Here the reviewer(s) should list 2-3 major concerns about the research (if they exist). This may relate to:
Experimental design: were the techniques appropriate for the hypothesis? Were suitable controls included? Were new techniques/mutants/cell lines etc. sufficiently characterised to allow the study to be replicated? Were robust statistical methods used?
- The experimental design is very impressive, considering the large number of comparisons included in the experiment. A major concern for us was the lack of biological replicate (or the clear description of them). The methods section indicates that each infection experiment was carried out in a 100 mL jar, but it seems like there was one jar per treatment combination (it is possible we are missing something in our reading). We feel it is important to clearly state how many jars were prepared per treatment and if it was only one jar per treatment to discuss why this was the case. We understand that this is large experiment, but biological replication would greatly increase the reliability of the results and it is important to recognize and discuss it. This comes especially into play when the authors discuss the variability in results between the current experiment and published results from a previous experiment. If there is biological replication in both experiments, readers can evaluate whether the observed variation was within a normal range of variation for this system. Likewise, error bars in Figure 2 would make the results easier to evaluate. If we have interpreted the text properly and biological replication was not included, the authors should consider explaining why this was the case and how they account for it in the results.
- In the introduction, methods, and results section the authors refer to parasite "strains," but switch to parasite "isolates" in the discussion where it becomes clear that this distinction is rather important to interpreting the results of the study. When the term "parasiteThe description of the parasite isolates indicate that they are not genetically identical - this would make the results very difficult to replicate. It would be helpful to use more controlled isolates (known strain composition) and do 16S monitoring to determine if strain composition changes over time (selection?).
- Is there different infection rates between the strains in the mothers? Is P5 for example more infectious in general?
Do the results support the authors’ conclusions and, if not, what additional experiments would you suggest?
- The authors mention the applicability of this study to the experimental assessment of vaccines in the abstract , in the last sentence of the introduction, and in the discussion. However the connection feels rather tenuous. Vertebrates in which vaccines are used have very different immune mechanisms than the inverts in this study. These differences may require a very different framework to evaluate specific and nonspecific efficiency of vaccines. The authors may consider discussing these differences in more details and give a clear example of how this study relates to vaccine assessment.
- The discussion states that this study is not concerned with the molecular methods involved in the immune priming. Despite this, the molecular mechanisms for immune priming may still be discussed in relation to the results found here. Perhaps the mechanisms for immune priming in invertebrates are not specific enough to differentiate between strains and would therefore support the results found in this study.
- It would be helpful if the authors discuss future directions for this work.