Deriving a Chronic Guideline Value for Nickel in Tropical and Temperate Marine Waters.

The absence of chronic toxicity data for tropical marine waters has limited our ability to derive appropriate water quality guideline values for metals in tropical regions. To aid environmental management, temperate data are usually extrapolated to other climatic (e.g., tropical) regions. However, differences in climate, water chemistry, and endemic biota between temperate and tropical systems make such extrapolations uncertain. Chronic nickel (Ni) toxicity data were compiled for temperate (24 species) and tropical (16 species) marine biota and their sensitivities to Ni compared. Concentrations to cause a 10% effect for temperate biota ranged from 2.9 to 20 300 µg Ni/L, with sea urchin larval development being the most sensitive endpoint. Values for tropical data ranged from 5.5 to 3700 µg Ni/L, with copepod early-life stage development being the most sensitive test. There was little difference in temperate and tropical marine sensitivities to Ni, with 5% hazardous concentrations (95% confidence interval) of 4.4 (1.8-17), 9.6 (1.7-26), and 5.8 (2.8-15) µg Ni/L for temperate, tropical, and combined temperate and tropical species, respectively. To ensure greater taxonomic coverage and based on guidance provided in Australia and New Zealand, it is recommended that the combined data set be used as the basis to generate a jurisdiction-specific water quality guideline of 6 µg Ni/L for 95% species protection applicable to both temperate and tropical marine environments. Environ Toxicol Chem 2020;39:2540-2551. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

The cleavage pattern of calanoid copepods-a case study.

Many crustacean groups show stereotyped cleavage patterns during early ontogeny. However, these patterns differ between the various major crustacean taxa, and a general mode is difficult to extract. Previous studies suggested that also copepods undergo an early cleavage with a more or less stereotyped pattern of blastomere divisions and fates. Yet, copepod embryology has been largely neglected. The last investigation of this kind dates back more than a century and the results are somewhat contradictory when compared with those of other researchers. To overcome these problems, we studied the early development of a so far undescribed calanoid copepod species, Skistodiaptomus sp., applying histochemical staining, confocal laser scanning microscopy, and bifocal 4D microscopy. The blastomere arrangement of the four-cell stage of this species varies to a large degree. It can either form a typical radial pattern with the four blastomeres lying in one plane or a tilted orientation of the axes connecting the sister cells of the previous division. In both cases, a stereotyped division pattern is maintained inside each quadrant during subsequent cleavages. In addition, we found two types of blastomere arrangements with a mirror symmetry. Most divisions within the quadrants follow the perpendicularity rule until the eighth cleavage. Deviations from this rule occur only in the narrow regions where the different quadrants touch and near the site of gastrulation. Gastrulation is initiated around the descendants of one individually identifiable blastomere of the 16-cell stage. This cell divides in a specific manner forming a characteristic cell arrangement, the gastrulation triangle. This gastrulation triangle initiates the internalization process of the gastrulation and it is encircled by another characteristic cell type, the crown cells. Our observations reveal several similarities to the early development of Calanus finmarchicus, another calanoid species. These relate to blastomere arrangements and divisions and the pattern of gastrulation. As Calanoida represent a basal or near basal branch of the copepod tree, this description will provide the ground for reconstruction of the cleavage pattern of the last common ancestor of Copepoda.

Embryos of an Antarctic zooplankton require anoxia for dormancy, are permeable to lipophilic chemicals, and reside in sediments containing PCBs.

Zooplankton in Antarctic maritime lakes face challenges imposed by anthropogenic chemicals. Studies on temperate species suggest that lipophilic chemicals will accumulate in dormant embryos of Antarctic zooplankton and decrease hatching success, thereby threatening centuries of accumulated genetic diversity that would increase population resilience in the face of climate change. We evaluated the potential for lakes to act as sinks for legacy pollutants in the maritime Antarctic by testing sediments for polychlorinated biphenyls (PCBs) previously identified in soil, flora and fauna of lake catchments. Direct tests of embryo permeability to chemicals are confounded by potential adhesion of chemicals to the embryo surface and limited biomass available. Therefore, in order to assess the potential for lipophilic chemicals to penetrate and passively accumulate in dormant embryos of Antarctic lacustrine zooplankton, we evaluated the effect of anoxia on post-diapause development in the calanoid copepod, Boeckella poppei, and then used chemical anoxia induced by rotenone as a reporter for permeability of these embryos to moderately lipophilic chemicals. The data presented demonstrate that embryos of B. poppei from Antarctic lake sediments will passively accumulate moderately lipophilic chemicals while lying dormant in anoxic sediments. Implications for legacy POPs in sediments of Antarctic maritime lakes are discussed.

Chromatin Diminution in Cyclops kolensis Lill. (Copepoda, Crustacea) as a Radical Way to Inactivate Redundant Genome in Somatic Cells.

Chromatin diminution (CD) is a phenomenon of programmed DNA elimination which takes place in early embryogenesis in some eukaryotes. The mechanism and biological role of CD remain largely unknown. During CD in the freshwater copepod Cyclops kolensis, the genome of cells of the somatic lineage is reorganized and reduced in size by more than 90% without affecting the genome of germline cells. Although the diploid chromosome number is unchanged, chromosome size is dramatically reduced by CD. The eliminated DNA consists primarily of repetitive sequences and localizes within granules during the elimination process. In this review, we provide an overview of CD in C. kolensis including both cytological and molecular studies.

Larval neurogenesis in the copepod Tigriopus californicus (Tetraconata, Multicrustacea).

Arthropod early neurogenesis shows distinct patterns that have been interpreted in an evolutionary framework. For instance, crustaceans and Hexapoda form the taxon Tetraconata and share the differentiation of specific neural precursors, the neuroblasts, a character which sets them apart from Chelicerata and Myriapoda. Neuroblasts are relatively large stem cells that generate ganglion mother cells by asymmetric divisions. Ganglion mother cells typically divide once to give rise to neurons and glia cells. In hexapods, neuroblasts segregate from the neuroectoderm before they begin their characteristic proliferative activity. In the crustaceans studied so far, neuroblasts remain in the neuroectoderm. Yet, detailed studies on early neurogenesis of crustaceans at the cellular level are largely restricted to some malacostracan and branchiopod species. Crustaceans are very diverse and likely paraphyletic with respect to hexapods. Hence, knowledge about neural differentiation in other crustacean taxa might contribute to the understanding of evolution of neurogenesis in Tetraconata. Here, we describe the early neurogenesis during naupliar development of the copepod Tigriopus californicus. We show that neuroblasts are present that generate ganglion mother cells, which in turn divide to give rise to neurons of the ventral nerve cord. These two neural precursor cell types and their specific arrangement correspond to what has been found in other crustaceans. One obvious difference concerns the relative size of the neuroblasts, which are not much larger than their progeny. Our results complement the picture of neural differentiation in crustaceans and suggest that superficially located neuroblasts are likely the ancestral condition in Tetraconata.

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.

Exocrine glands of Lepeophtheirus salmonis (Copepoda: Caligidae): Distribution, developmental appearance, and site of secretion.

Exocrine glands of blood-feeding parasitic copepods are believed to be important in host immune response modulation and inhibition of host blood coagulation, but also in the production of substances for integument lubrication and antifouling. In this study, we aimed to characterize the distribution of different types of salmon louse (Lepeophtheirus salmonis) exocrine glands and their site of secretion. The developmental appearance of each gland type was mapped and genes specifically expressed by glands were identified. Three types of tegumental (teg 1-3) glands and one labial gland type were found. The first glands to appear during development were teg 1 and teg 2 glands. They have ducts extending both dorsally and ventrally suggested to be important in lubricating the integument. Teg 1 glands were found to express two astacin metallopeptidases and a gene with fibronectin II domains, while teg 2 glands express a heme peroxidase. The labial glands were first identified in planktonic copepodids, with reservoirs that allows for storage of glandular products. The last gland type to appear during development was named teg 3 and was not seen before the preadult I stage when the lice become more virulent. Teg 3 glands have ducts ending ventrally at the host-parasite contact area, and may secrete substances important for the salmon lice virulence. Salmon lice teg 3 and labial glands are thus likely to be especially important in the host-parasite interaction. Proteins secreted from the salmon louse glands to its salmonid host skin or blood represents a potential interface where the host immune system can meet and elicit effective responses to sea lice antigens. The present study thus represents a fundamental basis for further functional studies and identification of possible vaccine candidates. J. Morphol. 277:1616-1630, 2016. © 2016 Wiley Periodicals, Inc.

Transcriptome analysis of the couch potato (CPO) protein reveals an expression pattern associated with early development in the salmon louse Caligus rogercresseyi.

The couch potato (CPO) protein is a key biomolecule involved in regulating diapause through the RNA-binding process of the peripheral and central nervous systems in insects and also recently discovered in a few crustacean species. As such, ectoparasitic copepods are interesting model species that have no evidence of developmental arrest. The present study is the first to report on the cloning of a putative CPO gene from the salmon louse Caligus rogercresseyi (CrCPO), as identified by high-throughput transcriptome sequencing. In addition, the transcription expression in larvae and adults was evaluated using quantitative real-time PCR. The CrCPO cDNA sequence showed 3261 base pairs (bp), consisting of 713bp of 5' UTR, 1741bp of 3' UTR, and an open reading frame of 807bp encoding for 268 amino acids. The highly conserved RNA binding regions RNP2 (LFVSGL) and RNP1 (SPVGFVTF), as well the dimerization site (LEF), were also found. Furthermore, eight single nucleotide polymorphisms located in the untranslated regions and one located in the coding region were detected. Gene transcription analysis revealed that CrCPO has ubiquitous expression across larval stages and in adult individuals, with the highest expression from nauplius to copepodid stages. The present study suggests a putative biological function of CrCPO associated with the development of the nervous system in salmon lice and contributes molecular evidence for candidate genes related to host-parasite interactions.

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.

Unusual augmentation of germline genome size in Cyclops kolensis (Crustacea, Copepoda): further evidence in support of a revised model of chromatin diminution.

Embryonic chromatin diminution, the selective excision of large amounts of heterochromatic DNA from presomatic cell lineages, provides an example of an unusually large augmentation of the germline genome and raises questions regarding the source of the increased amount of DNA and its relevance to the biology of the organism. DNA levels in adult germ cell nuclei of the copepod Cyclops kolensis were determined by DNA-Feulgen cytophotometry and compared with those of somatic nuclei of adults and both pre- and postdiminuted embryos from the same mothers. Almost 75 pg DNA/nucleus is excised by diminution, resulting in the return of each generation to the approximately 1 pg DNA/nucleus level found for adult soma. To account for the increase in DNA levels of germ cells observed here, we propose alternative hypotheses to the original model of chromatin diminution: (1) repetitive endocycles or (2) proliferation of genetic elements. Specific tests for these hypotheses using next-generation sequencing and quantitative cytophotometry, as well as the functional significance of germ cell DNA augmentation to the copepod, are discussed.

Characterisation of two vitellogenins in the salmon louse Lepeophtheirus salmonis: molecular, functional and evolutional analysis.

The salmon louse Lepeophtheirus salmonis Krøyer affects a variety of wild salmonoid hosts, but is also an important pest in aquaculture, which is a globally important and rapidly growing industry. Salmon lice have large reproductive outputs, and knowledge of reproductive processes may be crucial for the control of this parasite. Here, we report on the characterisation of 2 vitellogenins (LsVit1 and LsVit2), which are the precursors of salmon-louse egg-yolk glycoprotein. The structure of LsVit1 and LsVit2 was examined and compared to that in other oviparous animals. Phylogenetic analysis of LsVit1 and LsVit2 confirmed the view that crustaceans are a polyphyletic group. Transcriptional and translational analysis demonstrated production of LsVit1 and LsVit2 in the subcuticular tissue of the adult female lice. LsVit1 and LsVit2 could also be found in maturing oocytes and developing embryos and early larval stages. LsVit2 was found to be processed into 2 smaller fragments, whereas LsVit1 was found to be full length when deposited into the oocytes. Degradation of LsVit1 and LsVit2 was characterised through embryogenesis and the early non-feeding larval stages. Finally, protein content and the level of free amino acids were analysed in embryos and larval stages and their role in nutrition and osmoregulation discussed. In conclusion, our results confirm the role of vitellogenins in reproduction as providers of embryonic and larval nutrition.

Food quality effects on copepod growth and development: implications for bioassays in ecotoxicological testing.

We evaluated effects of six algal species in 25 combinations on growth and reproduction of the harpacticoid copepod Nitocra spinipes. In the first lifecycle test, Rhodomonas salina, Phaeodactylum tricornutum, and Dunaliella tertiolecta were used. The results showed that R. salina was the best food, whereas P. tricornutum (0% development success) and D. tertiolecta (41.7% malformations) were poor food items. In the second lifecycle test, a mixture of R. salina, Tetraselmis suecica, and Thalassiosira weisflogii (selected from screening tests) was tested together with a mono-diet of R. salina. Also in this test, copepods fed R. salina performed better (i.e. had higher survival and reproductive success) compared with the other treatment. We conclude that R. salina is appropriate to use as food in toxicity testing with N. spinipes, whereas some of the algae commonly used as feed in ecotoxicological tests with other copepods had detrimental effects on the development, reproduction, and survival of N. spinipes.

Heterochromatin endoreduplication prior to gametogenesis and chromatin diminution during early embryogenesis in Mesocyclops edax (Copepoda: Crustacea).

The segregation of progenitor somatic cells from those of the primordial germ cells that sequester and retain elevated levels of DNA during subsequent developmental events, poses an interesting, alternative pathway of chromosome behavior during the reproductive cycle of certain species of cyclopoid copepods and several other organisms. Separation of maternal and paternal chromosome sets during very early cleavages (gonomery) is often a feature following marked elevations of DNA levels in germ cells for some of these species. Here, we report on the accumulation of large amounts of DNA in germ line nuclei of both female and male juveniles and adults of a freshwater copepod, Mesocyclops edax (Forbes, 1890). We also report the robust uptake of 3H-thymidine by germ cells prior to gametogenesis in this species. By using cytophotometric analysis of the DNA levels in both germ line cells and somatic cells from the same specimens we demonstrate that germ cell nuclei accumulate high levels of DNA prior to the onset of gametogenesis. These elevated amounts coincide with the levels of heterochromatic DNA discarded during chromatin diminution. A new model is proposed of major cytological events accompanying the process of chromatin diminution in M. edax.

Respiration rates of subitaneous eggs from a marine calanoid copepod: monitored by nanorespirometry.

The oxygen consumption rate during embryogenesis of Acartia tonsa subitaneous eggs were measured at different temperatures (10, 15, 17, 21, 24 and 28 degrees C) with nanorespirometry. The oxygen consumption was constant during the embryogenesis but increased rapidly at hatching time. The mean +/- SD oxygen consumption rate increased exponentially with temperature and ranged from 0.09 +/- 0.04 (10 degrees C) to 0.54 +/- 0.09 nmol O(2) egg(-1) h(-1) (28 degrees C). The mean +/- SD Q(10)-value was 2.51 +/- 0.15. Calculations of energy consumption during embryogenesis ranged from 1.86 to 18.28 mJ depending on temperature and development time. We conclude that the effect of temperature on oxygen consumption rate was far less important than the prolonged development time when calculating the energy consumed during embryogenesis.

[The peculiarities of the radiation mutagenesis of Cyclops kolensis and Cyclops insignis (Crustacea, Copepoda)].

The frequency of the chromosomes aberrations (FA) of Cyclops kolensis before chromatin diminution (CD), which were inducted by gamma-irradiation in 50 times exceeds the FA of Cyclops kolensis after CD, during the CD, reducing the genome of C. kolensis in 15 times. During the embryogenesis the FA of Cyclops insignis doesn't change. The obtained results show us a low level of the spontaneous mutagenesis of C. kolensis and C. insignis embrios. The FA of Cyclops kolensis is correlated with the doses 1, 2, 3, 5 Gy gamma-irradiation. The similarity of the CD mechanism and of the mechanism of the chromosome aberrations is supposed.

DNA-Feulgen cytophotometric determination of genome size for the freshwater-invading copepod Eurytemora affinis.

Variation in nuclear DNA content within some eukaryotic species is well documented, but causes and consequences of such variation remain unclear. Here we report genome size of an estuarine and salt-marsh calanoid copepod, Eurytemora affinis, which has recently invaded inland freshwater habitats independently and repeatedly in North America, Europe, and Asia. Adults and embryos of E. affinis from the St. Lawrence River drainage were examined for somatic cell DNA content and the presence or absence of embryonic chromatin diminution, using Feulgen-DNA cytophotometry to determine a diploid or 2C genome size of 0.6-0.7 pg DNA/cell. The majority of somatic cell nuclei, however, have twice this DNA content (1.3 pg/nucleus) in all of the adults examined and possibly represent a population of cells arrested at the G2 stage of the cell cycle or associated with some degree of endopolyploidy. Both suggestions contradict assumptions that DNA replication does not occur in adult tissues during the determinate growth characteristic of copepods. Absence of germ cell nuclei with markedly elevated DNA values, commonly found for species of cyclopoid copepods that show chromatin diminution, indicates that E. affinis lacks this trait. The small genome size and presumed absence of chromatin diminution increase the potential utility of E. affinis as a model for genomic studies on mechanisms of adaptation during freshwater invasions.

A marine diatom-derived aldehyde induces apoptosis in copepod and sea urchin embryos.

The diatom-derived aldehyde 2-trans-4-trans-decadienal (DD) was tested as an apoptogenic inducer in both copepod and sea urchin embryos, using terminal-deoxynucleotidyl-transferase-mediated dUTP nick-end labelling (TUNEL), DNA fragmentation profiling (laddering) and an assay for caspase-3 activity. DD induced TUNEL positivity and DNA laddering, but not caspase-like activation, in copepod embryos spawned by females fed for 10-15 days the diatom diet Thalassiosira rotula Meunier (in vivo), or when newly spawned eggs were exposed for 1 h to 5 micro g ml(-1) DD (in vitro). To our knowledge, this is the first time that evidence for an apoptotic process in copepods has been obtained by cytochemical (TUNEL) and biochemical (DNA fragmentation) approaches. The absence of caspase-like activity in copepod embryos suggests that caspase-independent programmed cell death occurs in these organisms. In sea urchin embryos, DD induced apoptosis and also activated a caspase-3-like protease. The saturated aldehyde decanal induced apoptosis at higher concentrations and after a longer incubation period than DD, indicating that alpha,beta-unsaturation of the molecule, coupled with the aldehyde group, is responsible for the greater biological activity of DD. Since diatoms are an important food source for marine herbivores such as copepods and sea urchins, these findings may help explain why unsaturated aldehydes often induce reproductive failure, with important ecological consequences at the population level.

Use of the confocal laser scanning microscope in studies on the developmental biology of marine crustaceans.

Confocal Laser Scanning Microscope techniques have been applied to study the developmental biology of marine copepods and decapod larvae. The lipophylic probes DiI and DiOC(6) were used to study both the external and internal morphology of these crustaceans, whereas the same DiOC(6) and the specific nuclear probe Hoechst 33342 were used to study embryonic development of copepods in vivo. To distinguish viable from non-viable copepod embryos, the vital dye dichlorodihydrofluorescein diacetate (H(2)DCFDA) was used. Major advantages and difficulties in the use of these non-invasive techniques in studies of the reproductive biology of marine crustaceans are discussed.