crustacea, Copepoda)--the dominant zooplankter of the North Atlantic Ocean.

Assessing the impact of global warming on the food web of the North Atlantic will require difficult-to-obtain physiological data on a key copepod crustacean, Calanus finmarchicus. The de novo transcriptome presented here represents a new resource for acquiring such data. It was produced from multiplexed gene libraries using RNA collected from six developmental stages: embryo, early nauplius (NI-II), late nauplius (NV-VI), early copepodite (CI-II), late copepodite (CV) and adult (CVI) female. Over 400,000,000 paired-end reads (100 base-pairs long) were sequenced on an Illumina instrument, and assembled into 206,041 contigs using Trinity software. Coverage was estimated to be at least 65%. A reference transcriptome comprising 96,090 unique components ("comps") was annotated using Blast2GO. 40% of the comps had significant blast hits. 11% of the comps were successfully annotated with gene ontology (GO) terms. Expression of many comps was found to be near zero in one or more developmental stages suggesting that 35 to 48% of the transcriptome is "silent" at any given life stage. Transcripts involved in lipid biosynthesis pathways, critical for the C. finmarchicus life cycle, were identified and their expression pattern during development was examined. Relative expression of three transcripts suggests wax ester biosynthesis in late copepodites, but triacylglyceride biosynthesis in adult females. Two of these transcripts may be involved in the preparatory phase of diapause. A key environmental challenge for C. finmarchicus is the seasonal exposure to the dinoflagellate Alexandrium fundyense with high concentrations of saxitoxins, neurotoxins that block voltage-gated sodium channels. Multiple contigs encoding putative voltage-gated sodium channels were identified. They appeared to be the result of both alternate splicing and gene duplication. This is the first report of multiple NaV1 genes in a protostome. These data provide new insights into the transcriptome and physiology of this environmentally important zooplankter.

Functional genomics resources for the North Atlantic copepod, Calanus finmarchicus: EST database and physiological microarray.

The copepod, Calanus finmarchicus is a keystone species for the North Atlantic. Because of recent changes in the geographic distribution of this species, there are questions as to how this organism responds physiologically to environmental cues. Molecular techniques allow for examination and new understanding of these physiological changes. Here, we describe the development of a microarray for high-throughput studies of the physiological ecology of C. finmarchicus. An EST database was generated for this species using a normalized cDNA library derived from adult and sub-adult individuals. Sequence data were clustered into contigs and annotated using Blastx. Target transcripts were selected, and unique, 50 base-pair, oligomer probes were generated for 995 genes. Blast2GO processing provided detailed information on gene function. The selected targets included broad representation of biological processes, cellular components, and molecular functions. The microarray was tested in two sets of comparisons: adult females maintained at different food concentrations and field-caught sub-adults showing differences in lipid storage. Up-regulated and down-regulated transcripts were identified for both comparisons. Only a small subset of the genes up-regulated in low food individuals were also up-regulated in lipid-poor animals; no overlap was seen between the genes down-regulated in the two comparisons.

Habitat temperature is an important determinant of cholesterol contents in copepods.

Effects of habitat and acclimation temperature on cholesterol contents were examined in oceanic and inshore species of copepods. The cholesterol content of five species of thermally acclimated copepods was determined, and nine species (representing six families) were sampled to assess the role of habitat temperature. The species selected have maximum habitat temperatures (and temperature tolerances) that vary at least twofold. Levels of dietary cholesterol required to achieve maximum growth were also studied at different acclimation temperatures in a eurythermal copepod. Both eggs and copepodites of Calanus finmarchicus had higher cholesterol levels at the warm acclimation temperature (16 degrees C) than at the cooler temperature (6 degrees C). Neither Acartia tonsa, Acartia hudsonica, Temora longicornis nor Eurytemora affinis altered cholesterol contents with acclimation temperature. Maximum growth rates were achieved at fourfold higher concentrations of dietary cholesterol in warm-acclimated Eurytemora affinis than in cold-acclimated animals. The most consistent trend is the positive relationship between cholesterol content and habitat temperature. Species residing in warmer habitats (e.g. Centropages typicus, Eurytemora affinis) had approximately twice the cholesterol of species living in colder waters (e.g. Calanus glacialis, Euchaeta norvegica). A similar pattern was observed for comparisons of species within genera (Calanus, Acartia and Centropages), with the species abundant at lower latitudes having more cholesterol than the northern congener. These data indicate that habitat temperature is an important determinant of cholesterol content, and cholesterol endows membranes with the stability required for a range of body temperatures.

A cholesterol-enriched diet enhances egg production and egg viability without altering cholesterol Content of biological membranes in the copepod Acartia hudsonica.

Copepods may lack the capacity for de novo synthesis of cholesterol, while at the same time their dietary levels of sterol vary. We tested the hypothesis that copepods maintain the cholesterol contents of their biological membranes despite varying dietary levels of cholesterol. Acartia hudsonica were acclimated for 5 d to phytoplankton alone or phytoplankton supplemented with cholesterol, at a level sufficient to induce a maximal response on egg production rates. Biological membranes were prepared from the copepods and cholesterol contents assayed. Egg production and hatch rates were measured (the former to confirm that supplemented cholesterol was being assimilated). Analyses of marker enzymes indicate that the majority of membrane-associated cholesterol in the copepod resides in the plasma membrane. In membranes fractions, cholesterol normalized to protein or activity of Na+/K+-ATPase is not significantly different for supplemented and unsupplemented groups (29 and 33 mu g cholesterol mg(-1) protein, respectively; 0.24 and 0.25 mg cholesterol U(-1) Na+/K+-ATPase, respectively). At the same time, acclimating animals to a diet enriched with cholesterol enhances egg production by up to 1.8-fold and egg viability by 1.5-fold. We conclude that a cholesterol-enriched diet stimulates both egg production and hatching rates without altering cholesterol contents of plasma membranes in the copepod A. hudsonica.