Peer review of: A drug-inducible sex-separation technique for insects (2019)

Reviewers' comments: 
 
Reviewer #1 (Remarks to the Author): 
 
Key results: The authors devise a novel sex-selection cassette in Drosophila melanogaster that 
permits isolation of a single sex when raised on antibiotics. The sex-selection method uses a 
transgene cassette including the antibiotic resistance genes NeoR and PuroR, which confer 
resistance to geneticin and puromycin, respectively. The authors engineered these antibiotic 
resistance genes to incorporate the introns TraF and DsxM, which are spliced in a sex-specific 
manner to produce a functional NeoR copy in females and a functional PuroR copy in males. This 
cassette is not detrimental to organismal fitness, and as sex-specific introns are common to a 
number of insect species, a similar approach can be applied to other organisms, making this sex-
selection method a potentially important tool in control of pest insect populations. 
 
Originality and significance: The authors highlight that this sex-selection method is of immense 
use in insect population control, which frequently relies on large numbers of sterile males. 
Although the narrative is presented from an angle of importance to insect control, it would 
increase the significance of the work if the value of this selection cassette to fly geneticists was 
also highlighted in the Introduction and Discussion. (Admirably, the authors have already 
deposited plasmids and stocks in the relevant repositories.) 
 
Data and methodology: As mentioned in the below comments, some graphs could be altered to 
improve legibility and ease of data interpretation. Methodology is recorded in sufficient detail to 
reproduce results. 
Appropriate use of statistics and treatment of uncertainties: The use of statistics in all data is 
appropriate. 
 
Suggested improvements (all minor): 
 
There are additional genetic methods of selection for females in Drosophila. Ashburner et al. 
(Drosophila, A Laboratory Handbook, pp. 135-6) describe Y-linked conditional or binary expression 
systems. McMahan et al. 2013 (doi 10.1534/g3.113.006411) describe a UAS::rpr transgene on the 
Y chromosome that can be used to male progeny in crosses with a GAL4 transgene. 
 
Acronyms such as SIT, RIDL, TSL and IIT that are used relatively infrequently can be written out in 
their entirety to improve readability. 
 
Insects have sexes, not genders. 
 
[Numbers below refer to line numbers] 
31: Change “geneticin supplements” to “geneticin-supplemented food” for clarity 
41: In “Since then, SIT was successfully implemented…” change “was” to “has been” 
61: Missing the word “transgene” after “conditional lethal” 
94: Omit “the” before “first instar larvae” 
169: Clarify that only males were recovered from all three of the transgenic lines 
250: Although the system is resistant to loss-of-function mutations, over extended use it could 
select for gain-of-function mutants in which the tra or dsx introns can be spliced out in the 
undesired sex. 
268: “the” should come before “functional” in “they harbor functional the sex-sorter gene cassette” 
294: Missing “the” before “Tet-Off system” 
305: The authors mention that they did not anticipate that the sex selection cassette would be 
detrimental to fly fitness, and instead they chose to assess the effect of different integration sites 
on fitness. However, they did assess fitness of homozygous copies of the cassette and found 
comparable fitness to wt flies, so this can be omitted. 
 

In the Materials and Methods, fly genotypes are not italicized in several places. 
 
382: Capitalize “biological” in “Carolina Biological Supply Company” 
415: The wording “both males are equal at female mating” is confusing. Perhaps “both males have 
equal mating success?” 
 
Figure 1: 
608: “Insure” should be changed to “ensure” in “To insure that functional antibiotic-resistance 
proteins…” 
 
Figure 2: 
C and D could be changed to dot plots or box-and-whisker plots to improve interpretation of data. 
 
Figure S1: 
It would help in interpreting the data if A and B were displayed as dot plots or box-and-whisker 
plots rather than bar graphs. It would be easier to compare the sex ratios in C and D if they were 
displayed as stacked bar graphs as in Figure 2A and B. 
B: Change the Y axis to % survival (Transgenic/wild-type) 
C and D: Puromycin and geneticin sections should be moved so that they are on the same side in 
both C and D. “Gender percentage” as a Y axis title is a bit confusing and could be changed. 
 
Table S3: “SexingLine” should be changed to “sex-sorter line” to be consistent with the text. 
 
 
 
 
Reviewer #2 (Remarks to the Author): 
 
This manuscript by Kandul et al. provide a sophisticated and intelligent design to cause sex 
separation in Drosophila melanogaster by the use of different antibiotics. While this is a clever 
approach, this is a strategy that should be strictly restricted to lab use!!!! To propose this 
approach for application in pest control is unreasonable and irresponsible!! Insecticide and 
antibiotica resistance are two major world wide problems, which should not be made even more 
severe by any means! The proposition to release antibiotica resistant insects is therefore 
absolutely irresponsible! First, the animals will die at some point and their DNA will enter the 
environment for bacteria to take this DNA up. Secondly, the antibiotica resistance will provide the 
insect carriers with a potential selectable advantage. Thus, rare survivors might have under certain 
circumstances an advantage and the transgenes could be selected and their presence increase in 
the population. While this is unlikely, this will still provide NGOs an argument against such 
approaches. So far transgenic SIT approaches use only neutral (fluorescent markers) or negative 
(sterility lethality) transgenes. And this is good so, as there will be no positive natural selection 
possible! Scientists in this field should be careful in what to promote and what NOT to promote! 
They should not offer any unnecessary arguments for opponents of such approaches. 
 
Besides that I will review the manuscript in the following based on scientific standards, as it is a 
good piece of science and could be published as a Drosophila technique. Maybe then not in nature 
Communications but elsewhere. 
 
Major revisions: 
1. Sell it as Drosophila technique not as SIT approach. 
 
2. Why is it, that having two copies reduces the amount of antibiotics to get sex separation 
(starting line 176). This is not straight forward. Having more should require more and not less. At 
least if it is leakiness of expression!!! Or is this a homozygousing effect? More sex-specifc splicing 
when locus homozygous? That could be tried by getting two copies that are at differnt loci. Thus 

combining #2 and #3 in a transheterozygous condition! 
 
3. The competition experiments are not competitive! They need to be carried out correctly or 
deleted. Giving two males to 10 virgin females is not a competitive situation. Giving 10 males of 
one kind and 10 males of the other kind to 10 virgins would be a competitive situation. Virgin 
Drosophila females, once old enough, will mate even with males that almost don't move and are 
not competitive at all. THus either drop all sentences to competitiveness of the males or do the 
experiments correctly. 
 
Minor edits: 
 
Use of articles and singular/plural should be checked carefully thoughout the mansucript (also 
Figure legends!!) 
 
Line 28: tra and dsx are recessive lethal alleles and not capitalized. Please check this throughout 
the mansucript, as this is changing again and again. Also check the Figure legends. 
 
Line 49: References 8 and 9 do not refer to IIT. They are referring to an approach originally called 
Eliminate Dengue (in Australia) and is now called "World Mosquito Program". However this is not 
IIT as the spread of Wolbachia is intended. This is more a gene drive scenario for population 
replacement! IIT is the approach by Stephen Dobson (Mosquito Mate). Thus publications by him 
should be cited in this respect! 
 
Line 59: There is a sole focus on mosquitoes. What about references to Tephritid fruit flies (pupal 
colours) or Tse-Tse flies (hatch time) in respect to sexing methods! 
 
Line 70: The first paper showing this type of sexing is not cited: Fu et al. Nat Biotechnol. 2007 
Mar;25(3):353-7. Reference 30 is too old to be correct here! This one could be replaced by Mesa 
et al. 2018: Comparison of classical and transgenic genetic sexing strains of Mediterranean fruit fly 
(Diptera: Tephritidae) for application of the sterile insect technique. 
doi.org/10.1371/journal.pone.0208880. 
 
Lines 95 to 97. While their is resistance to loss of function mutations, their could be mutations that 
interfere with sex-specific splicing and thereby disrupt the sexing. To present this as completely 
resistant to mutations is wrong and should not be done! 
 
Line 115 and elsewhere (e.g. line 272): piggyBac (in italics) Not capitalized or any other way! 
 
Lines 160 to 161: Splicing is not necessary affecting expression! Thus it is not the expression of 
..... genes, but "the production of ....resistance can be limited... 
 
Lines 229- : The assay used is not conclusive for competition! See above! Thus either do a real 
competitive experiment or remove this statements! 
 
Line 244: there is an "over " missing: advantages over 
 
Line 252: Yes LOFs will selected against, but mutations in the construct causing the loss of sex-
specific splicing could generate a problem. Might be more rare indeed but should be mentioned! 
 
Line 255: What are Y-autosome or X-autosomes? (probably chrosmosomes or translocations?) 
 
Line 281: NO, the Drosophila tra intron is not conserved in other species. Actually in Drosophila 
female tra splicing is performed by Sxl and not by TRA itself, as in other species such as C. 
capitata. Thus if the authors had used the C.c. tra intron yes it could be transferable (as shown in 
the not cited Fu et al., 2007) but not the D.m. tra!!! 

 
Lines 287 to 289: The promotion of using insecticide resistance genes in such approaches is 
ludicrous! Float the environment even more with things we don't want to have there! No No No !!!! 
 
Lines 291 - : the positive selection also has its drawbacks, as it can be used as an argument that 
these insects have an advantage and could be selected in case rare SIT survivors carry these 
genes! Negative selection has here a great advantage as selling point for release! 
 
Lines 311- : Drop sentences to mating competitivess or do the experiments correctly! 
 
Line 328 Point 7: No!!! Floating the environment with antibiotica resistance genes is NOT sellable! 
PERIOD! 
 
Line 362: Where exactly were the introns integrated. Any thought or specific consideration on the 
exact sequence position as made in the non-cited Fu et al., 2007? Sequences outside the intron 
might influence the efficiency of sex-specific splicing! 
 
Line 370: "Sp" is a dominant mutation! 
 
Lines 410- : One and one male for 10 females is not a competitive situation!! This is not a 
competition experiment! 
 
Line 439: Only one line was deposited 
 
Figure 1 legend. Text under (b) is refering still to (a). There is no (b) text, which could be: survival 
of females and/or males carrying the respective constructs on antibiotic selection (pur, blue) (gen, 
green) 
 
Figure S1: This should become a main Figure. This provides the clear data! 
Panel b) transgenic / wild type. This cannot be true. This cannot be a ratio! A ratio compared to 0 
does not work. It is probably survival percentage or something similar! 
 
Line 652: 1 mg/mL NOT 0.1!!!!!