Observing copepods through a genomic lens

Background: Copepods outnumber every other multicellular animal group. They are critical components of the world's freshwater and marine ecosystems, sensitive indicators of local and global climate change, key ecosystem service providers, parasites and predators of economically important aquatic animals and potential vectors of waterborne disease. Copepods sustain the world fisheries that nourish and support human populations. Although genomic tools have transformed many areas of biological and biomedical research, their power to elucidate aspects of the biology, behavior and ecology of copepods has only recently begun to be exploited. Discussion: The extraordinary biological and ecological diversity of the subclass Copepoda provides both unique advantages for addressing key problems in aquatic systems and formidable challenges for developing a focused genomics strategy. This article provides an overview of genomic studies of copepods and discusses strategies for using genomics tools to address key questions at levels extending from individuals to ecosystems. Genomics can, for instance, help to decipher patterns of genome evolution such as those that occur during transitions from free living to symbiotic and parasitic lifestyles and can assist in the identification of genetic mechanisms and accompanying physiological changes associated with adaptation to new or physiologically challenging environments. The adaptive significance of the diversity in genome size and unique mechanisms of genome reorganization during development could similarly be explored. Genome-wide and EST studies of parasitic copepods of salmon and large EST studies of selected free-living copepods have demonstrated the potential utility of modern genomics approaches for the study of copepods and have generated resources such as EST libraries, shotgun genome sequences, BAC libraries, genome maps and inbred lines that will be invaluable in assisting further efforts to provide genomics tools for copepods. Summary: Genomics research on copepods is needed to extend our exploration and characterization of their fundamental biological traits, so that we can better understand how copepods function and interact in diverse environments. Availability of large scale genomics resources will also open doors to a wide range of systems biology type studies that view the organism as the fundamental system in which to address key questions in ecology and evolution

HERITABLE LIFE HISTORY VARIATION IN WIDELY SEPARATED POPULATIONS OF MESOCYCLOPS EDAX (CRUSTACEA: COPEPODA)

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GENETIC DIFFERENTIATION OF LIFE HISTORY TRAITS IN POPULATIONS OF MESOCYCLOPS EDAX (CRUSTACEA: COPEPODA)

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Genome Sizes of Cyclopoid Copepods (Crustacea): Evidence of Evolutionary Constraint

Genome sizes for 36 species of cyclopoid copepods were determined by DNA-Feulgen cytophotometry of nuclei from adults collected from diverse habitats and locales in North America, South America, Europe, and Asia. Genome sizes are small, show a 20-fold range (C = 0.10-2.02 pg DNA), and vary in a discontinuous fashion. The genomes of cyclopoid copepods are remarkably small and constant within each species, unlike the large and variable genomes of marine calanoid species. These differences may reflect the evolutionary antiquity of marine copepods in relation to marine, brackish, and freshwater copepods, as well as differences in mechanisms used to modulate genome size. The small genome sizes of contemporary cyclopoids provide substantive evidence of evolutionary constraint, possibly favouring small genomes, rapid replication rates and accelerated development as adaptive strategies for survival in often fragmented, stressful, and changing habitats

Changes in Nuclear Morphology Associated With Elevated DNA Levels During Gametogenesis in Cyclopoid Copepods With Chromatin Diminution

Most species of freshwater cyclopoid copepods follow a conventional course of DNA replication during gametogenesis, but certain species regularly undergo chromatin diminution during early embryogenesis, a process that is accompanied by the exclusion of large amounts of heterochromatic DNA from progenitor somatic cells and selective retention of this DNA by primordial germ cells after their segregation from the soma. We have used scanning microdensitometry and image analysis cytometry of individual Feulgen-stained nuclei to determine the DNA levels of individual somatic cell nuclei, oocytes, spermatocytes, and sperm for seven species, including Acanthocyclops brevispinosus, Acanthocyclops vernalis, Ectocyclops phaleratus, Eucyclops agilis, Eucyclops ensifer, Macrocyclops albidus, and Thermocyclops decipiens. The oocyte nuclei of these species have twice the DNA content of their diploid somatic cell nuclei. In specimens of Cyclops strenuus, Mesocyclops edax, Mesocyclops longisetus, Mesocyclops longisetus curvatus, and Metacyclops mendocinus, marked increases in DNA levels were noted in both female and male germ cells before meiosis. The appearance of enlarged nuclei with densely stained chromocenters is a distinguishing feature of oocytes and spermatocytes of cyclopoid species that exhibit excessive accumulations of DNA during gametogenesis and subsequently undergo chromatin diminution. The net increase in DNA content of the prediminution nuclei is 6-10 times the DNA level of their somatic cell nuclei and is largely attributable to increases in the amount of DNA associated with their heterochromatic chromocenters. The identification of a morphologically distinctive type of germ cell and its dramatic accumulation of large amounts of DNA before meiosis are discussed in terms of the selective elimination of heterochromatin during early cleavage stages in these cyclopoid species

Endopolyploidy in Cyclopoid Copepods

Somatic tissues in mature copepods are determinate in growth, not undergoing mitosis; cell number remains constant throughout adult life. Here we report evidence of polyploidization for eight species of cyclopoid copepods. Using static Feulgen-DNA cytophotometry, we measured individual somatic nuclei in populations of Eucyclops ensifer from Brazil and of Eucyclops agilis from Ohio, U.S.A. Small but potentiality significant percentages of the adult somatic cells in these species, as well as in Bryocyclops caroli, Halicyclops tagea, Macrocyclops albidus, Mesocyclops edax, Mesocyclops thermocyclopoides, and Thermocyclops decipiens, contained at least twice (4C) the amount of DNA found in their diploid (2C) cells. These species have 2C DNA values that are representative of the range of genome sizes in cyclopoid copepods which, as a group, have much smaller genomes than calanoid copepods. Polyploidy may be a previously unrecognized mechanism whereby copepods alter the DNA content or nucleotype during specific stages in development. DNA reduplication preceding a cycle of endomitosis may result in a doubling of the functional genome, thus providing additional template for mRNA transcription related to specific functions

Gonomery and Chromatin Diminution in Mesocyclops Longisetus (Copepoda)

The segregation of progenitor somatic cells from those of the primordial germ cells during early cleavage divisions occurs in certain copepods exhibiting the phenomenon of chromatin diminution during early embryogenesis. These species provide an interesting alternative to the usual reproductive strategy of other species of freshwater cyclopoid copepods. Levels of DNA for the soma and germ cells of Mesocyclops longisetus have been determined for individual nuclei by using Feulgen-DNA cytophotometry to monitor changes of DNA amounts during gametogenesis and early cleavage stages of embryogenesis. Germ cell nuclei of both female and male adults contain marked elevations of DNA, far in excess of expected 4C DNA level for their replication prior to meiosis. The elevated amounts of DNA in these germ cells are equivalent to the elevated DNA content found during the gonomeric divisions observed in embryos. Following the gonomeric divisions there is roughly a 40% loss of germ cell heterochromatin during the chromatin diminution stages of embryogenesis. The role of this excised DNA remains unclear

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

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

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