Developmental stages in diapausing eggs: an investigation across monogonont rotifer species

C. Boschetti, F. Leasi, and C. Ricci- 2011

Review by Rachel K Johnston

Background

Asexual Female Rotifer

Figure 1. Asexual female rotifer (B. manjavacas) carrying amictic eggs. Image by R.K. Johnston (2011).

Rotifers are a phylum of microscopic, aquatic animals with the unique ability to survive unfavorable environments and extreme stress through dormancy. Bdelloid rotifers can become quiescent at any point in their life span, desiccating their bodies and halting metabolism. In contrast, monogonont rotifers can only enter dormancy at a specific developmental stage known as a resting egg or diapausing embryo. Resting eggs hatch into amictic females that reproduce asexually (Fig. 1). Those offspring can either continue asexual reproduction or under proper conditions, produce mictic eggs. Unfertilized, mictic eggs will hatch into males (Fig. 2), but fertilized mictic eggs become resting eggs which can desiccate and remain dormant for many years (Fig. 3). These dormant eggs will then hatch once satisfactory environmental conditions are met. Previous work investigating the stage at which diapausing embryos arrest is contradictory largely not well understood. Boschetti et al. (2011) used a nuclear stain and confocal microscopy to determine the stage of embryogenesis at which nine different monogonont rotifer species enter dormancy, and whether this stage is species specific or common across taxa.

Mictic Female + Males

Figure 2. A female rotifer (B. manjavacas) bearing mictic eggs and two male rotifers. Males are a fraction of the size of females and only live a couple days, dying after fertilizing a female. Image by R.K. Johnston (2012).

Rotifer Life Cycle

Figure 3. The life cycle of monogonont rotifers. Reproduced from Hoff and Snell, 1987

Methods

Resting eggs from nine different monogonont rotifer species were collected from around the world. The eggs were rehydrated, fixed with paraformaldehyde and permeabilized using detergents. DAPI, a fluorescent stain, was then used to stain the nuclei of the eggs. The stained eggs were imaged on a confocal microscope, and software was used to create a three-dimensional image and count the number of cells present. Mann-Whitney U and Kruskal-Wallis statistical tests were performed to find any significant differences in cells number between taxa.

Results and Conclusions

Boschetti 2011 Fig 1

Figure 4. Average number of cells per resting egg in nine different rotifer species. Number of cells was determined by DAPI nuclear staining and confocal microscopy. Results indicate two significantly different developmental stages in which embryo development can be arrested. Reproduced from Boschetti et al, 2011.

The number of nuclei counted in each resting egg varied greatly from 18 to 150. However, the number of cells and therefore the stage of developmental arrest could be categorized into two significantly distinct groups (P=0.000).  Brachionus plicatilis, B. manjavacas, and Epiphanidae senta resting eggs contained an average of 45 to 68 cells with high variation within taxa (Fig. 4). All other species contained an average of 20 to 25 cells in each resting egg, with very low variation between or within taxa. One species, Lecanidae bulla was not successfully stained and was thus removed from analysis. The group with the larger number of cells may indicate a difference of a single mitotic event before arresting.

Boschetti 2011 Fig 2

Figure 5. Nuclei of resting eggs stained with DAPI and imaged at 361-365nm. (a) B. calyciflorus (b) R. frontalis (c) B. plicatilis (d) E. senta. Colors indicate depth of field and scale bars are 25um. Reproduced from Boschetti et al, 2011.

Regardless of the number of cells, all resting eggs displayed a similar patterning of the nuclei. All resting eggs contained a central group of large nuclei with a surrounding layer of smaller cells (Fig. 5). This morphological appearance is indicative of the gastrulation stage of embryogenesis. These findings directly contradict earlier studies claiming that embryos completed mitotic before entering diapause. This is an important distinction because rotifers are eutelic, meaning they have a fixed number of cells that stop dividing once that number is reached. Because of this, it is important to understand whether the divisions stop before or after diapause.

Further Investigations

While this paper reached significant conclusions about resting egg development, it leaves many additional questions that should be addressed. First, there should be exploration into a possible correlation between the stage an egg reaches before arresting and hatching time or viability. If an egg progresses further through development before entering dormancy, it is possible that egg will hatch faster or have a higher chance of survival. Second, Boschettie et al were unable to stain the membranes of the cells, only the nuclei. There fore, it is still unknown whether resting eggs contain distinct cells or syncytia- a multinucleate fusion of cells. Finally, it is possible that resting eggs could store maternal RNAs for use until diapause and then begin transcribing their own RNAs after reactivation. The possibility of vastly different environmental conditions for the mother as opposed to the hatching egg may drive this kind of differential gene regulation. Recent advances in rotifer transcriptomics (Snell 2014) make this a viable line of investigation.

Broader Implications

gulf oil spill

Figure 6. Environmental disasters such as the BP gulf oil spill can have devastating and long term consequences for aquatic environments. Rotifers are used in toxicology screens as an indicator species for the overall health of marine systems. Photo courtesy of National Geographic.

As a major food source for larval fish, rotifers are an integral part of many aquatic ecosystems. Because any change in rotifer population can have major downstream effects in the food web, they are often used as an indicator species in environmental studies and are the subject of many toxicology screens (Rico-martinez et al. 2013). Also, rotifers have gained popularity as a model organism in the study of aging mechanisms and aging interventions (Snell et al. 2014). Because of the vital use of rotifers in other areas of research, it is essential to more fully understand their developmental process.

Critique

Neonate- Edited

Figure 7. Neonate rotifers (B. manjavacas) newly hatched from resting eggs. Video taken by R.K. Johnston (2013).

There are several strengths in this study. First, Boschetti et al were able to provide strong evidence to answer a question that has had many conflicting arguments in the past. They used a simple yet elegant experimental design to address questions about an ecologically relevant species. Also, their investigation explored rotifers of several different taxa from around the world. This supports the idea that their results are indicative of an underlying biological phenomenon rather than something driven by a particular species or environment. However, there are several weaknesses to Broschetti et al’s approach. Because the staining of L. bulla failed, they were unable to include that species in their statistical analysis. Also, there was a huge variance (30 to 90) in cell number within B. plicatilis samples, which they were unable to explain. Finally, because there were few genetic tools available, Boschetti et al were unable to explore any genetic factors in resting egg development. Recent developments in rotifer genetics such as RNAi (Snell et al. 2014) and plasmid expression (Liu et al. 2013) may provide the tools necessary to revisit these questions from a genetic approach.

Summary

Reproductive Females- Edited

Figure 8. Asexual female rotifers (B. manjavacas) carrying amictic eggs. Video taken by R.K. Johnston (2013).

Because rotifers are an ecologically important model organism for many other types of research, it is vital to understand the stages and mechanisms of their development. Boschetti et al (2011) used nuclear staining and confocal microscopy to determine that rotifer resting eggs across several species arrest development at the gastrulation stage. However, whether the embryo is arresting during early or late gastrulation seems to be species specific.

 

 

References

Boschetti, C., F. Leasi, and C. Ricci. “Developmental stages in diapausing eggs: an investigation across monogonont rotifer species.” Hydrobiologia 662 (2011): 149-155.

Hoff, F.H. and T.W. Snell. Plankton Culture Manual. Dade City: Florida Aqua Farms, Inc, 1987.

Liu, B.R. et al. “Delivery of Nucleic Acids, Proteins, and Nanoparticles by Arginine-rich Cell-Penetrating Peptides in Rotifers.” Marine Biotechnology 15 (2013): 584-595.

Rico-Martinez, R., T.W. Snell, and T.L. Shearer. “Synergistic toxicity of Macondo crude oil and dispersant Corexit 9500A to the Brachionus plicatilis species complex (rotifera).” Environmental Pollution 173 (2013): 5-10.

Snell, T.W., R.K. Johnston, K.E. Gribble, and D.B. Mark Welch. “Rotifers as experimental tools for investigating aging.” Invertebrate Reproduction and Development. In press.

Snell, T.W., R.K. Johnston, et al. “Joint inhibition of TOR and JNK pathways interacts to extend the lifespan of Brachionus manjavacas (rotifera).” Experimental Gerontology 52 (2014): 55-69.

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