Analysis of the transcriptional pathways associated with the induction of quiescent embryonic arrest in the calanoid copepod Acartia tonsa.

The copepod species Acartia tonsa (Dana)(Crustacea) have the unique ability to induce quiescent embryonic dormancy if adverse environmental conditions occur; a characteristic shared by 41 other species belonging to the superfamily Centropagoida in the Calanoida order. However, the transcriptional changes characterizing this process are not known. Here, we compare the transcriptome of embryos in arrested quiescence with the normal development to identify pathways and differentially regulated transcripts involved in quiescent embryogenesis. Quiescence was induced by incubating eggs at 4 °C with anoxia for 26 h(hr), while eggs undergoing normal immediate development were incubated at 16.9 °C in normoxia for 7 h (where gastrulation occurs) or 14 h (where organogenesis occurs) before collecting for RNA extraction and analysis by RNA-sequencing. Results indicate that the expression profile of the quiescent embryo is not as different from the normal embryonic gastrulation as initially expected: None of the mapped transcripts is uniquely expressed in quiescence. Moreover, in quiescence a large proportion of the annotated transcripts display expression values halfway in-between the normal, immediate developmental stages of gastrulation and organogenesis. In depth comparison between the organogenesis stage and quiescent samples, reveal a high degree of divergence, confirming that a developmental arrest has been induced through quiescence. Specifically: Stress response transcripts are prominent in the quiescent phase with a transcript like the mammalian autophagy gene Sequestosome-1/p62 (SQSTM) being upregulated. The present analysis provides a better understanding of the molecular mechanisms characterizing the quiescent embryonic state of A. tonsa.

Embryogenesis of a calanoid copepod analyzed by transcriptomics.

The calanoid copepod Acartia tonsa (Dana) has attracted interest because of its use as a copepod model organism as well as its potential economic role as live fish larval feed. While the adult genome and transcriptome of A. tonsa has been investigated, no studies have been performed investigating the genome-wide transcriptional changes during the normal subitaneous embryogenesis. Thus, the aim of the current study was to investigate said transcriptional changes throughout A. tonsa embryonic development. RNA extraction and de novo transcriptome assembly for the subitaneous embryogenesis of the copepod was conducted. The assembly includes for the first-time samples describing quiescent development and overall helps establishing a framework for future studies on the molecular biology of our species of interest. Among the findings reported, sequences annotated to well-known developmental genes, were identified. At the same time are described the molecular changes and gene expression levels throughout the entire 42 h the embryonic development lasts. In conclusion, here we present the most complete genome-wide transcriptional map of early copepod embryonic development to date, enabling further use of A. tonsa as a model organism for crustacean development. Keywords: enrichment of pathways; subitaneous embryogenesis, comparative genomics; transcriptome assembly; invertebrate genomics.

Biowaste and by-products as rearing substrates for black soldier fly (Hermetia illucens) larvae: Effects on larval body composition and performance.

Black soldier fly (Hermetia illucens) larvae can convert biowaste and by-products into body mass high in protein (~40% dry matter, DM) and lipid (~30% DM). However, the type of rearing substrate also affects the larval body composition and thus its nutritional value. Hitherto, it remains unclear how and to what extent the larval body composition can be altered by the substrate. This study was therefore performed to examine the possibilities of modifying larval body composition using different rearing substrates. To investigate this, 5-days old larvae were reared for seven days on different locally available waste and by-products: brewer's spent grain, mitigation mussels (Mytilus edulis), rapeseed cake, and shrimp waste meal (Pandalus borealis). Larval composition and performance were compared to larvae reared on a commercial chicken feed as well as a mixed feed (mixture of chicken feed and by-products, with a similar macronutrient composition to chicken feed). Larval body weight was recorded daily to determine growth over time whereas larvae and substrates were sampled at the start and end of the trial and analysed for their nutritional composition. The type of rearing substrate affected both larval body composition and growth performance. There was a clear relation between the nutritional composition of the substrate and larvae for certain fatty acids. Larvae reared on marine-based waste substrates contained a higher share of omega-3 fatty acids than larvae reared on the other substrates, indicating an accumulation of omega-3 fatty acids from the substrate. There was a strong positive linear correlation between the ash content in the substrate and larvae whereas larval lipid, protein, amino acid, and chitin content seemed more affected by larval development. Overall, this study showed that the rearing substrate affects larval composition and development, and that larval composition of certain nutrients can be tailored depending on further food and feed applications.

Particles as carriers of matter in the aquatic environment: Challenges and ways ahead for transdisciplinary research.

A diverse array of natural and anthropogenic particles found in the aquatic environment, can act as carriers of co-transported matter (CTM), such as nutrients, genetic material and contaminants. Thus, understanding carrier particle transport will increase our understanding of local and global fluxes of exogenous CTM (affiliated with the particle) and endogenous CTM (an inherent part of the particle). In the present contribution, researchers from multiple disciplines collaborated to provide perspectives on the interactions between carrier particles and CTM, and the fundamentals of transport of particles found in the aquatic environment and the generic spherical smooth particles, often used to make predictions about particle behavior in suspension. Evidently, the particles in the aquatic environment show a great variety of characteristics and vary greatly from each other as well as from the generic particle. However, in spite of these differences, many fundamental concepts apply to particles in general. We emphasize the importance of understanding the basic concepts of transport of particle-associated CTM, and the main assumptions in the generic-founded models, which are challenged by the diverging characteristics of particles found in the aquatic environment, as paramount moving forward. Additionally, we identified the need for a conceptual and semantic link between different scientific fields of particle research and initiated the formation of a consistent terminology. Disciplinary and organizational (academic and funding) barriers need to be overcome to enable individual researchers to move beyond their knowledge sphere, to stimulate future interdisciplinary collaborations and to avoid research silos. Hereby, we can foster faster and better progress of evolving research fields on new and emerging anthropogenic carrier particles, and stimulate the development of solutions to the technological and environmental challenges.

The Whole Genome Sequence and mRNA Transcriptome of the Tropical Cyclopoid Copepod Apocyclops royi.

Copepoda is one of the most ecologically important animal groups on Earth, yet very few genetic resources are available for this Subclass. Here, we present the first whole genome sequence (WGS, acc. UYDY01) and the first mRNA transcriptome assembly (TSA, Acc. GHAJ01) for the tropical cyclopoid copepod species Apocyclops royi Until now, only the 18S small subunit of ribosomal RNA gene and the COI gene has been available from A. royi, and WGS resources was only available from one other cyclopoid copepod species. Overall, the provided resources are the 8th copepod species to have WGS resources available and the 19th copepod species with TSA information available. We analyze the length and GC content of the provided WGS scaffolds as well as the coverage and gene content of both the WGS and the TSA assembly. Finally, we place the resources within the copepod order Cyclopoida as a member of the Apocyclops genus. We estimate the total genome size of A. royi to 450 Mb, with 181 Mb assembled nonrepetitive sequence, 76 Mb assembled repeats and 193 Mb unassembled sequence. The TSA assembly consists of 29,737 genes and an additional 45,756 isoforms. In the WGS and TSA assemblies, >80% and >95% of core genes can be found, though many in fragmented versions. The provided resources will allow researchers to conduct physiological experiments on A. royi, and also increase the possibilities for copepod gene set analysis, as it adds substantially to the copepod datasets available.

The Genome and mRNA Transcriptome of the Cosmopolitan Calanoid Copepod Acartia tonsa Dana Improve the Understanding of Copepod Genome Size Evolution.

Members of the crustacean subclass Copepoda are likely the most abundant metazoans worldwide. Pelagic marine species are critical in converting planktonic microalgae to animal biomass, supporting oceanic food webs. Despite their abundance and ecological importance, only six copepod genomes are publicly available, owing to a number of factors including large genome size, repetitiveness, GC-content, and small animal size. Here, we report the seventh representative copepod genome and the first genome and the first transcriptome from the calanoid copepod species Acartia tonsa Dana, which is among the most numerous mesozooplankton in boreal coastal and estuarine waters. The ecology, physiology, and behavior of A. tonsa have been studied extensively. The genetic resources contributed in this work will allow researchers to link experimental results to molecular mechanisms. From PCR-free whole genome sequence and mRNA Illumina data, we assemble the largest copepod genome to date. We estimate that A. tonsa has a total genome size of 2.5 Gb including repetitive elements we could not resolve. The nonrepetitive fraction of the genome assembly is estimated to be 566 Mb. Our DNA sequencing-based analyses suggest there is a 14-fold difference in genome size between the six members of Copepoda with available genomic information. This finding complements nucleus staining genome size estimations, where 100-fold difference has been reported within 70 species. We briefly analyze the repeat structure in the existing copepod whole genome sequence data sets. The information presented here confirms the evolution of genome size in Copepoda and expands the scope for evolutionary inferences in Copepoda by providing several levels of genetic information from a key planktonic crustacean species.

Molecular physiology of copepods - from biomarkers to transcriptomes and back again.

Planktonic copepods are a diverse and abundant group of small (~mm sized) aquatic animals that play a critical role in linking the base of the food chain with higher trophic levels. These invertebrates are a primary food source for marine fish larvae. Their ubiquitous presence is thus of vital importance for recruitment of fish stocks and also as promising live feed for finfish production in aquaculture. This paper reviews the application of molecular approaches to understanding copepod physiology, particularly in non-parasitic species. The review includes both targeted gene approaches and untargeted transcriptomic approaches, with suggestions for best practices in each case. Issues particularly relevant to studies of copepods include heterogeneity within species, morphologically cryptic species, experimental artifacts associated with sample handling, and limited annotation of copepod genes and transcripts. The emergence of high-throughput sequencing and associated increased availability of genomic and transcriptomic databases have presented a huge opportunity to advance knowledge of copepod physiology. The research community can leverage this opportunity through efforts to maintain or improve data accessibility, database annotation, and documentation of analytical pipelines.

n-3 PUFA biosynthesis by the copepod Apocyclops royi documented using fatty acid profile analysis and gene expression analysis.

The cyclopoid copepod Apocyclops royi (Lindberg 1940) is one of two dominant mesozooplankton species in brackish Taiwanese aquaculture ponds. Periodically low n-3 polyunsaturated fatty acid (PUFA) content in seston could potentially be a limiting factor for zooplankton diversity. Apocyclops royi's potential ability to biosynthesize n-3 PUFA was investigated through a short-term feeding experiment on four species of microalgae. Furthermore, we analyzed the expression of genes encoding putative fatty acid elongase (ELO) and desaturase (FAD) enzymes in A. royi on long-term diets of the PUFA-poor Dunaliella tertiolecta and the PUFA-rich Isochrysis galbana The copepods exhibited high contents of docosahexaenoic acid (DHA, C22:6n-3) (>20% of total fatty acid) even when DHA-starved for two generations, and no significant differences were found in absolute DHA content between treatments. Transcripts correlating to the four enzymes Elovl4, Elovl5, Fad Δ5 and Fad Δ6 in the n-3 PUFA biosynthetic pathway were identified. Gene expression analysis revealed a significantly higher expression of two desaturases similar to Fad Δ6 in copepods fed PUFA-lacking algae compared to copepods fed algae with high PUFA content. These findings suggest a highly active n-3 PUFA biosynthesis and capability of DHA production in A. royi when fed low-PUFA diets.

Eggs of the copepod Acartia tonsa Dana require hypoxic conditions to tolerate prolonged embryonic development arrest.

BACKGROUND: Copepods make up the largest zooplankton biomass in coastal areas and estuaries and are pivotal for the normal development of fish larva of countless species. During spring in neritic boreal waters, the copepod pelagic biomass increases rapidly from near absence during winter. In the calanoid species Acartia tonsa, a small fraction of eggs are dormant regardless of external conditions and this has been hypothesized to be crucial for sediment egg banks and for the rapid biomass increase during spring. Other eggs can enter a state of induced arrest called quiescence when external conditions are unfavourable. While temperature is known to be a pivotal factor in the transition from developing to resting eggs and back, the role of pH and free Oxygen in embryo development has not been systematically investigated. RESULTS: Here, we show in a laboratory setting that hypoxic conditions are necessary for resting eggs to maintain a near-intact rate of survival after several months of induced resting. We further investigate the influence of pH that is realistic for natural sediments on the viability of resting eggs and document the effect that eggs have on the pH of the surrounding environment. We find that resting eggs acidify their immediate surroundings and are able to survive in a wide range of pH. CONCLUSIONS: This is the first study to demonstrate the importance of hypoxia on the survival capabilities of A. tonsa resting eggs in a controlled laboratory setting, and the first to show that the large majority of quiescent eggs are able to tolerate prolonged resting. These findings have large implications for the understanding of the recruitment of copepods from sediment egg banks, which are considered the primary contributor of nauplii seeded to pelagic populations in nearshore habitats in late spring.

Timing of embryonic quiescence determines viability of embryos from the calanoid copepod, Acartia tonsa (Dana).

Like 41 other calanoid copepods, Acartia tonsa, are capable of inducing embryonic quiescence when experiencing unfavorable environmental conditions. The ecdysone-signaling cascade is known to have a key function in developmental processes like embryogenesis and molting of arthropods, including copepods. We examined the role of ecdysteroid-phosphate phosphatase (EPPase), ecdysone receptor (EcR), ß fushi tarazu transcription factor 1 (ßFTZ-F1), and the ecdysteroid-regulated early gene E74 (E74), which represent different levels of the ecdysone-signaling cascade in our calanoid model organism. Progression of embryogenesis was monitored and hatching success determined to evaluate viability. Embryos that were induced quiescence before the gastrulation stage would stay in gastrulation during the rest of quiescence and exhibited a slower pace of hatching as compared to subitaneous embryos. In contrast, embryos developed further than gastrulation would stay in gastrulation or later stages during quiescence and showed a rapid pace in hatching after quiescence termination. Expression patterns suggested two peaks of the biological active ecdysteroids, 20-hydroxyecdysone (20E). The first peak of 20E was expressed in concert with the beginning of embryogenesis originating from yolk-conjugated ecdysteroids, based on EPPase expression. The second peak is suggested to originate from de novo synthesized 20E around the limb bud stage. During quiescence, the expression patterns of EPPase, EcR, ßFTZ-F1, and E74 were either decreasing or not changing over time. This suggests that the ecdysone-signaling pathway play a key role in the subitaneous development of A. tonsa embryogenesis, but not during quiescence. The observation is of profound ecological and practical relevance for the dynamics of egg banks.

Seasonal genetic variation associated with population dynamics of a poecilogonous polychaete worm.

Poecilogonous species show variation in developmental mode, with larvae that differ both morphologically and ecologically. The spionid polychaete Pygospio elegans shows variation in developmental mode not only between populations, but also seasonally within populations. We investigated the consequences of this developmental polymorphism on the spatial and seasonal genetic structure of P. elegans at four sites in the Danish Isefjord-Roskilde-Fjord estuary at six time points, from March 2014 until February 2015. We found genetic differentiation between our sampling sites as well as seasonal differentiation at two of the sites. The seasonal genetic shift correlated with the appearance of new size cohorts in the populations. Additionally, we found that the genetic composition of reproductive individuals did not always reflect the genetic composition of the entire sample, indicating that variance in reproductive success among individuals is a likely explanation for the patterns of chaotic genetic patchiness observed during this and previous studies. The heterogeneous, unpredictable character of the estuary might maintain poecilogony in P. elegans as a bet-hedging strategy in the Isefjord-Roskilde-Fjord complex in comparison with other sites where P. elegans are expected to be fixed to a certain mode of development.

A simple and fast method for extraction and quantification of cryptophyte phycoerythrin.

The microalgal pigment phycoerythrin (PE) is of commercial interest as natural colorant in food and cosmetics, as well as fluoroprobes for laboratory analysis. Several methods for extraction and quantification of PE are available but they comprise typically various extraction buffers, repetitive freeze-thaw cycles and liquid nitrogen, making extraction procedures more complicated. A simple method for extraction of PE from cryptophytes is described using standard laboratory materials and equipment. The cryptophyte cells on the filters were disrupted at -80 °C and added phosphate buffer for extraction at 4 °C followed by absorbance measurement. The cryptophyte Rhodomonas salina was used as a model organism. •Simple method for extraction and quantification of phycoerythrin from cryptophytes.•Minimal usage of equipment and chemicals, and low labor costs.•Applicable for industrial and biological purposes.

Changes in free amino acid content during naupliar development of the Calanoid copepod Acartia tonsa.

Changes in free amino acids (FAA) were investigated in the potentially important live feed and neritic copepod species Acartia tonsa during naupliar development. Total content of FAA in A. tonsa nauplii was around 17% of dry weight at first development stage, and declined to 6% for later stages. Relative to body-volume and biomass, the FAA content indicated possible volume-dependent changes. However, changes in FAA with osmolytic activity could not account for this decline in FAA content, but suggests that the decline reflected degradation of residual FAAs from the embryonic stage. Glutamic acid revealed the largest change in relative abundance during naupliar development and declined from 29.0% at first nauplius stage to 7.1% at later stages. The high FAA pool in early naupliar stages may be necessary for naupliar development due to an absence of feeding at first development stages. The high FAA content in early nauplii indicates that A. tonsa is a valuable source for nutritional energy for first-feeding fish larvae and should be further exploited for aquaculture purposes. Enhancements to FAA abundances in nauplii through manipulation of maternal diets could be of future interest, as copepod nauplii can contain a substantial pool of FAAs at first development stage.

Are invertebrates relevant models in ageing research? Focus on the effects of rapamycin on TOR.

Ageing is the organisms increased susceptibility to death, which is linked to accumulated damage in the cells and tissues. Ageing is a complex process regulated by crosstalk of various pathways in the cells. Ageing is highly regulated by the Target of Rapamycin (TOR) pathway activity. TOR is an evolutionary conserved key protein kinase in the TOR pathway that regulates growth, proliferation and cell metabolism in response to nutrients, growth factors and stress. Comparing the ageing process in invertebrate model organisms with relatively short lifespan with mammals provides valuable information about the molecular mechanisms underlying the ageing process faster than mammal systems. Inhibition of the TOR pathway activity via either genetic manipulation or rapamycin increases lifespan profoundly in most invertebrate model organisms. This contribution will review the recent findings in invertebrates concerning the TOR pathway and effects of TOR inhibition by rapamycin on lifespan. Besides some contradictory results, the majority points out that rapamycin induces longevity. This suggests that administration of rapamycin in invertebrates is a promising tool for pursuing the scientific puzzle of lifespan prolongation.

Dietary supplementation of essential fatty acids in larval pikeperch (Sander lucioperca); short and long term effects on stress tolerance and metabolic physiology.

The present study examined the effects of feeding pike perch larvae Artemia, enriched with either docosahexanoic acid (DHA), arachidonic acid (ARA), oleic acid (OA), olive oil (OO) or a commercial enrichment DHA Selco (DS) on tissue lipid deposition, stress tolerance, growth and development, and metabolic rate. There was higher tissue retention of ARA than DHA at comparable inclusion levels. No differences were observed between diets on the percentage contribution of ARA or DHA to the fatty acid profile of tissues (head and trunk). Total fatty acid content (mgg(-1)) was significantly higher in the head, reflecting its high content of neural tissue. Observations on larval erratic behaviour and mortality following exposure to salinity stress suggested that high inclusions levels of DHA had an alleviating effect, while ARA did not. Particularly larval groups reared for 16 days on diets enriched with OO and OA had mortality rates approaching 100% within two hours. Interestingly, this tendency, although not as pronounced, was also apparent in juvenile fish after 120 days of rearing on a common diet. Standard metabolic rate in larvae on an OO enriched diet was significantly elevated, but otherwise no groups had significant changes to their respiratory physiology. In addition to increased stress challenge sensitivity, early feeding with OA had long term impact on pike perch neural development indicated by a smaller brain size in juvenile fish. In conclusion, lack of DHA in the diet of pikeperch larvae suggests that this long chain polyunsaturated fatty acid is involved in processes that increase stress tolerance and that lack of dietary DHA in early larval stage caused increased stress sensitivity and long-term impaired neural development, while it does not appear to affect metabolic rate at rest.

Do Acartia tonsa (Dana) eggs regulate their volume and osmolality as salinity changes?

Subitaneous eggs from an euryhaline calanoid copepod Acartia tonsa were challenged by changes in salinity within the range from full strength salinity, down to zero and up to >70 psu. Egg volume changed immediately, increasing from 2.8 × 10(5) µm(3) at full strength salinity (35 psu) to 3.8 × 10(5) µm(3) at 0 psu and back to its initial volume when gradually being returned to full strength salinity. Egg osmolality followed the molality of the surrounding water when challenged within a salinity range from 2 to 50 psu. Egg respiration was not affected when eggs kept at 35 psu was exposed to low salinity (2 psu). These results suggest that eggs are unable to regulate their volume or osmolality when challenged with changes in salinity. Gradual changes in salinity from 35 to 2 psu and back did not harm the eggs (embryos), since the hatching success remained unaffected by such changes in salinity. In contrast, extreme hyper-saline conditions (76 psu) made the eggs implode and killed the embryo. We propose that the embryo is protected from salinity stress by its plasma membrane and that water exchange driven by osmosis is restricted to the perivitelline space of the egg, which acts as a perfect osmometer in the salinity range of 5-35 psu. We hypothesize further that the embryo is able to keep its volume and osmolality constant due to the impermeability of the inner plasma membrane of the egg or by a combination of osmoregulation and reduced permeability of the inner plasma membrane.