C. Boschetti, F. Leasi, and C. Ricci- 2011
Review by Rachel K Johnston
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.
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
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.
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.
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.
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.
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.
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.
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