Pharyngeal Pumping and Tissue-Specific Transgenic P-Glycoprotein Expression Influence Macrocyclic Lactone Susceptibility in Caenorhabditis elegans

Round 1

Reviewer 1 Report

Gerhard et al use heterologous tissue-directed expression of the Parascaris univalens PgP efflux pumps in Caenorhabditis elegans to guage the effects of this drug eflux pump on susceptibility to the macrolide lactones (MLs) Ivermectin (IVM) and Moxidectin (MOX). Measurements of nematode thrashing in response to treatment the MLs confirm that heterologous tissue-specific expression is likely to have occurred, although there are some reservations about the confocal microscopy visualization of the tissue specificity of the epidermal expression of the PgP i.e. while the intestine-specific expression of PgP is well demonstrated by confocal microscopy, 3-D images are needed to demonstrate the claimed epidermal-specific expression of pGp. The dramatic ~30-fold increase in susceptibility to IVM on feeding with bacteria, or mimicking this with 5-HT, for the strain proposed to express PgP in the epidermis is consistent with tissue specificity i.e. feeding exposes a readily traversed intestinal barrier. The much smaller (probably biologically insignificant) increase in susceptibility for MOX on feeding indicates the expected low specificity of PgP for this compound and that intestinal uptake is likely to be minor, points confirmed experiments in which Pgp was heertologously expressed in the intestine. In contrast, intestinally expressed PgP gave a small 2.5-3.5-fold decrease  in susceptibility to IVM compared with the control host strain on feeding behaviour that exposes the intestine, consistent with intestinal efflux via the recombinant PgP.

There are some problems with this paper that need to be addressed. Tables 1 and 2 could better presented as supplementary materials and in single page form. Figures 2 and 3 and especially the summary presented Figure 4  better show what needs to be communicated. The value  (Figure 1; WT strain 5-HT+) of 2.08 (1.39-1.83) is clearly incorrect. The tables demonstrate that the effects of feeding versus 5-HT treatment are essentially identical. The figures could be improved with better colour matches or use of alternate line styles especially in Figure 3 a,b versus c,d. The figure legends uses captialized A,B,C,D etc while the figures use a,b,c,d etc.

 The value and readability of this paper could be much improved by shortening it, (e.g. by using a combined results and discussion section) and focusing the discussion on the results presented in Figure 4.

Minor changes

Abstract

Line10: change " are essential drugs" to "are drugs used widely"

Line 13 change "efflux pumps P-glycoproteins" to "P-glycoprotein efflux pumps,"

Introduction

Line34: delete principally

Line 43  change "not yet sufficiently" to "insufficiently

Line 60 delete "with"

Line 62 replace "of" with "for"

Line 66 delete "it"

Line 70 delete "still"

Line 71 milbemycins not milbemycines (check rest of text)

Line 76 replace "to" with "for"

Line 79 replace "permeability" with "permeation"

Line 82 replace "prevent" with "restrict effective"

Line 86 remove "exemplarily" and in rest of textl-

Line 87 replace "tissue-specific effect of" with "the effect of tissue-specific "

Replace ivermectin and moxidectin with IVM and MOX after first use e.g. line88

Line 92 change from "limiting exposure mostly" to "mostly limiting exposure"

Results

Line 101 changed "exploited" to "used"

Lines 102 - this is presented in a ponderous fashion. Break into shorter sentences - one for each construct.

Line 128 replace "by OP51 or 5-HT or absence" with "by feeding OP51 or 5-HT treatment or the absence"

Line 129 replace "under" with "using"

Line 136 replace "for the WT in" with of the WT for IVM"

Line 141 and 142 delete  8.0-fold is incorrect - should be 10.4-fold . Delete "to a level .... bacteria

Line 197 Generally should be In general

Discussion Line 254 replace " contribute to reducing" to "can reduce'

Lines 288-289 This is probably an overstatement for MOX

Line 296 use concurs rather than high concordance (thorought text)

Line 343 replace "are, to some extent" with "may be"

Line 364 replace "would support" with "suggest"

Line 375 remove "only'

Material and methods

Line 460 replace "Trashing" with Thrashing"

Figure legends Line 713 "Verification" with "Confirmation"

Line 518 replace "primers" with Olignucleotide primers"

Line 518 insert "detected" to give "bands detected for"

Line 475 replace "in" with "of"

Line 459 replace "Concentration-response" with Dose-response"

Line 767 delete sentence starting "Prior to ..."

Line 798 Delete hypothetic and change line to "Schematic illustration of hypothetical..."

Line 800 replace" i.e. the epidermis and intestine intestine prohibit" with "i.e. at the epidermis and intestine block"

In figure replace "stimulation" with "stimulated"

Y axis legend of Figure 4 does not make sense.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

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Comments for author File: Comments.pdf

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Please consider simplifying sentences. A number of examples have been noted but there are likely to be others where principles such a shortening sentences and reducing both language and grammatical complexity could be usefully applied in order to improve readability and your communication with readers.

Also  missing bracket in supplementary table 2 3rd line of data