Review 1: "Changing Species Dynamics and Species-specific Associations Observed between Anopheles and Plasmodium Genera in Diebougou Health District, Southwest Burkina Faso"

RR\ID Evidence Scale rating by reviewer:

• Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.

***************************************

Review: The authors provide compelling evidence for changes in Plasmodium parasites species composition in human population in the study locality (Diebougou health district, Burkina Faso) spanning a period of six years from being predominantly Pf and now more of non-falciparum species (Po, Pm) mixed with Pf or occurring alone. This was also reflected in the parasite composition in the major vector species Anopheles gambiae sl and An. funestus sl. It is likely a result of the effect of control measures/interventions which have been known to alter vector species distribution; however, the present findings on changes in parasite populations has been minimally explored, and in my view very informative. The data provide useful insights supporting the plasticity of An. funestus sl in the changing malaria dynamics as described in literature in other settings in Africa.

The conclusions drawn are largely supported by the data presented. However, as alluded by the authors the evidence is mainly associative and does not prove causality although there is strength in the evidence provided from the data. I will caution against inference on preferential ‘transmission’ of non-falciparum species between An. funestus and An. gambiae s.l. which is not supported by the current data. I would rather limit it to preferential ‘detection’.

The mechanisms that underlie differential parasite presence/detection between the species may not be explained by competence alone and other contributing factors cannot be ruled out. For instance, could the degree of feeding on non-human hosts among the vectors influence the outcome of parasite infection? Blood meals and depending on the host types and sequence can impact parasite transmission through the vectors. What if this is captured as a covariate and the dataset subjected to detailed analysis rather than just proportional comparisons? The contribution of non-human blood feeding in the outcomes in my view should be accounted for.

The study nonetheless generates many important unanswered hypotheses for further consideration. For instance, do the vectors show differential attractive patterns to bite humans when infected? Are differences contingent on specific parasite species?  If this is the case, then the vectors could be exposed to different parasite composition in a human blood meal. It will be interesting to correlate the outcome of mixed infection (e.g. Po/Pf) in abdomen and head/thorax in individual mosquitoes for clues on differential transmission.