ABC transporter functions as a pacemaker for sequestration of plant glucosides in leaf beetles

1) Providing experimental evidence (Western blot) that the RNAi data are not a result of off-target effects.

Experimentally, we excluded off-target effects based on the analysis of co-silencing-effects on non-target genes using qPCR, SDS-Page and Western blot. We have included Figure 3–figure supplement 1: “Effects of cpmrp silencing on the protein level of the glandular tissue of C. populi” to provide additional experimental evidence that the RNAi data are not a result of off-target effects.

2) The indication of concentrations when calculating the Km values.

We have revised Figure 4B and indicated the injected salicin in relation to the oocyte volume to calculate substrate concentrations.

3) Including comparative discussion considering possible cpmrp homologs in other insects.

We agree that a comparable discussion of cprmp homologs could extend our sequestration model to other insects but it is rather limited and speculative to date. In insect databases there are predicted ABC transporter sequences homologous to CpMRP identified in Chrysomela populi (the highest amino acid sequence similarity (75%) with 59% identity was found to the predicted ATP-binding cassette transporter of Tribolium castaneum (TC014776)). However, these sequences have been neither functionally characterized nor localized in the insects to date. Based on the uniform morphology of insect epidermal glands only, we would speculate the presence of cpmrp homologs in class 3 gland cells of insects (e.g., dermal glands of Tenebrio molitor, sternal glands of Periplaneta americana, pheromone gland of Harpobittacus australis (Noirot and Quennedy, 1974)). This need to be further investigated. In our manuscript we therefore focused on the discussion of the proposed sequestration model including CpMRP homologs of other leaf beetles (C. lapponica and P. cochleariae) and their general relevance in exocrine glands of Chrysomelina species.

4) Providing more information on the beetles used in the Materials and methods section, such as: origin of the beetles, rearing methods and the food used, number of beetles used to start the colony, and information on the P. cochleariae beetles that were used in the experiments.

We have provided all information for collection and rearing of the leaf beetle species used in the study.

5) The paper is not very clearly written and the lack of detail in the description of the results was a problem. Similarly, the authors need to use punctuation to clarify their meaning in many places. The English language throughout the text should be improved.

We have edited the Results and Discussion parts. Particularly, the last paragraphs of this section were completely revised. We have included a conclusion in order to provide a clear take-home message for a broad readership. We have corrected typos and we have improved the English language throughout the manuscript.

6) Finally the first three words of the title do not seem to connect to the contents of the manuscript and distract from the message of the manuscript and its title.

We have changed the title to read: “ABC transporter functions as a pacemaker for the sequestration of plant glucosides in leaf beetles”