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.

New distinct compartments in the G2 phase of the cell cycle defined by the levels of γH2AX.

Induction of DNA double strand breaks leads to phosphorylation and focus-formation of H2AX. However, foci of phosphorylated H2AX (γH2AX) appear during DNA replication also in the absence of exogenously applied injury. We measured the amount and the number of foci of γH2AX in different phases of the cell cycle by flow cytometry, sorting and microscopy in 4 malignant B-lymphocyte cell lines. There were no detectable γH2AX and no γH2AX-foci in G1 cells in exponentially growing cells and cells treated with PARP inhibitor (PARPi) for 24 h to create damage and reduce DNA repair. The amount of γH2AX increased immediately upon S phase entry, and about 10 and 30 γH2AX foci were found in mid-S phase control and PARPi-treated cells, respectively. The γH2AX-labeled damage caused by DNA replication was not fully repaired before entry into G2. Intriguingly, G2 cells populated a continuous distribution of γH2AX levels, from cells with a high content of γH2AX and the same number of foci as S phase cells (termed "G2H" compartment), to cells that there were almost negative and had about 2 foci (termed "G2L" compartment). EdU-labeling of S phase cells revealed that G2H was directly populated from S phase, while G2L was populated from G2H, but in control cells also directly from S phase. The length of G2H in particular increased after PARPi treatment, compatible with longer DNA-repair times. Our results show that cells repair replication-induced damage in G2H, and enter mitosis after a 2-3 h delay in G2L.

Temperature and developmental responses of body and cell size in Drosophila; effects of polyploidy and genome configuration.

Increased adult body size in Drosophila raised at lower temperatures could be attributed both to an increase in the cell volume and cell number. It is not clear, however, whether increased cell size is related to (or even caused by) increased nuclear volume and genome size (or configuration). Experiments with Drosophila melanogaster stocks (Oregon-R and w1118) raised at 16, 22, 24, and 28°C resulted in larger adult body and wing size with lower temperature, while eye size was less affected. The increase in wing size reflected an increase in cell size in both males and females of both stocks. The nucleus size, genome size, and DNA condensation of adult flies, embryos, and Schneider 2 cells (S2 cells, of larval origin) were estimated by flow cytometry. In both adult flies and S2 cells, both nucleus size and DNA condensation varied with temperature, while DNA content appears to be constant. From 12% to 18% of the somatic cells were tetraploid (4C) and 2-5% were octoploid (8C), and for the Oregon strain we observed an increase in the fraction of polyploid cells with decreasing temperature. The observed increase in body size (and wing size) at low temperatures could partly be linked with the cell size and DNA condensation, while corresponding changes in the haploid genome size were not observed.

Appraising the suitability of succinimidyl and lipophilic fluorescent dyes to track proliferation in non-quiescent cells by dye dilution.

Successful completion of the cell cycle usually results in two identical daughter progeny. This process of generational doubling is termed proliferation and when it occurs in a regulated fashion the benefits range from driving embryonic development to mounting a successful immune response. However when it occurs in a dis-regulated fashion, it is one of the hallmarks of cancer and autoimmunity. These very reasons make proliferation a highly informative parameter in many different biological systems. Conventional flow cytometry (CFC) is a high-throughput, fluorescence-based method for measuring the phenotype and function of cells. The application of CFC to measuring proliferation requires a fluorescent dye able to mark live cells so that when they divide, the daughter progeny receives approximately half the fluorescence of the parent. In measurement space, this translates into peaks of fluorescence decreasing by approximately half, each corresponding to a round of division. It is essential that these peaks can be resolved from one another otherwise it is nearly impossible to obtain accurate quantitative proliferation data. Peak resolution is affected by many things, including instrument performance, the choice of fluorescent dye and the inherent properties of the cells under investigation. There are now many fluorescent dyes available for tracking proliferation by dye dilution differing in their chemistry and spectral properties. Here we provide a method for assessing the performance of various candidate dyes with particular emphasis on situations where the cell type is non-quiescent. We have shown previously that even under optimised instrument and labelling conditions, the heterogeneity of non-quiescent cells makes it impossible to obtain an input width below the threshold for peak resolution without reducing the fluorescence distribution using a cell sorter. Moreover, our method also measures how the dye performs post-labelling in terms of loss/transfer to other cells and how the dye is inherited across the cytokinetic plane. All of these factors will affect peak resolution both in non-quiescent and primary cell types.

Multigenerational genomic responses to dietary phosphorus and temperature in Daphnia.

Temperature and nutrient availability are both hypothesized to affect organisms at the cellular and genomic levels. In this multigenerational study, Daphnia magna (D. magna) and Daphnia pulex (D. pulex) were maintained at high (20 °C) and low (10 °C) temperatures and nourished with phosphorus (P)-sufficient (50 µmol/L) and P-deficient (2 µmol/L) algae for up to 35 generations to assess the multigenerational impacts on genome size and nucleus size. Analysis by flow cytometry revealed significant increases in nucleus size for both species as well as genome size for D. magna in response to a low temperature. The degree of endoreplication, measured as cycle value, was species specific and responded to temperature and dietary composition. Under dietary P deficiency, D. magna, but not D. pulex, showed an apparent reduction in haploid genome size (C-value). These genomic responses are unlikely to reflect differences in nucleotide numbers, but rather structural changes affecting fluorochrome binding. While the ultimate and proximate causes of these responses are unknown, they suggest an intriguing potential for genomic responses that merits further research.

Larger Daphnia at lower temperature: a role for cell size and genome configuration?

Experiments with Daphnia magna and Daphnia pulex raised at 10 and 20 °C yielded larger adult size at the lower temperature. This must reflect increased cell size, increased cell numbers, or a combination of both. As it is difficult to achieve good estimates on cell size in crustaceans, we, therefore, measured nucleus and genome size using flow cytometry at 10 and 20 °C. DNA was stained with propidium iodide, ethidium bromide, and DAPI. Both nucleus and genome size estimates were elevated at 10 °C compared with 20 °C, suggesting that larger body size at low temperature could partly be accredited to an enlarged nucleus and thus cell size. Confocal microscopy observations confirmed the staining properties of fluorochromes. As differences in nucleotide numbers in response of growth temperature within a life span is unlikely, these results seem accredited to changed DNA-fluorochrome binding properties, presumably reflecting increased DNA condensation at low temperature. This implies that genome size comparisons may be impacted by ambient temperature in ectotherms. It also suggests that temperature-induced structural changes in the genome could affect cell size and for some species even body size.