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.     

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.     

The effects of chromatin diminution on the pattern of C-banding in the chromosomes of Acanthocyclops vernalis Fischer (Copepoda: Crustacea)

Chromatin diminution is the loss of selected regions of pre-somatic cell chromosomes during early development, resulting in the removal of a large amount of the genomic DNA from the pre-somatic cells. In copepods, diminution is characterized by the formation of heterochromatically staining regions, or H-segments, which contain the chromatin to be lost. The removal of H-segments during diminution also must represent a major restructuring of the chromosomes which contained them. In order to examine the effects of diminution on the morphology and structure of the chromosomes, the C-banding technique was used. This procedure revealed that most C-bands present in the pre-diminution complement were absent in the post-diminution set. Additionally, in order to explore further the possible composition of the DNA contained in H-segments, a comparison, based on the relationship of C-bands to highly-repetitive DNA in chromosomes, was made between pre-diminution C-bands and H-segments. This comparison showed that not all H-segments are at chromosomal locations which produce a C-band, indicating that H-segments are perhaps not entirely composed of genetically inert DNA, as is currently supposed.     

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.     

A simple and effective method for assessing chromatin diminution values in copepods using qPCR

The value of chromatin diminution (CD) in different species of freshwater cyclopoid copepods can differ significantly. The biological and evolutionary roles of these differences remain unclear. To expand the knowledge on CD distribution and magnitude in this group of copepods, a quick method for its evaluation was required. This study proposes a simple approach for CD assessment in copepods using quantitative realtime PCR (qPCR). The magnitude of changes in the genome size was assessed by comparing fluorescence curves of qPCR fragments of target genes for pre- and post-diminution materials. The method was tested on four cyclopoid copepods species. In Cyclops kolensis, CD was assessed as 95.3 ± 1.2; in Acanthocyclops vernalis it was assessed at 94.6 ± 0.8%; at C. insignis, it was 82.3 ± 5.2%; and for the first time, CD was found in Megacyclops viridis at 91.1 ± 2.6%. The advantages of our approach are its rapidity, simplicity and minimal requirements of materials studied.     

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.     

Climate change and the phytoplankton spring bloom: warming and overwintering zooplankton have similar effects on phytoplankton

Indoor mesocosms were used to study the combined effect of warming and of different densities of overwintering mesozooplankton (mainly copepods) on the spring development of phytoplankton in shallow, coastal waters. Similar to previous studies, warming accelerated the spring phytoplankton peak by ca. 1 day °C^?1 whereas zooplankton did not significantly influence timing. Phytoplankton biomass during the experimental period decreased with warming and with higher densities of overwintering zooplankton. Similarly, average cell size and average effective particle size (here: colony size) decreased both with zooplankton density and warming. A decrease in phytoplankton particle size is generally considered at typical footprint of copepod grazing. We conclude that warming induced changes in the magnitude and structure of the phytoplankton spring bloom cannot be understood without considering grazing by overwintering zooplankton.     

Do phosphorus requirements for RNA limit genome size in crustacean zooplankton?

As for most other organisms, genome size in zooplankton differs widely. This may have a range of consequences for growth rate, development, and life history strategies, yet the causes of this pronounced variability are not settled. Here we propose that small genome size may be an evolutionary consequence of phosphorus (P) allocation from DNA to RNA under P deficiency. To test this hypothesis we have compared the two major groups of zooplankton, copepods and cladocerans, that have overlapping niches and body size. Relative to the cladocerans, copepods have a more complex life history and a lower mass-specific P content, while cladocerans tend to have higher P and RNA contents and higher specific growth rates and frequently experience P-limited growth, likely due to a shortage of P for ribosome synthesis. Cladocerans also generally have smaller genomes than copepods (1C = 0.17-0.63 pg DNA.cell-1 vs. 1C = 0.10-10 pg DNA.cell-1). Furthermore, cladocerans have a higher slope of the relationship of body size with DNA content (1.5 vs. 0.28 in copepods) and present almost 15-fold higher RNA:DNA ratios (24.8 in cladocerans vs. 1.6 in copepods). Hence, small genome size in cladocerans could reflect an evolutionary pressure towards "efficient" genomes to conserve a key element needed to maximize growth rate. We do not claim that this is a universal cause of genome size variability, but propose that streamlining of genomes could be related to P conservation rather than energy conservation. This could be relevant for a range of organisms that may suffer P-limited growth rates.     

Size paradigms in copepod communities: a re-examination

A longstanding view in zooplankton research has been that large copepods are the important members of most communities. It has also been thought that warm water communities contain smaller copepods than temperate waters, with cold polar waters containing large species. We present copepod size spectra from detailed microscopic analysis of tropical and temperate locations, to challenge these paradigms. While the size range of copepods does increase with decreasing temperature and with depth into oceanic waters, the fundamental attributes of the size spectra are similar. Small copepods and early developmental stages dominate all communities.     

Chromatin diminution at the border of the XX and XXI centuries

The size of genomes in eukaryotic organisms is one of the greatest mysteries of biology. As known from the middle of the XX century, the level of organization of a particular organism, does not depend on its genome size, i. e. on DNA amount in the nucleus. We believe that an actual function of non-coding DNA stands behind the phenomenon of chromatin diminution, known already for 100 years. Diminution of chromatin normally takes place in cells involved in body building and never occurs in developmental precursors of germ cells. Apparently, the former are cells, in which non-coding DNA is functionally significant. We cloned a fraction of DNA eliminated during chromatin diminution of Cyclops kolensis (Cyclopoida, Crustascea) and sequenced 90 clones totally making 32 kb. Taken together, the provided evidence has demonstrated a high organization ordering of DNA sequences restricted to the germ line. Chromatin diminution never takes place in human cells and in cells of the majority of animals. These cells may isolate non-coding DNA in other ways, making it unreactable for most enzymes and thus functionally cut off. Thus, a certain part of genome with a particular size and structure may serve for genetic isolation of species as shellfish or junk DNA are vital components rather than pieces of garbage.     

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.     

Gigantism, temperature and metabolic rate in terrestrial poikilotherms

The mechanisms dictating upper limits to animal body size are not well understood. We have analysed body length data for the largest representatives of 24 taxa of terrestrial poikilotherms from tropical, temperate and polar environments. We find that poikilothermic giants on land become two-three times shorter per each 10 degrees of decrease in ambient temperature. We quantify that this diminution of maximum body size accurately compensates the drop of metabolic rate dictated by lower temperature. This supports the idea that the upper limit to body size within each taxon can be set by a temperature-independent critical minimum value of mass-specific metabolic rate, a fall below which is not compatible with successful biological performance.     

Body size and sexual size dimorphism in calanoid copepods

Patterns of sexual size dimorphism and body size in calanoid copepods are examined. We hypothesize that favorable conditions for development will result in large body size and high sexual size dimorphism among populations of a given species and that differences in this allometric relationship among species is governed by the male's role in insemination. We confirm that there is a greater advantage to large female size, normally the larger sex, when compared to males, hence leading to selection for developmental patterns favoring high size dimorphism. Individuals from populations of four centropagid copepod species were measured; other sizes were obtained from published sources. In the four species we examined, the relationships between prosome length and both clutch size and the ability to produce multiple clutches with one insemination were determined. Results show a trend toward hyperallometry in all centropagid species examined: sexual size dimorphism increases with increasing size. Large females produce larger clutches and more additional clutches on one insemination. That hyperallometry is not observed in diaptomid copepods may result from the greater role the male plays in reproduction. Males are needed for each clutch produced, hence the selective pressure to be larger is greater than that in the centropagidae.     

Diet, prey selection, and growth of larval and juvenile burbot Lota lota (L.)

The diet, prey selection, and growth of larval and juvenile Oneida Lake burbot, Lota lota (L.), was investigated from the time of first hatch in mid-April until their disappearance from the pelagic zone at the beginning of June. Mean length increased exponentially from 3–20 mm on 18 April to 15–00 mm on 29 May. The number and size of prey in burbot stomachs, and the prey size most selected by burbot increased as they grew. Prey width, not prey length, limited the size of prey ingested. The smallest burbot ate and selected highly for the largest prey they could ingest, but older burbot did not. As burbot grew, prey selection was highest first for the rotifer Asplanchna , followed by copepod nauplii, then cyclopoid copepods. Daphnia and calanoid copepods and cyclopoid copepods were selected to similar degrees by 29 May. For a given size, however, cyciopoid copepods were selected more strongly than any other prey type from 18 to 29 May. There were no significant differences in prey selection among offshore, nearshore, and onshore habitats, nor among depths in the offshore habitat.     

Factors influencing the degree of sexual size dimorphism within and among calanoid copepod species

Populations of Diaptomus leptopus (Copepoda: Calanoida) and other calanoid copepods exhibit varying degrees of sexual size dimorphism. We examined whether intraspecific or interspecific variation in dimorphism could be explained by allometry, and we examined the relationship between adult size attained and development rate to determine any relationship between the two. We compared the degree of sexual size dimorphism in D. leptopus and in other calanoid copepods inhabiting temporary and permanent habitats. Allometry did not explain variation in sexual size dimorphism within or among populations or among species. Permanence of habitat affected the degree of dimorphism: dimorphism was greater within and among species inhabiting temporary environments. Non-significant differences in development rate were found among populations and significant differences were found between sexes of D. leptopus when reared under identical laboratory conditions: males developed more rapidly than females but there was no general relationship between development rate and adult size. Potential adaptive hypotheses to explain the differences between populations inhabiting temporary and permanent habitats 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.     

Chromatin diminution in Ascaris suum: nucleotide sequence of the eliminated satellite DNA.

We have determined the prototype sequence of the DNA which is eliminated in the course of chromatin diminution in Ascaris suum. This DNA which is virtually absent from somatic cells but retained in the germ line consists predominantly of highly repetitive sequences which are variants of an AT rich 123 base pair repeat unit. Both major and minor variants have been sequenced. The overall structure of this germ line limited DNA corresponds to the segmental organization characteristic of satellite DNAs. Possible correlations between the mechanism of chromatin diminution and some properties of the satellite sequence are discussed.     

Inter‐ and intraspecific variation in body‐ and genome size in calanoid copepods from temperate and arctic waters

The tendency of ectotherms to get larger in the cold (Bergmann clines) has potentially great implications for individual performance and food web dynamics. The mechanistic drivers of this trend are not well understood, however. One fundamental question is to which extent variation in body size is attributed to variation in cell size, which again is related to genome size. In this study, we analyzed body and genome size in four species of marine calanoid copepods, Calanus finmarchicus, C. glacialis, C. hyperboreus and Paraeuchaeta norvegica, with populations from both south Norwegian fjords and the High Arctic. The Calanus species showed typical interspecific Bergmann clines, and we assessed whether they also displayed similar intraspecific variations—and if correlation between genome size and body size differed between species. There were considerable inter‐ as well as intraspecific variations in body size and genome size, with the northernmost populations having the largest values of both variables within each species. Positive intraspecific relationships suggest a functional link between body and genome size, although its adaptiveness has not been settled. Impact of additional drivers like phylogeny or specific adaptations, however, was suggested by striking divergences in body size – genome size ratios among species. Thus, C. glacialis and C. hyperboreus, had fairly similar genome size despite very different body size, while P. norvegica, of similar body size as C. hyperboreus, had the largest genome sizes ever recorded from copepods. The inter‐ and intraspecific latitudinal body size clines suggest that climate change may have major impact on body size composition of keystone species in marine planktonic food webs.     

Ocean carbon sequestration: Particle fragmentation by copepods as a significant unrecognised factor?

Ocean biology helps regulate global climate by fixing atmospheric CO₂ and exporting it to deep waters as sinking detrital particles. New observations demonstrate that particle fragmentation is the principal factor controlling the depth to which these particles penetrate the ocean's interior, and hence how long the constituent carbon is sequestered from the atmosphere. The underlying cause is, however, poorly understood. We speculate that small, particle-associated copepods, which intercept and inadvertently break up sinking particles as they search for attached protistan prey, are the principle agents of fragmentation in the ocean. We explore this idea using a new marine ecosystem model. Results indicate that explicitly representing particle fragmentation by copepods in biogeochemical models offers a step change in our ability to understand the future evolution of biologically-mediated ocean carbon storage. Our findings highlight the need for improved understanding of the distribution, abundance, ecology and physiology of particle-associated copepods.     

Temperature-mediated changes in zooplankton body size: large scale temporal and spatial analysis

Climate warming has been linked with changes in the spatiotemporal distribution of species and the body size structure of ecological communities. Body size is a master trait underlying a host of physiological, ecological and evolutionary processes. However, the relative importance of environmental drivers and life history strategies on community body size structure across large spatial and temporal scales is poorly understood. We used detailed data of 83 copepod species, monitored over a 57-year period across the North Atlantic, to test how sea surface temperature, thermal and day length seasonality relate to observed latitudinal-size clines of the zooplankton community. The genus Calanus includes dominant taxa in the North Atlantic that overwinter at ocean depth. Thus we compared the copepod community size structure with and without Calanus species, to partition the influence of this life history strategy. The mean community body size of copepods was positively associated with latitude and negatively associated with temperature, suggesting that these communities follow Bergmann's rule. Including Calanus species strengthens these relationships due to their larger than average body sizes and high seasonal abundances, indicating that the latitudinal-size cline may be adaptive. We suggest that seasonal food availability prevents high abundance of smaller-sized copepods at higher latitudes, and that active vertical migration of dominant pelagic species can increase their survival rate over the resource-poor seasons. These findings improve our understanding of the impacts that climate warming has on ecological communities, with potential consequences for trophic interactions and biogeochemical processes that are well known to be size dependent.