De novo transcriptomes of six calanoid copepods (Crustacea): a resource for the discovery of novel genes.

This study presents eight new high-quality de novo transcriptomes from six co-occurring species of calanoid copepods, the first published for Neocalanus plumchrus, N. cristatus, Eucalanus bungii and Metridia pacifica and additional ones for N. flemingeri and Calanus marshallae. They are ecologically-important members of sub-arctic North Pacific marine zooplankton communities. 'Omics data for this diverse and numerous taxonomic group are sparse and difficult to obtain. Total RNA from single individuals was used to construct gene libraries that were sequenced on an Illumina Next-Seq platform. Quality filtered reads were assembled with Trinity software and validated using multiple criteria. The study's primary purpose is to provide a resource for gene expression studies. The integrated database can be used for quantitative inter- and intra-species comparisons of gene expression patterns across biological processes. An example of an additional use is provided for discovering novel and evolutionarily-significant proteins within the Calanoida. A workflow was designed to find and characterize unannotated transcripts with homologies across de novo assemblies that have also been shown to be eco-responsive.

Larval fish counteract ram and suction to capture evasive prey.

A simple hydrodynamic model of predator-prey interactions between larval clownfish and copepod prey is used to elucidate how larval fish capture highly evasive copepods. Fish larvae are considered to be suction feeders; however, video observations revealed that successful captures by clownfish larvae were preceded by rapidly accelerating lunges (ram), while the role of suction to draw prey into the fish's mouth was less clear. Simulations were made of the fish's strike, varying strengths of ram and suction to characterize optimal strategies for copepod capture given known evasive capabilities. Our results suggest that, contrary to expectations, suction feeding is dominant only in older larvae, whereas ram feeding is the dominant mode for early larvae. Despite the relatively weak suction produced by smaller larvae, it still plays a crucial role in prey capture through hydrodynamic stealth. Escape-triggering water deformations from the strike can be cancelled through controlled suction. Experimental data obtained from larval clownfish agree with model results, suggesting that the primary role of suction in early larvae is providing hydrodynamic stealth rather than capture.

Post-diapause transcriptomic restarts: insight from a high-latitude copepod.

BACKGROUND: Diapause is a seasonal dormancy that allows organisms to survive unfavorable conditions and optimizes the timing of reproduction and growth. Emergence from diapause reverses the state of arrested development and metabolic suppression returning the organism to an active state. The physiological mechanisms that regulate the transition from diapause to post-diapause are still unknown. In this study, this transition has been characterized for the sub-arctic calanoid copepod Neocalanus flemingeri, a key crustacean zooplankter that supports the highly productive North Pacific fisheries. Transcriptional profiling of females, determined over a two-week time series starting with diapausing females collected from > 400 m depth, characterized the molecular mechanisms that regulate the post-diapause trajectory. RESULTS: A complex set of transitions in relative gene expression defined the transcriptomic changes from diapause to post-diapause. Despite low temperatures (5-6 °C), the switch from a "diapause" to a "post-diapause" transcriptional profile occurred within 12 h of the termination stimulus. Transcriptional changes signaling the end of diapause were activated within one-hour post collection and included the up-regulation of genes involved in the 20E cascade pathway, the TCA cycle and RNA metabolism in combination with the down-regulation of genes associated with chromatin silencing. By 12 h, females exhibited a post-diapause phenotype characterized by the up-regulation of genes involved in cell division, cell differentiation and multiple developmental processes. By seven days post collection, the reproductive program was fully activated as indicated by up-regulation of genes involved in oogenesis and energy metabolism, processes that were enriched among the differentially expressed genes. CONCLUSIONS: The analysis revealed a finely structured, precisely orchestrated sequence of transcriptional changes that led to rapid changes in the activation of biological processes paving the way to the successful completion of the reproductive program. Our findings lead to new hypotheses related to potentially universal mechanisms that terminate diapause before an organism can resume its developmental program.

Diapause vs. reproductive programs: transcriptional phenotypes in a keystone copepod.

Many arthropods undergo a seasonal dormancy termed "diapause" to optimize timing of reproduction in highly seasonal environments. In the North Atlantic, the copepod Calanus finmarchicus completes one to three generations annually with some individuals maturing into adults, while others interrupt their development to enter diapause. It is unknown which, why and when individuals enter the diapause program. Transcriptomic data from copepods on known programs were analyzed using dimensionality reduction of gene expression and functional analyses to identify program-specific genes and biological processes. These analyses elucidated physiological differences and established protocols that distinguish between programs. Differences in gene expression were associated with maturation of individuals on the reproductive program, while those on the diapause program showed little change over time. Only two of six filters effectively separated copepods by developmental program. The first one included all genes annotated to RNA metabolism and this was confirmed using differential gene expression analysis. The second filter identified 54 differentially expressed genes that were consistently up-regulated in individuals on the diapause program in comparison with those on the reproductive program. Annotated to oogenesis, RNA metabolism and fatty acid biosynthesis, these genes are both indicators for diapause preparation and good candidates for functional studies.

t-Distributed Stochastic Neighbor Embedding (t-SNE): A tool for eco-physiological transcriptomic analysis.

High-throughput RNA sequencing (RNA-Seq) has transformed the ecophysiological assessment of individual plankton species and communities. However, the technology generates complex data consisting of millions of short-read sequences that can be difficult to analyze and interpret. New bioinformatics workflows are needed to guide experimentation, environmental sampling, and to develop and test hypotheses. One complexity-reducing tool that has been used successfully in other fields is "t-distributed Stochastic Neighbor Embedding" (t-SNE). Its application to transcriptomic data from marine pelagic and benthic systems has yet to be explored. The present study demonstrates an application for evaluating RNA-Seq data using previously published, conventionally analyzed studies on the copepods Calanus finmarchicus and Neocalanus flemingeri. In one application, gene expression profiles were compared among different developmental stages. In another, they were compared among experimental conditions. In a third, they were compared among environmental samples from different locations. The profile categories identified by t-SNE were validated by reference to published results using differential gene expression and Gene Ontology (GO) analyses. The analyses demonstrate how individual samples can be evaluated for differences in global gene expression, as well as differences in expression related to specific biological processes, such as lipid metabolism and responses to stress. As RNA-Seq data from plankton species and communities become more common, t-SNE analysis should provide a powerful tool for determining trends and classifying samples into groups with similar transcriptional physiology, independent of collection site or time.

Predatory posture and performance in a precocious larval fish targeting evasive copepods.

Predatory fishes avoid detection by prey through a stealthy approach, followed by a rapid and precise fast-start strike. Although many first-feeding fish larvae strike at non-evasive prey using an S-start, the clownfish Amphiprion ocellaris feeds on highly evasive calanoid copepods from a J-shaped position, beginning 1 day post-hatch (dph). We quantified this unique strike posture by observing successful predatory interactions between larval clownfish (1 to 14 dph) and three developmental stages of the calanoid copepod Bestiolina similis The J-shaped posture of clownfish became less tightly curled (more L-shaped) during larval development. Larvae were also less tightly curled when targeting adult copepods, which are more evasive than younger copepod stages. Strike performance measured as time to capture and as peak speed improved only slightly with larval age. Therefore, the J-posture may allow first-feeding larvae to minimize disturbance during their approach of sensitive prey, and may represent an alternative predatory strategy to the prototypical S-start.

Going with the flow: hydrodynamic cues trigger directed escapes from a stalking predator.

In the coevolution of predator and prey, different and less well-understood rules for threat assessment apply to freely suspended organisms than to substrate-dwelling ones. Particularly vulnerable are small prey carried with the bulk movement of a surrounding fluid and thus deprived of sensory information within the bow waves of approaching predators. Some planktonic prey have solved this apparent problem, however. We quantified cues generated by the slow approach of larval clownfish ( Amphiprion ocellaris) that triggered a calanoid copepod ( Bestiolina similis) to escape before the fish could strike. To estimate water deformation around the copepod immediately preceding its jump, we represented the body of the fish as a rigid sphere in a hydrodynamic model that we parametrized with measurements of fish size, approach speed and distance to the copepod. Copepods of various developmental stages (CII-CVI) were sensitive to the water flow caused by the live predator, at deformation rates as low as 0.04 s-1. This rate is far lower than that predicted from experiments that used artificial predator-mimics. Additionally, copepods localized the source, with 87% of escapes directed away (greater than or equal to 90°) from the predator. Thus, copepods' survival in life-threatening situations relied on their detection of small nonlinear signals within an environment of locally linear deformation.

Directional Hydrodynamic Sensing by Free-Swimming Organisms.

Many aquatic organisms detect the presence of moving objects in their environment, such as predators, by sensing the hydrodynamic disturbances the movements produce. The resultant water flow is readily detectable by stationary organisms, but free-swimming organisms are carried with the surrounding water and may not detect the bulk surrounding flow, which limits the available information about the source. We have developed a theory that clarifies what information is contained in disturbances generated by an attacking predator that is available to a free-swimming organism and might be extracted from local flow deformations alone. The theory shows that, depending on how well the deformations can be measured in space and time, an organism can reduce the range of possible locations, speeds, sizes, and arrival times of the predator. We apply the theory to planktonic copepods that have mechanosensory hairs along a pair of antennules. The study reveals the presence of "blind spots," potential ambiguities in resolving from which of two sides a predator attacks, and whether it generates a bow wave or suction. Our findings lead to specific testable hypotheses concerning optimal escape strategies, which are helpful for interpreting the behavior of evasive prey and designing free-swimming robots with sensory capabilities.

A deep transcriptomic resource for the copepod crustacean Labidocera madurae: A potential indicator species for assessing near shore ecosystem health.

Coral reef ecosystems of many sub-tropical and tropical marine coastal environments have suffered significant degradation from anthropogenic sources. Research to inform management strategies that mitigate stressors and promote a healthy ecosystem has focused on the ecology and physiology of coral reefs and associated organisms. Few studies focus on the surrounding pelagic communities, which are equally important to ecosystem function. Zooplankton, often dominated by small crustaceans such as copepods, is an important food source for invertebrates and fishes, especially larval fishes. The reef-associated zooplankton includes a sub-neustonic copepod family that could serve as an indicator species for the community. Here, we describe the generation of a de novo transcriptome for one such copepod, Labidocera madurae, a pontellid from an intensively-studied coral reef ecosystem, Kane'ohe Bay, Oahu, Hawai'i. The transcriptome was assembled using high-throughput sequence data obtained from whole organisms. It comprised 211,002 unique transcripts, including 72,391 with coding regions. It was assessed for quality and completeness using multiple workflows. Bench-marking-universal-single-copy-orthologs (BUSCO) analysis identified transcripts for 88% of expected eukaryotic core proteins. Targeted gene-discovery analyses included searches for transcripts coding full-length "giant" proteins (>4,000 amino acids), proteins and splice variants of voltage-gated sodium channels, and proteins involved in the circadian signaling pathway. Four different reference transcriptomes were generated and compared for the detection of differential gene expression between copepodites and adult females; 6,229 genes were consistently identified as differentially expressed between the two regardless of reference. Automated bioinformatics analyses and targeted manual gene curation suggest that the de novo assembled L. madurae transcriptome is of high quality and completeness. This transcriptome provides a new resource for assessing the global physiological status of a planktonic species inhabiting a coral reef ecosystem that is subjected to multiple anthropogenic stressors. The workflows provide a template for generating and assessing transcriptomes in other non-model species.

Complementary mechanisms for neurotoxin resistance in a copepod.

Toxin resistance is a recurring evolutionary response by predators feeding on toxic prey. These adaptations impact physiological interaction and community ecology. Mechanisms for resistance vary depending on the predator and the nature of the toxin. Potent neurotoxins like tetrodotoxin (TTX) and saxitoxin (STX) that are highly toxic to humans and other vertebrates, target conserved voltage-gated sodium channels (NaV) of nerve and muscle, causing paralysis. The copepod Calanus finmarchicus consumes the STX-producing dinoflagellate, Alexandrium fundyense with no effect on survival. Using transcriptomic approaches to search for the mechanism that confers resistance in C. finmarchicus, we identified splice variants of NaVs that were predicted to be toxin resistant. These were co-expressed with putatively non-resistant form in all developmental stages. However its expression was unresponsive to toxin challenge nor was there any up-regulation of genes involved in multi-xenobiotic resistance (MXR) or detoxification (phases I or II). Instead, adults consistently regulated genes encoding digestive enzymes, possibly to complement channel resistance by limiting toxin assimilation via the digestive process. The nauplii, which were more susceptible to STX, did not regulate these enzymes. This study demonstrates how deep-sequencing technology can elucidate multiple mechanisms of toxin resistance concurrently, revealing the linkages between molecular/cellular adaptations and the ecology of an organism.

Escapes in copepods: comparison between myelinate and amyelinate species.

Rapid conduction in myelinated nerves keeps distant parts of large organisms in timely communication. It is thus surprising to find myelination in some very small organisms. Calanoid copepods, while sharing similar body plans, are evenly divided between myelinate and amyelinate taxa. In seeking the selective advantage of myelin in these small animals, representatives from both taxa were subjected to a brief hydrodynamic stimulus that elicited an escape response. The copepods differed significantly in their ability to localize the stimulus: amyelinate copepods escaped in the general direction of their original swim orientation, often ending up closer to the stimulus. However, myelinate species turned away from the stimulus and distanced themselves from it, irrespective of their original orientation. We suggest that faster impulse conduction of myelinated axons leads to better precision in the timing and processing of sensory information, thus allowing myelinate copepods to better localize stimuli and respond appropriately.

Evolution of rapid nerve conduction.

Rapid conduction of nerve impulses is a priority for organisms needing to react quickly to events in their environment. While myelin may be viewed as the crowning innovation bringing about rapid conduction, the evolution of rapid communication mechanisms, including those refined and enhanced in the evolution of myelin, has much deeper roots. In this review, a sequence is traced starting with diffusional communication, followed by transport-facilitated communication, the rise of electrical signaling modalities, the invention of voltage-gated channels and "all-or-none" impulses, the emergence of elongate nerve axons specialized for communication and their fine-tuning to enhance impulse conduction speeds. Finally within the evolution of myelin itself, several innovations have arisen and have been interactively refined for speed enhancement, including the addition and sealing of layers, their limitation by space availability, and the optimization of key parameters: channel density, lengths of exposed nodes and lengths of internodes. We finish by suggesting several design principles that appear to govern the evolution of rapid conduction. This article is part of a Special Issue entitled SI: Myelin Evolution.

Choreographed swimming of copepod nauplii.

Small metazoan paddlers, such as crustacean larvae (nauplii), are abundant, ecologically important and active swimmers, which depend on exploiting viscous forces for locomotion. The physics of micropaddling at low Reynolds number was investigated using a model of swimming based on slender-body theory for Stokes flow. Locomotion of nauplii of the copepod Bestiolina similis was quantified from high-speed video images to obtain precise measurements of appendage movements and the resulting displacement of the body. The kinematic and morphological data served as inputs to the model, which predicted the displacement in good agreement with observations. The results of interest did not depend sensitively on the parameters within the error of measurement. Model tests revealed that the commonly attributed mechanism of 'feathering' appendages during return strokes accounts for only part of the displacement. As important for effective paddling at low Reynolds number is the ability to generate a metachronal sequence of power strokes in combination with synchronous return strokes of appendages. The effect of feathering together with a synchronous return stroke is greater than the sum of each factor individually. The model serves as a foundation for future exploration of micropaddlers swimming at intermediate Reynolds number where both viscous and inertial forces are important.

The evolution of vertebrate and invertebrate myelin: a theoretical computational study.

Multilayered, lipid-rich myelin increases nerve impulse conduction velocity, contributes to compact nervous systems, and reduces metabolic costs of neural activity. Based on the hypothesis that increased impulse conduction velocity provides a selective advantage that drives the evolution of myelin, we simulated a sequence of plausible intermediate stages of myelin evolution, each of which providing an enhancement of conduction speed. We started with the expansion of insulating glial coverage, which led first to a single layer of myelin surrounding the axon and then to multiple myelin wraps with well-organized nodes. The myelinated fiber was modeled at three levels of complexity as the hypothesized evolutionary progression became more quantitatively exacting: 1) representing the fiber as a mathematically-tractable uniform active cylinder with the effect of myelination approximated by changing its specific capacitance (C(m)); 2) representing it as a chain of simple, cable-model compartments having alternating nodal and internodal parameters subject to optimization, and 3) representing it in a double cable model with the axon and myelin sheath treated separately. Conduction velocity was optimized at each stage. To maintain optimal conduction velocities, increased myelin coverage of axonal surface must be accompanied by an increase in channel density at the evolving nodes, but along with increases in myelin thickness, a reduction in overall average channel density must occur. Leakage under the myelin sheath becomes more of a problem with smaller fiber diameters, which may help explain the tendency for myelin to occur preferentially in larger nerve fibers in both vertebrates and invertebrates.

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.

The "Lillie transition": models of the onset of saltatory conduction in myelinating axons.

Almost 90 years ago, Lillie reported that rapid saltatory conduction arose in an iron wire model of nerve impulse propagation when he covered the wire with insulating sections of glass tubing equivalent to myelinated internodes. This led to his suggestion of a similar mechanism explaining rapid conduction in myelinated nerve. In both their evolution and their development, myelinating axons must make a similar transition between continuous and saltatory conduction. Achieving a smooth transition is a potential challenge that we examined in computer models simulating a segmented insulating sheath surrounding an axon having Hodgkin-Huxley squid parameters. With a wide gap under the sheath, conduction was continuous. As the gap was reduced, conduction initially slowed, owing to the increased extra-axonal resistance, then increased (the "rise") up to several times that of the unmyelinated fiber, as saltatory conduction set in. The conduction velocity slowdown was little affected by the number of myelin layers or modest changes in the size of the "node," but strongly affected by the size of the "internode" and axon diameter. The steepness of the rise of rapid conduction was greatly affected by the number of myelin layers and axon diameter, variably affected by internode length and little affected by node length. The transition to saltatory conduction occurred at surprisingly wide gaps and the improvement in conduction speed persisted to surprisingly small gaps. The study demonstrates that the specialized paranodal seals between myelin and axon, and indeed even the clustering of sodium channels at the nodes, are not necessary for saltatory conduction.

Meningeal-like organization of neural tissues in calanoid copepods (Crustacea).

Meninges, the connective tissue of the vertebrate central nervous system (CNS), have not been recognized in invertebrates. We describe the ultrastructure of the adult brain, antennules, and cord in five marine copepods: Calanus finmarchicus, Gaussia princeps, Bestiolina similis, Labidocera madurae, and Euchaeta rimana. In all of these locations we identified cell types with characteristics of the typical cells of vertebrate meninges and of their peripheral nervous system (PNS) connective tissue counterpart: fibroblasts, having flattened twisting processes with labyrinthine cavities communicating with the extracellular space, and macrophages, containing prominent lysosomes, well-developed endoplasmic reticulum, Golgi apparatus, and indented heterochromatin. The vertebrate distinction between electron-dense cells in the most external connective tissues (dura mater and epineurium) versus electron-lucent cells in the more internal connective tissues (pia-arachnoid and endoneurium-perineurium) was also found in the copepod CNS and PNS. Similar to the vertebrate organization, electron-dense cell networks penetrated from the outer layer (subcuticle) to surround inner substructures of the copepod nervous systems, and electron-lucent networks penetrated deeply from the brain and nerve surfaces to form intertwined associations with neural cells. Moreover, the association of these cells with basement membranes, glycocalyx, and fibrils of collagen in copepods conforms to a meningeal organization. The primary deviation from the vertebrate ultrastructural organization was the often tight investment of axons by the meningeal-like cells, with an intercalated basement membrane. Together, these data suggest that the tissues investing the copepod nervous system possess an organization that is analogous in many respects to that of vertebrate meninges.

In silico characterization of the insect diapause-associated protein couch potato (CPO) in Calanus finmarchicus (Crustacea: Copepoda).

Couch potato (CPO) is an RNA-binding protein involved in the regulation of nervous system development and adult diapause in insects. Within insects, this protein is highly conserved, yet it has not been identified in another large arthropod group, the Crustacea. Here, functional genomics was used to identify putative CPO homologs in the copepod Calanus finmarchicus, a planktonic crustacean that undergoes seasonal diapause. In silico mining of expressed sequence tag (EST) and 454 pyrosequencing data resulted in the identification of two full-length CPO proteins, each 205 amino acids long. The two C. finmarchicus CPOs (Calfi-CPO I and II) are identical in sequence with the exception of three amino acids, and are predicted to possess a single RNA recognition motif (RRM). Sequence comparison of the two Calfi-CPOs with those of insects shows high levels of amino acid conservation, particularly in their RRMs. Using the C. finmarchicus sequences as queries, ESTs encoding partial CPOs were identified from two other crustaceans, the parasitic copepod Lernaeocera branchialis and shrimp Penaeus monodon. Surprisingly, no convincing CPO-encoding transcripts were identified from crustacean species with very large (>100,000) EST datasets (e.g. Litopenaeus vannamei, Daphnia pulex and Lepeophtheirus salmonis), suggesting that CPO transcript/protein may be expressed at very low levels or absent in some crustaceans. RNA-Seq data suggested stage-specific expression of CPO in C. finmarchicus, with few transcripts present in eggs (which represent mixed embryonic stages) and adults, and high levels in nauplii and copepodites; stages exhibiting high CPO expression are consistent with a role for it in neuronal development.

Novel organization and development of copepod myelin. i. ontogeny.

Nerve impulse conduction is greatly increased by myelin, a multilayered membranous sheath surrounding axons. Best known from and most extensively investigated among vertebrates, a few invertebrates, including some superfamilies of copepod, have functionally and structurally similar myelin-like sheaths surrounding their axons. We examined the development of myelin ultrastructure in Bestiolina similis, a paracalanoid copepod. Development occurred in a novel way: initial myelination always appeared first as a partial layer, which in later stages came to encircle an axon completely. This partial myelin first appeared in a single pair of reidentifiable fibers, at the second naupliar stage. Two additional pairs of reidentifiable fibers also became partially myelinated by the third naupliar stage. The number of myelin layers in this trio of axon pairs increased with development, but, at any one stage, each axon had the same number of layers along its entire length. These axons disappeared after the copepodite metamorphosis. After metamorphosis, the fiber that took over as largest in the nerve cord became the most heavily myelinated and was identified as the lateral dorsal giant fiber. The rate of myelination was also characterized in the antennular nerve as a representative of the peripheral nervous system. As axons became larger, they were more likely to be partially, and then completely, myelinated, the latter having a lower ratio of axon core to fiber diameter than the former. Copepod myelin is an instructive example of convergent evolution, with far-reaching consequences for nervous system functioning and the behavior that nervous systems subserve.