Effects of petrogenic pollutants on North Atlantic and Arctic Calanus copepods: From molecular mechanisms to population impacts.

Oil and gas industries in the Northern Atlantic Ocean have gradually moved closer to the Arctic areas, a process expected to be further facilitated by sea ice withdrawal caused by global warming. Copepods of the genus Calanus hold a key position in these cold-water food webs, providing an important energetic link between primary production and higher trophic levels. Due to their ecological importance, there is a concern about how accidental oil spills and produced water discharges may impact cold-water copepods. In this review, we summarize the current knowledge of the toxicity of petroleum on North Atlantic and Arctic Calanus copepods. We also review how recent development of high-quality transcriptomes from RNA-sequencing of copepods have identified genes regulating key biological processes, like molting, diapause and reproduction in Calanus copepods, to suggest linkages between exposure, molecular mechanisms and effects on higher levels of biological organization. We found that the available ecotoxicity threshold data for these copepods provide valuable information about their sensitivity to acute petrogenic exposures; however, there is still insufficient knowledge regarding underlying mechanisms of toxicity and the potential for long-term implications of relevance for copepod ecology and phenology. Copepod transcriptomics has expanded our understanding of how key biological processes are regulated in cold-water copepods. These advances can improve our understanding of how pollutants affect biological processes, and thus provide the basis for new knowledge frameworks spanning the effect continuum from molecular initiating events to adverse effects of regulatory relevance. Such efforts, guided by concepts such as adverse outcome pathways (AOPs), enable standardized and transparent characterization and evaluation of knowledge and identifies research gaps and priorities. This review suggests enhancing mechanistic understanding of exposure-effect relationships to better understand and link biomarker responses to adverse effects to improve risk assessments assessing ecological effects of pollutant mixtures, like crude oil, in Arctic areas.

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.

Physiological acclimatization in high-latitude zooplankton.

How individual organisms adapt to nonoptimal conditions through physiological acclimatization is central to predicting the consequences of unusual abiotic and biotic conditions such as those produced by marine heat waves. The Northeast Pacific, including the Gulf of Alaska, experienced an extreme warming event (2014-2016, "The Blob") that affected all trophic levels and led to large-scale changes in the community. The marine copepod Neocalanus flemingeri is a key member of the subarctic Pacific pelagic ecosystem. During the spring phytoplankton bloom this copepod builds substantial lipid stores as it prepares for its nonfeeding adult phase. A 3-year comparison of gene expression profiles of copepods collected in Prince William Sound in the Gulf of Alaska between 2015 and 2017 included two high-temperature years (2015 and 2016) and one year with very low phytoplankton abundances (2016). The largest differences in gene expression were between high and low chlorophyll years, and not between warm and cool years. The observed gene expression patterns were indicative of physiological acclimatization. The predominant signal in 2016 was the down-regulation of genes involved in glycolysis and its incoming pathways, consistent with the modulation of metabolic rates in response to prolonged low food conditions. Despite the down-regulation of genes involved in metabolism, there was no evidence of suppression of protein synthesis based on gene expression or behavioural activity. Genes involved in muscle function were up-regulated, and the copepods were actively swimming and responsive to stimuli at collection. However, genes involved in fatty acid metabolism were down-regulated in 2016, suggesting reduced lipid accumulation.

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.

Diapause within the Context of Life-History Strategies in Calanid Copepods (Calanoida: Crustacea).

Post-embryonic diapause in copepods is an adaptation that allows species in the copepod family Calanidae to thrive in high-latitude environments by transforming a short spring phytoplankton bloom into large numbers of lipid-rich individuals capable of surviving a long period of starvation. The copepods, with their high-energy lipid reservoirs, are a critical food source for higher trophic levels, making the Calanidae a key component of high-latitude marine ecosystems. The physiological ecology of the developmental program remains poorly understood. However, new studies using high-throughput RNA sequencing approaches are giving detailed access to physiological status by generating gene expression profiles for both field-collected and laboratory-incubated individuals. These are beginning to characterize the diapause phenotype, elucidate the transcriptional and physiological progression through the diapause program, and illustrate the effects of organism-environment interactions. This paper reviews gene expression profiling studies on the life cycle and diapause program of Neocalanus flemingeri Miller (1988) that were conducted as part of a long-term observation program in the northern Gulf of Alaska. It summarizes recent findings and relates them to the ecology of this species and to that of other calanids.

Regional heterogeneity impacts gene expression in the subarctic zooplankter Neocalanus flemingeri in the northern Gulf of Alaska.

Marine pelagic species are being increasingly challenged by environmental change. Their ability to persist will depend on their capacity for physiological acclimatization. Little is known about limits of physiological plasticity in key species at the base of the food web. Here we investigate the capacity for acclimatization in the copepod Neocalanus flemingeri, which inhabits the Gulf of Alaska, a heterogeneous and highly seasonal environment. RNA-Seq analysis of field-collected pre-adults identified large regional differences in expression of genes involved in metabolic and developmental processes and response to stressors. We found that lipid synthesis genes were up-regulated in individuals from Prince William Sound and down-regulated in the Gulf of Alaska. Up-regulation of lipid catabolic genes in offshore individuals suggests they are experiencing nutritional deficits. The expression differences demonstrate physiological plasticity in response to a steep gradient in food availability. Our transcriptional analysis reveals mechanisms of acclimatization that likely contribute to the observed resilience of this population.

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.

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.

Physiological characterization of the emergence from diapause: A transcriptomics approach.

Organisms inhabiting high-latitude environments have evolved adaptations, such as diapause to time reproduction and growth to optimize their survival. However, the physiological regulation of the timing of complex life histories is poorly understood, particularly for marine copepods, that diapause at depth. A member of the pelagic community of the sub-Arctic Pacific Ocean, Neocalanus flemingeri enters diapause in June. Egg production occurs in winter/spring. In order to characterize the transition from diapause to egg release, females were collected in late September from 400-700 m depth, incubated in the dark at 4-5 °C and sampled for RNASeq at weekly intervals. The diapause phenotype showed down-regulation of protein turnover and up-regulation of stress genes. Activation of the reproductive program was marked by the up-regulation of genes involved in germline development. Thereafter, progress through phases of oocyte development could be linked to changes in gene expression. At 5 weeks, females showed up-regulation of spermatogenesis, indicating that stored sperm had been in a quiescent stage and completed their maturation inside the female. Gene expression profiles provide a framework to stage field-collected females. The 7-week progression from diapause to late oogenesis suggests that females typically spawning in January initiated the reproductive program in November.

The effects of food stoichiometry and temperature on copepods are mediated by ontogeny.

Climate change is warming the oceans, increasing carbon dioxide partial pressure and reducing nutrient recycling from deep layers. This will affect carbon (C) and phosphorus (P) availability in the oceans, thus, altering the balance between the nutrient content of consumers and their food resource. The combined effects of food quality and temperature have been investigated for adult copepods; however, nauplii, the early developmental stages of copepods, often far outnumber adults, grow more rapidly and have a higher phosphorus body content and demand than later life stages. Consequently, ontogeny may affect how copepods respond to the combined stressors of increasing temperature and altered food stoichiometry. We conducted temperature-controlled experiments (24, 28 and 32 °C) where Parvocalanus crassirostris was fed either a P-replete or a P-limited phytoplankton food source. Reduced survival of nauplii and copepodites at the highest temperature was ameliorated when fed P-replete food. At higher temperatures, copepodite growth remained stable, but internal C:P stoichiometry diverged in the direction of phytoplankton C:P, suggesting that increased temperature affected copepodite stoichiometric homeostasis. In contrast, naupliar P content increased with temperature and naupliar growth was P limited, suggesting nauplii required additional phosphorus at higher temperatures. We conclude that resource stoichiometry plays a key role in how copepod survival and growth are impacted by temperature, and that ontogeny mediates these responses. Our results suggest that as the extent of warming oceans and phytoplankton nutrient limitation increase, copepod survival and the growth of early life stages may decline.

Prediction of a peptidome for the ecotoxicological model Hyalella azteca (Crustacea; Amphipoda) using a de novo assembled transcriptome.

Due to its sensitivity to many environmental and anthropogenic stressors, including a wide range of chemical compounds, Hyalella azteca, a freshwater amphipod, has emerged as one of the most commonly used invertebrates for ecotoxicological assessment.Peptidergic signaling systems are key components in the control of organism-environment interactions, and there is a growing literature suggesting that they are targets of a number of aquatic toxicants.Interestingly, and despite its model species status in the field of ecotoxicology, little is known about the peptide hormones of H. azteca.Here, a transcriptome was produced for this species using the de novo assembler Trinity and mined for sequences encoding putative peptide precursors; the transcriptome was assembled from 460,291,636 raw reads and consists of 133,486 unique transcripts.Seventy-six sequences encoding peptide pre/preprohormones were identified from this transcriptome, allowing for the prediction of 202 distinct peptides, which included members of the allatostatin A, allatostatin B, allatostatin C, allatotropin, bursicon, CCHamide, corazonin, crustacean cardioactive peptide, crustacean hyperglycemic hormone/molt-inhibiting hormone, ecdysis-triggering hormone, eclosion hormone, elevenin, FMRFamide-like peptide, glycoprotein hormone, GSEFLamide, inotocin, leucokinin, myosuppressin, neuropeptide F, orcokinin, orcomyotropin, pigment dispersing hormone, proctolin, pyrokinin, red pigment concentrating hormone, RYamide, short neuropeptide F, SIFamide, sulfakinin, tachykinin-related peptide and trissin families.These peptides expand the known peptidome for H. azteca approximately nine-fold, forming a strong foundation for future studies of peptidergic control, including disruption by aquatic toxicants, in this important ecotoxicological model.

De novo transcriptome assembly of the calanoid copepod Neocalanus flemingeri: A new resource for emergence from diapause.

Copepods, small planktonic crustaceans, are key links between primary producers and upper trophic levels, including many economically important fishes. In the subarctic North Pacific, the life cycle of copepods like Neocalanus flemingeri includes an ontogenetic migration to depth followed by a period of diapause (a type of dormancy) characterized by arrested development and low metabolic activity. The end of diapause is marked by the production of the first brood of eggs. Recent temperature anomalies in the North Pacific have raised concerns about potential negative effects on N. flemingeri. Since diapause is a developmental program, its progress can be tracked using through global gene expression. Thus, a reference transcriptome was developed as a first step towards physiological profiling of diapausing females using high-throughput Illumina sequencing. The de novo transcriptome, the first for this species was designed to investigate the diapause period. RNA-Seq reads were obtained for dormant to reproductive N. flemingeri females. A high quality de novo transcriptome was obtained by first assembling reads from each individual using Trinity software followed by clustering with CAP3 Assembly Program. This assembly consisted of 140,841transcripts (contigs). Bench-marking universal single-copy orthologs analysis identified 85% of core eukaryotic genes, with 79% predicted to be complete. Comparison with other calanoid transcriptomes confirmed its quality and degree of completeness. Trinity assembly of reads originating from multiple individuals led to fragmentation. Thus, the workflow applied here differed from the one recommended by Trinity, but was required to obtain a good assembly.

Molecular Characterization of Copepod Photoreception.

Copepod crustaceans are an abundant and ecologically significant group whose basic biology is guided by numerous visually guided behaviors. These behaviors are driven by copepod eyes, including naupliar eyes and Gicklhorn's organs, which vary widely in structure and function among species. Yet little is known about the molecular aspects of copepod vision. In this study we present a general overview of the molecular aspects of copepod vision by identifying phototransduction genes from newly generated and publicly available RNA-sequencing data and assemblies from 12 taxonomically diverse copepod species. We identify a set of 10 expressed transcripts that serve as a set of target genes for future studies of copepod phototransduction. Our more detailed evolutionary analyses of the opsin gene responsible for forming visual pigments found that all of the copepod species investigated express two main groups of opsins: middle-wavelength-sensitive (MWS) opsins and pteropsins. Additionally, there is evidence from a few species (e.g., Calanus finmarchicus, Eurytemora affinis, Paracyclopina nana, and Lernaea cyprinacea) for the expression of two additional groups of opsins-the peropsins and rhodopsin 7 (Rh7) opsins-at low levels or distinct developmental stages. An ontogenetic analysis of opsin expression in Calanus finmarchicus found the expression of a single dominant MWS opsin, as well as evidence for differences in expression across development in some MWS, pteropsin, and Rh7 opsins, with expression peaking in early naupliar through early copepodite stages.

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.

Vertical gradients in species richness and community composition across the twilight zone in the North Pacific Subtropical Gyre.

Although metazoan animals in the mesopelagic zone play critical roles in deep pelagic food webs and in the attenuation of carbon in midwaters, the diversity of these assemblages is not fully known. A metabarcoding survey of mesozooplankton diversity across the epipelagic, mesopelagic and upper bathypelagic zones (0-1500 m) in the North Pacific Subtropical Gyre revealed far higher estimates of species richness than expected given prior morphology-based studies in the region (4,024 OTUs, 10-fold increase), despite conservative bioinformatic processing. Operational taxonomic unit (OTU) richness of the full assemblage peaked at lower epipelagic-upper mesopelagic depths (100-300 m), with slight shoaling of maximal richness at night due to diel vertical migration, in contrast to expectations of a deep mesopelagic diversity maximum as reported for several plankton groups in early systematic and zoogeographic studies. Four distinct depth-stratified species assemblages were identified, with faunal transitions occurring at 100 m, 300 m and 500 m. Highest diversity occurred in the smallest zooplankton size fractions (0.2-0.5 mm), which had significantly lower % OTUs classified due to poor representation in reference databases, suggesting a deep reservoir of poorly understood diversity in the smallest metazoan animals. A diverse meroplankton assemblage also was detected (350 OTUs), including larvae of both shallow and deep living benthic species. Our results provide some of the first insights into the hidden diversity present in zooplankton assemblages in midwaters, and a molecular reappraisal of vertical gradients in species richness, depth distributions and community composition for the full zooplankton assemblage across the epipelagic, mesopelagic and upper bathypelagic zones.