Culture conditions
We cultured a population of different D. magna clones (D. magna clone Elias from Mount Sinai, Egypt, D. magna clone L7 form Lake Ring, Denmark kindly provided by L. Orsini, and D. magna clone FT442 from Finland and kindly provided by Dieter Ebert) to study and compare the cellular changes during development in sexually and asexually produced embryos preventing clonal specificities and ambiguities from inbreeding effects [29].
All animals of the culture and the experiments were raised in 1 L glass jars (WECK®, Germany) filled with a modified version of ADaM medium (2.3 mg CaCl2 × 2 H20; 2.2 mg NaHCO3; 0.1 mg SeO2; 12.5 mg sea salt, filled up to 1 L with ultrapure water, refer to Klüttgen, et al. [49]) in temperature controlled incubators at 20 °C ± 0.1 °C and under respective light conditions (for asexually produced embryos: 16:8 day:night; for sexually produced embryos: 8:16 day:night). Animals were fed the algae Acutodesmus obliquus ad libitum > 1.5 g C/L. Animal remnants and exuviae were removed every other day and medium was exchanged every week. Unless otherwise stated, all animals were bred in a clone specific manner.
All clonal females were kept in low densities (about 30 adult females per 800 mL of ADaM in a 1 L jar, Weck®; Germany) producing asexual embryos via parthenogenesis. Sexual reproduction in the respective clones was initiated under standardized conditions (20 °C ± 0.1 °C, with a shortened photoperiod 8 h: 16 h light: dark cycle) and via crowding. At densities of more than 50 adult animals and under limited food conditions < 1 g C/L) in 800 mL ADaM this initiated sexual reproduction.
Ovulation monitoring in sexually and asexually reproducing Daphnia
To determine how development differs for embryos that are destined to diapause versus those that are not, we monitored the time point of ovulation in sexually and asexually reproducing females. These embryos are distinguishable within the ovary of adult females (Fig. 5), which were individually transferred into 50 mL snap cap vials filled with 40 mL ADaM and algal fed Acutodesmus obliquus ad libitum. To ensure fertilisation, we co-cultured one male of a different clone together with one sexually reproducing female. At 15 min intervals, we checked and documented the time point of ovulation. When the animals reached the respective stage for our microscopic observations, they were fixed in 4% PFA-TX (formaldehyde 37%; Merck, Germany; diluted in phosphate buffered saline 0.1 M, pH 7.4; with 0.05% Triton X; Serva, Germany) and stored at 4 °C until further processing. Sexually produced embryos that entered diapause were encapsulated in the maternal ephippia and were cast off during the next molting cycle, at 74 h post ovulation. These ephippia were collected and transferred into dry, cold (4 °C) and fully dark conditions in a refrigerator until processing.
Cell number changes during developmental progression
In asexually produced embryos, we counted the number of cells hourly from 4 h until 25 h post ovulation. In sexually produced embryos, we counted the number of cells every two hours from 12 h until 40 h post ovulation. We also collected embryos at 44 h, 46 h, 48 h and 56 h after ovulation, before the ephippia were shed. Once the ephippia were cast off, they were collected within 24 h and transferred into dark and cold conditions as described above and collected to be fixed at 408 h post ovulation and 27 months post ovulation.
Fixed embryos were squashed, mounted in Vectashield+DAPI and coverslipped (H-120, Vectalaboratories, Burlington USA) in one single step to ensure that no cells were lost during the preparation procedure. DAPI (4′,6′-diamidino-2-phenylindole) fluorescence staining of the nucleus, by strongly adhering to adenine-thymine rich regions of the nuclear DNA, was documented using a Zeiss Axiophot fluorescent microscope equipped with an Olympus XC10 monochrome digital camera together with the imaging software CellSense (Olympus, Germany).
A single composite image showing all squashed cells of a single embryo was acquired from individual images using the stitching function and cell numbers were determined using the counting function in CellSense (Olympus, Germany). Every time point was collected at least three times.
Cell division curve modeling
Logistic curves of cell number over time were fitted to the original experimental data with R Studio according to the Eq. (1):
$$ y=\frac{a_1}{1+{e}^{-\left({a}_2+{a}_3\times t\right)}} $$
(1)
Initial starting parameters were manually set based on the approximated curves.
-
y=cell number in one embryo
-
a1=the first parameter, first a rough approximation of the maximal cell number (e.g. 4000 cells in sexually produced embryos and 8000 cells in asexually produced embryos). Later this value was replaced by the modeled parameter.
-
a2=the second parameter of the logistic function
-
a3=the third parameter of the logistic function
-
t=hours post ovulation
In detail, a linear model was fitted to the data, using the logit-function to linearize the sigmoid growth curves in Eq. (2).
$$ \mathrm{lm}\left(\mathrm{logit}\left(\frac{y}{a_1}\right)\sim t\right) $$
(2)
The parameters intercept and slope were extracted from the linear model using the coef()-function and used as approximated values for the parameters a2 and a3.
The final logistic model was established by inserting the calculated rough parameters in the formula and fitting it to the data, using the nls()-function in R, giving the final coefficients for the calculation shown in Eq. (3).
$$ {a}_{1/2/3}=\mathrm{coef}\left(\mathrm{lm}\left(\mathrm{logit}\left(\frac{y}{a_1}\right)\sim t\right)\right)\left[1/2/3\right] $$
(3)
The logistic model was created by inserting the calculated parameters in the formula. Original data points and the modeled growth curves were plotted in a single plot.
The cell division rate was calculated as the derivative with respect to time to determine the time point of highest cell division rate in Eq. (4).
$$ {y}^{\prime }=\frac{\partial y}{\partial t} $$
(4)
The second derivative with respect to time was calculated to estimate the change of cell division rate, which is the ‘acceleration’ in cell division using Eq. (5).
$$ {y}^{\prime \prime }=\frac{\partial {y}^{\prime }}{\partial t}=\frac{\left(\frac{\partial y}{\partial t}\right)}{\partial t}=\frac{\partial y}{{\partial t}^2} $$
(5)
The maxima and minima of this function depicts the transition from the latent phase into the active phase (maxima) and the second transition from the active into the deceleration phase (minima).
Resuscitation of diapausing embryos
Diapausing embryos used for resuscitation were all 6 months of age (kept at 4 °C in dark conditions). The diapausing embryos were dissected from the collected ephippia and transferred to cell culture dishes filled with 2 mL sterile ADaM medium. Subsequently, embryos were exposed to a constant light source (a combination of Fluora and Biolux lamps; Osram, Germany) at 25 °C. Resuscitation and hatching from the ephippium is normally a process of 3 to 7 days in Daphnia.
Immunolabelling procedure and cytoskeletal staining
For indirect immunofluorescence, we chose three representative stages in both embryo types based on the cell number and embryonic morphology: stage I with ~ 1000 cells (i.e. at 20 °C ± 0.1 °C 10 h post ovulation in asexually produced embryos and 24 h post ovulation in sexually produced embryos); stage II with ~ 3500 cells (i.e. 15 h post ovulation in asexually produced embryos and > 27 months post ovulation dormant embryos); stage III (i.e. diapause termination indicated by the appearance of the appearance morphological features, > 7000 cell stage in asexually and sexually produced embryos). The embryos were fixed for 15 min (in 4% PFA-TX) and squashed on a poly-lysine coated object slide (VWR, Germany). Cover-slips were flipped off with a razor blade upon fixation in liquid nitrogen. All cell preparations were rinsed three times for 5 min in phosphate buffered saline (PBS; pH 7.4 and 0.1 M) and incubated for 2 h with primary antibodies at room temperature. Following three 5 min washes in PBS, the slides were incubated in the dark for 1 h at room temperature with the corresponding secondary antibodies. The slides were then rinsed 3 times in PBS, mounted and coverslipped in Vectashield+DAPI. Finally, coverslips were sealed with rapidly solidifying nail varnish. Preparations were kept in the dark at 4 °C until further analysis.
Labeling agents
For the immunolabeling procedure, mitotic activity was described with a specific antibody against the cell proliferation marker H3S10ph (06–570; Millipore, Germany) raised in rabbit and diluted to 1:150 in PBS. To detect microtubules, α-tubulin was visualized using a monoclonal FITC (fluorescein isothiocyanate) labelled antibody raised in mouse (F2168; Sigma, Germany) at a dilution of 1:70 in PBS. Specificity of H3S10ph and α-tubulin primary antibody binding has been validated by Gómez et al. [29]. Centrosomal γ-tubulin was detected with the help of a polyclonal antibody raised in rabbit (AB11317; Abcam, Germany) diluted 1:30 in PBS. Primary antibodies were detected with the respective secondary antibodies i.e. goat anti-rabbit IgG (Alexa 594, Dianova Germany), goat anti-mouse IgG (Alexa 488, Dianova Germany), diluted 1:150 in PBS. Fluorescent images were taken as described above.
Polymerized/F-actin microfilaments were directly stained with Phalloidin (Abcam, AB176756; Molecular Probes, Germany) diluted 1:150 in PBS for 2 h at room temperature. Fluorescent images were taken as described above.
Temperature dependence of cytoskeletal changes
Temperature decline is known to affect cytoskeletal integrity [26, 27]. To ensure that potential cytoskeletal changes are independent of temperature, we exposed resting embryos to 20 °C and 4 °C under dark conditions. Again, the cytoskeleton was stained with Phalloidin and α-tubulin (F2168; Sigma, Germany) as described above. We monitored potential cytoskeletal decay on day 0, day 18, day 25, day 30 and day 90.
Data analysis and image composition
The morphological changes during development of both embryo types were documented using a stereo microscope (SZX 16 Olympus) equipped with a digital camera (Colorview III, Olympus, Germany) controlled by software Cell^D (Olympus, Germany). Images of one focal plane with adjusted contrast and brightness were assembled using Adobe Photoshop CS6. All data shown in this study were collected from at least six independent replicates.