Chromatin diminution in the copepod Mesocyclops edax: diminution of tandemly repeated DNA families from somatic cells.

Chromatin diminution, i.e., the loss of selected chromosomal regions during the differentiation of early embryonic cells into somatic cells, has been described in taxa as varied as ciliates, copepods, insects, nematodes, and hagfish. The nature of the eliminated DNA has been extensively studied in ciliate, nematode, and hagfish species. However, the small size of copepods, which makes it difficult to obtain enough DNA from early embryonic cells for cloning and sequencing, has limited such studies. Here, to identify the sequences eliminated from the somatic cells of a copepod species that undergoes chromatin diminution, we randomly amplified DNA fragments from germ line and somatic line cells of Mesocyclops edax, a freshwater cyclopoid copepod. Of 47 randomly amplified germ line clones, 45 (96%) contained short, tandemly repeated sequences composed of either 2 bp CA-repeats, 8 bp CAAATAGA-repeats, or 9 bp CAAATTAAA-repeats. In contrast, of 83 randomly amplified somatic line clones, only 47 (57%) contained such short, tandemly repeated sequences. As previously observed in some nematode species, our results therefore show that there is partial elimination of chromosomal regions containing (CAAATAGA and CAAATTAAA) repeated sequences during the chromatin diminution observed in the somatic cells of M. edax. We speculate that chromatin diminution might have evolved repeatedly by recruitment of RNAi-related mechanisms to eliminate nonfunctional tandemly repeated DNA sequences from the somatic genome of some species.     

Changes in nuclear morphology associated with elevated DNA levels during gametogenesis in cyclopoid copepods with chromatin diminution

Abstract. 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.     

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.     

The relationship between genome size, development rate, and body size in copepods

Freshwater cyclopoid copepods exhibit at least a fivefold range in somatic genome size and a mechanism, chromatin diminution, which could account for much of this interspecific variation. These attributes suggest that copepods are well suited to studies of genome size evolution. We tested the nucleotypic hypothesis of genome size evolution, which poses that variation in genome size is adaptive due to the bulk effects of both coding and noncoding DNA on cell size and division rates, and their correlates. We found a significant inverse correlation between genome size and developmental (growth) rate in five freshwater cyclopoid species at three temperatures. That is, species with smaller genomes developed faster. Species with smaller genomes had significantly smaller bodies at 22 °C, but not at cooler and warmer temperatures. Species with smaller genomes developed faster at all three temperatures, but had smaller bodies only at 22 °C. We propose a model of life history evolution that adds genome size and cell cycle dynamics to the suite of characters on which selection may act to mold life histories and to influence the distribution of traits among different habitats.     

Unusually high numbers of ribosomal RNA genes in copepods (Arthropoda: Crustacea) and their relationship to genome size.

We report on copy numbers of 18S ribosomal RNA genes in three species of copepods (Crustacea: Copepoda), two of which possess an unusual arrangement in which 5S genes are included within the 18S-5.8S-28S repeat unit. Slot blots of genomic and standard DNA were hybridized with an 18S rRNA gene probe constructed from one of the marine species and hybridization was quantified using chemiluminescence. Diploid 18S rRNA gene copy numbers are estimated as ca. 15 300 and 33 500 in the marine species Calanus finmarchicus (13.0 pg DNA in 2C adult nuclei) and C. glacialis (24.2 pg DNA), respectively, and ca. 840 and 730 in two freshwater populations of Mesocyclops edax (both ca. 3 pg DNA) from Virginia and Nova Scotia, respectively. The roughly proportional relationship between 2C somatic nuclear DNA contents and rRNA gene copy number in the sibling species C. finmarchicus and C. glacialis may reflect polytenic replication of entire genomes during abrupt speciation events. Copy numbers may also reflect differential losses during embryonic chromatin diminution.     

Patterns of genome size in the copepoda

Adult somatic nuclear DNA contents are reported for eleven cyclopoid species (Megacyclops latipes, Mesocyclops edax, M. longisetus, M. ruttneri, M. leuckarti, M. woutersi, Macrocyclops albidus, Cyclops strenuus, Acanthocyclops robustus, Diothona oculata, Thermocyclops crassus) and for the harpacticoid Tigriopus californicus and range from 0.50 to 4.1 pg DNA per nucleus. These diploid genome sizes are consistent with previously published values for four Cyclops species (0.28–1.8 pg DNA per nucleus), but are strikingly smaller than those reported for marine calanoids (4.32–24.92 pg DNA per nucleus). We discuss three explanations, none of them exclusive of another, to account for the smaller size and range of cyclopoid genome sizes relative to calanoid genome sizes: (1) higher prevalence of chromatin diminution in the Cyclopoida, (2) phylogenetic structure or older age of the Calanoida relative to Cyclopoida and (3) nucleotypic selection that may influence life history variation and fitness. Measurements of genome size were made on Feulgen stained, somatic cell nuclei, using scanning microdensitometry which is well suited to the sparse and heterogeneous populations of copepod nuclei. The importance of measuring large numbers of nuclei per specimen, possible sources of variation associated with cytophotometric measurements, and appropriate use of internal reference standards and stoichiometry of the Feulgen stained nuclei are discussed.     

Some conclusions on the role of redundant DNA and the mechanisms of eukaryotic genome evolution inferred from studies of chromatin diminution in Cyclopoida

The absence of progress in understanding the problem of redundant eukaryotic DNA is stated. This is caused primarily by the attempts to solve this problem either in terms of the traditional approaches (the general phenotypic parameters such as developmental rate, body size, etc. depend on the genome size) or by introducing such vague terms as egoistic, parasitic, or junk DNA. Studying chromatin diminution (CD) in copepods yielded two important conclusions. First, part of the genome of a certain size (94% in Cyclops kolensis first described by the authors) is not needed for somatic functions as it is eliminated during the early (third to seventh) cleavage divisions from the presumptive somatic cells. Second, this DNA is not redundant, let alone selfish or junk, relative to the germline cells. In this sense, it can be regarded as invariant (monomorphic) trait that characterizes the species. Analysis of cloned and sequenced DNA regions eliminated from the somatic cell genome by CD (i.e., confined to the germline), which was first carried out for C. kolensis, showed that the molecular structure of this DNA has at least two features of regular organization: a mosaic structure of repetitive sequences and high (sometimes up to 100%) homology between different repeats and subrepeats. We have suggested that the germline-restricted DNA forms a unique molecular portrait of the species genome, thus acting as a significant factor of genetic isolation. Yet, the phenomenon of CD proper as it occurs in Cyclopoida without disintegration of the chromosome structure) may be regarded as a model of reductional genome evolution, which has repeatedly occurred in the history of eukaryotes.     

Number and DNA content of nuclei in the free-living nematode <Emphasis Type="Italic">Panagrellus silusiae</Emphasis> at each stage during postembryonic development

Nuclei from the four major tissues of the nematode Panagrellus silusiae were enumerated and examined using Feulgen microspectrophotometry at each stage during postembryonic development. The number of nuclei in the hypodermis, nerve, and intestine remains fairly constant during maturation, but there is a slight increase (～57%) in the number of muscle nuclei. Thus, this organism is not stringently eutelic. The total number of somatic nuclei is about 600. DNA values of hypodermis and nerve nuclei were unimodal and adult nuclei had 2C amounts of DNA. The DNA distribution of muscle nuclei reflects the pattern expected for a tissue in which a portion of the nuclei are undergoing DNA synthesis. Intestinal nuclei accumulated DNA in the absence of nuclear division and in the adult the nuclei fall into discrete DNA classes which correspond to a geometric series of the 2C value. It is concluded that chromatin diminution does not occur in this species. In addition, the relationship in the different tissues of nuclear DNA content to nuclear volume and cell size is discussed.     

Number and DNA content of nuclei in the free-living nematode Panagrellus silusiae at each stage during postembryonic development

Nuclei from the four major tissues of the nematode Panagrellus silusiae were enumerated and examined using Feulgen microspectrophotometry at each stage during postembryonic development. The number of nuclei in the hypodermis, nerve, and intestine remains fairly constant during maturation, but there is a slight increase (57%) in the number of muscle nuclei. Thus, this organism is not stringently eutelic. The total number of somatic nuclei is about 600. DNA values of hypodermis and nerve nuclei were unimodal and adult nuclei had 2C amounts of DNA. The DNA distribution of muscle nuclei reflects the pattern expected for a tissue in which a portion of the nuclei are undergoing DNA synthesis. Intestinal nuclei accumulated DNA in the absence of nuclear division and in the adult the nuclei fall into discrete DNA classes which correspond to a geometric series of the 2C value. It is concluded that chromatin diminution does not occur in this species. In addition, the relationship in the different tissues of nuclear DNA content to nuclear volume and cell size is discussed.The study was supported by the National Research Council of Canada (Grant A-3491).     

Some conclusions on the role of redundant DNA and the mechanisms of eukaryotic genome evolution inferred from studies of chromatin diminution in Cyclopoida

The absence of progress in understanding the problem of redundant eukaryotic DNA is stated. This is caused primarily by the attempts to solve this problem either in terms of the traditional approaches (the general phenotypic parameters such as developmental rate, body size, etc. depend on the genome size) or by introducing such vague terms as egoistic, parasitic, or junk DNA. Studying chromatin diminution (CD) in copepods yielded two important conclusions. First, part of the genome of a certain size (94% in Cyclops kolensis first described by the authors) is not needed for somatic functions as it is eliminated during the early (fourth to seventh) cleavage divisions from the presumptive somatic cells. Second, this DNA is not redundant, let alone selfish or junk, relative to the germline cells. In this sense, it can be regarded as invariant (monomorphic) trait that characterizes the species. Analysis of cloned and sequenced DNA regions eliminated from the somatic cell genome by CD (i.e., confined to the germline), which was first carried out for C. kolensis, showed that the molecular structure of this DNA has at least two features of regular organization: a mosaic structure of repetitive sequences and high (sometimes up to 100%) homology between different repeats and subrepeats. We have suggested that the germline-restricted DNA forms a unique molecular portrait of the species genome, thus acting as a significant factor of genetic isolation. Yet, the phenomenon of CD proper as it occurs in Cyclopoida (without disintegration of the chromosome structure) may be regarded as a model of reductional genome evolution, which has repeatedly occurred in the history of eukaryotes.__________Translated from Genetika, Vol. 41, No. 4, 2005, pp. 466–479.Original Russian Text Copyright © 2005 by Akifyev, Grishanin.     

Evidence for endoreduplication: germ cell DNA levels prior to chromatin diminution in Mesocyclops edax.

We studied the functional significance of marked differences in the DNA content of somatic cells and germ line nuclei by static Feulgen-DNA cytophotometry for several species of microcrustaceans that exhibit chromatin diminution during very early stages of embryogenesis. Mature females and males showed many gonadal nuclei with elevated amounts of DNA that persist until dispersal of this "extra" DNA throughout the cytoplasm as fragments and coalescing droplets of chromatin during anaphase of the diminution division.     

Cytophotometric determination of genome size in two species of Cyclops of Lake Baikal (Crustacea: Copepoda, Cyclopoida) in ontogenetic development

The genome size of Cyclops in cells at early stages of cleavage (up to the fifth division) and in somatic cells was estimated by static digital Feulgen cytophotometry in order to study quantitative changes in DNA content during chromatin diminution. Described here cytophotometric method was approbated on five different digital-imaging systems in blood cells of four vertebrate species. In all cases, we observed a direct correlation between the data obtained with known from the literature on genome size and high reproducibility, which will allow these systems to be used in future work. We also optimized the conditions for DNA hydrolysis of both blood smears and for two species of Cyclops from the Moscow population as 30 min in 5 N HCl at 24°C. Here, we first revealed chromatin diminution in two endemic Baikal species of Cyclopoida: Acanthocyclops incolotaenia and Diacyclops galbinus. We estimated the extent of chromatin diminution in Diacyclops galbinus as 95.5–96.2%. Cytometric analysis of the third species, Mesocyclops leuckarti, did not reveal obvious chromatin diminution.     

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. © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 87 , 625–635.     

Temporal control of DNA replication and the adaptive value of chromatin diminution in copepods.

Chromatin diminution is a precisely controlled, highly repeatable, genome-wide deletion of noncoding heterochromatic segments from the presomatic line. The somatic line is reduced in size and reorganized; the germ line remains unaltered. Little is understood about its mechanistic underpinnings and adaptive significance in the nematodes, copepods, and hagfish in which it occurs. Here, we propose that microcrustacean copepods, whose cytology, development, and evolutionary ecology are well understood from an adaptationist point of view, provide the vehicle to test how chromatin diminution might orchestrate certain cell cycle dynamics, with the consequence of influencing the evolution of nuclear DNA contents, organismal development rates, and body size.     

Comparison of nuclear DNA content of rodlet cells and erythrocytes in some freshwater teleosts

DNA of rodlet cells and erythrocytes from three species of freshwater teleosts, Semotilus atromaculatus atromaculatus, Catostomus commersoni and Cyprinus carpio , was stained with the Feulgen reaction and examined by microdensitometry. Rodlet cells showed nuclear DNA content significantly different from erythrocytes of the same species, but the difference was less than a factor of C, assuming that erythrocytes reflect the normal 2C genome of somatic cells. In two species, S. atromaculatus and C. carpio , the rodlet cell nuclei contained less DNA than the erythrocytes; in C. commersoni they contained more. The identity of the rodlet cell is unknown; the results of these experiments lead to the rejection of the hypothesis that rodlet cells and erythrocytes of a species have the same DNA content, i.e. that the rodlet cell is a normal somatic component of fish tissue.     

The occurrence, role and evolution of chromatin diminution in nematodes

Chromatin diminution takes place in presomatic cells of some parasitic nematodes during early development. This phenomenon may play an important role in somatic cell differentiation, since the somatic cells of these species undergo an extensive genome reorganization during development via chromatin diminution and polyploidization, as explained here by Clara Goday and Sergio Pimpinelli.     

Chromosome diminution in Ascaris suum. Two-fold increase of nucleosomal histone to DNA ratios during development

The swine intestinal nematode, Ascaris suum, eliminates chromatin material from its primordial somatic cells during early embryogenesis. A technique for isolation of nuclei from pre- and post-diminution stage embryos has been developed and these isolated nuclei were used in investigations of nuclear events during diminution. The amount of DNA per nucleus determined by diphenylamine assays and isotope dilutions was 0.66 pg and 0.29 pg in pre- and post-diminution nuclei, respectively. Thus, A. suum loses 56% of its nuclear DNA during diminution. The loss of nuclear DNA enabled in vivo examination of histone to DNA ratios as a function of changes in DNA quantities. Ascaris histones were identified by acid extractability and tryptic fingerprint comparison with rat liver histones. Measurement of histone quantities was accomplished using linearity of Coomassie blue binding to histones separated in dodecyl sulfate gels. Ascaris nucleosomal histones levels were relatively constant in pre- and post-diminution nuclei. However, nucleosomal histone to DNA ratios approximately doubled during diminution.     

Chromatin diminution in nematodes

The process of chromatin diminution in Parascaris and Ascaris is a developmentally controlled genome rearrangement, which results in quantitative and qualitative differences in DNA content between germ line and somatic cells. Chromatin diminution involves chromosomal breakage, new telomere formation and DNA degradation. The programmed elimination of chromatin in presomatic cells might serve as an alternative way of gene regulation. We put forward a new hypothesis of how an ancient partial genome duplication and chromatin diminution may have served to maintain the genetic balance in somatic cells and simultaneously endowed the germ line cells with a selective advantage.     

Estimation of nuclear DNA content of various bamboo and rattan species

We determined the nuclear DNA content (genome size) of over 35 accessions each of bamboo and rattan species from Southeast Asia. The 2C DNA per nucleus was quantified by flow cytometry. The fluorescence of nuclei isolated from the leaves and stained with propidium iodide was measured. The genome size of the bamboo species examined was between 2.5 and 5.9 pg DNA per 2C nucleus. The genome size of the rattan species examined ranged from 1.8 to 10.5 pg DNA per 2C nucleus. This information will be useful for scientists working in diverse areas of plant biology such as biotechnology, biodiversity, genome analysis, plant breeding, physiology and molecular biology. Such data may be utilized to attempt to correlate the genome size with the ploidy status of bamboo species in cases where ploidy status has been reported.