of 12
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!!!!!
Reviewers' comments:
Reviewer #1 (Remarks to the Author):
Key results: The authors devise a novel sex-sel
ection cassette in Droso
phila melanogaster that
permits isolation of a single sex when raised
on antibiotics. The sex-selection method uses a
transgene cassette including the antibiotic resi
stance genes NeoR and PuroR, which confer
resistance to geneticin and puromycin, respectiv
ely. 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 fema
les and a functional PuroR copy in males.
This cassette is not detrimental to organismal fitn
ess, and as sex-specific
introns are common to a
number of insect species, a sim
ilar 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 frequen
tly relies on large numb
ers 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 Discu
ssion. (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 da
ta interpretation. Methodology is
recorded in suffi
cient detail to
reproduce results.
Appropriate use of statistics and treatment of uncer
tainties: The use of statistics in all data is
appropriate.
Suggested improvements (all minor):
There are additional genetic methods of selecti
on 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.
Thank you, we corrected the current text accordingly.
Acronyms such as SIT, RIDL, TSL and IIT that ar
e used relatively infrequently can be written
out in their entirety to improve readability.
Thank you for the detailed look at our manuscript.
Infrequently used acronyms are now spelled
out throughout the manuscript. At the same time
, SIT, RIDL, and IIT are the major methods for
insect control and as such are frequently
referred only by their acronyms in the press.
Insects have sexes, not genders.
This has been changed throughout the manuscript.
[Numbers below refer to line numbers]
31: Change “geneticin supplements” to
“geneticin-supplemented food” for clarity
This has been corrected.
41: In “Since then, SIT was successfully implemented...” change “was” to “has been”
This is no longer applicable with
the significant text changes.
61: Missing the word “transgene” after “conditional lethal”
This has been corrected.
94: Omit “the” before “first instar larvae”
This has been corrected.
169: Clarify that only males
were recovered from all thr
ee of the transgenic lines
This has been corrected.
250: Although the system is resi
stant to loss-of-function mutati
ons, 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.
This has been corrected, now we refer to loss-o
f-function mutations in th
e text. Gain-of-function
mutations indeed can happen but they are much
less frequent than loss-of-function mutations.
268: “the” should come before “functional” in
“they harbor functional the sex-sorter gene
cassette”
This has been corrected.
294: Missing “the” befo
re “Tet-Off system”
Sentence has been rearranged and is now correct.
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 c
opies of the cassette and found
comparable fitness to wt flies, so this can be omitted.
This has been deleted.
In the Materials and Methods, fly genotype
s are not italicized in several places.
This has been corrected.
382: Capitalize “biological
” in “Carolina Biologi
cal Supply Company”
This has been corrected.
415: The wording “both males are equal at fema
le mating” is confusing. Perhaps “both males
have equal mating success?”
This has been corrected.
Figure 1:
608: “Insure” should be changed to
“ensure” in “To insu
re that functional
antibiotic-resistance
proteins...”
This has been corrected.
Figure 2:
C and D could be changed to dot
plots or box-and-whisker plots
to improve interpretation of
data.
We added the data points to the bar gra
phs to improve presentation of the data.
Figure S1:
It would help in interpreting th
e 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 gra
phs as in Figure 2A and B.
B: Change the Y axis to % su
rvival (Transge
nic/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.
We updated the figures following your comments. Thank you.
Table S3: “SexingLine” should be changed to “sex-s
orter line” to be consistent with the text.
This has been corrected.
Reviewer #2 (Remarks to the Author):
This manuscript by Kandul et al. provide a sophist
icated and intelligent design to cause sex
separation in Drosophila melanogaster by the use of
different antibiotics. Wh
ile this is a clever
approach, this is a strategy that should be stri
ctly restricted to lab use!!!! To propose this
approach for application in pest control is
unreasonable and irresponsi
ble!! Insecticide and
antibiotica resistance are
two major world wide problems, whic
h should not be made even more
severe by any means! The proposition to releas
e antibiotica resistant
insects is therefore
absolutely irresponsible! First, the animals w
ill die at some point a
nd their DNA will enter the
environment for bacteria to take this DNA up.
Secondly, the antibiotica re
sistance will provide
the insect carriers with
a potential selectable ad
vantage. Thus, rare su
rvivors might have under
certain circumstances an advantage and the tran
sgenes 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 a
pproaches 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 fo
llowing based on scientific standards, as it is a
good piece of science and could be
published as a Drosophila t
echnique. Maybe then not in
nature Communications but elsewhere.
Major revisions:
1. Sell it as Drosophila tec
hnique not as SIT approach.
We understand your concerns raised by our work, a
nd agree that as originally presented, this
technology could have unexpected and dangerous c
onsequences. We have therefore adjusted the
paper accordingly. Please see th
e altered focus in the abstra
ct, introduction, and discussion,
which now also includes a caution against using this
technology in the field. We also suggest that
a simple strategy to safeguard against the potential
horizontal transfer and
spread of transgene in
prokaryotes. The insertion
of a regular introl to break the codi
ng sequence of the transgene that is
intended for the field releases
would block it splicing and f
unction in any prokaryote. We
successfully used this strategy to clone and
manipulate multiple genes that are otherwise ‘toxic’
for E. coli, such as restriction
endonucleases and T4
DNA ligase, etc.
(
https://www.nature.com
/articles/ncomms13100
), in the lab..
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 s
hould 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 trie
d by getting two copies
that are at differnt
loci. Thus combining #2 and #3 in
a transheterozygous condition!
We think this is the homozygousing effect th
at improves sex-specific splicing. Since both
expression level and sex specific splicing depend to the genome location, we analysed multiple
insertions of every tested transgene to identif
y the transgenic line, in which each transgene
operated properly. We found that a single copy of
PuroR
dsxM
or
NeoR
traF
were spliced in both
sexes at the tested insertion sites with an
antibiotic concentration of 0.4mg/mL; but
PuroR
dsxM
or
NeoR
traF
was splicied more efficiently in males or females, respectively, at one established
insertion line, and this became apparent at the hi
gher antibiotic concentration. We were able to
homozygous one of two ‘working’ (at 1.0mg/mL)
insertion lines of th
e sex-sorter cassette
(
PuroR
dsxM
+ NeoR
traF
) and found that two-copies
of the cassette operated properly at the lower
concetration (0.4mg/mL) than one copy did (
1.0mg/mL). The sex-specific spling of sex-
determination genes are known to
be self-reinforcing pr
ocess, therefore two
copies of the sex-
sorter cassette located at the homologous loca
tion could reinforce th
e correct splicing and
suppress the incorrect splicing by recruting SXL,
TRA, and other proteins to their genomic
locations. We agree that the combining the lin
es #2 and #3 of the se
x-sorter cassette in
transheterozygous conditions would
be an interesting test to t
ease apart the homologous location
effect. Unfortunately, it goes beyond
the scope of this paper.
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 situ
ation. Giving 10 males of
one kind and 10 males of the other kind to 10 virg
ins 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 sent
ences to competitiveness of the males or do the
experiments correctly.
We removed the male competition assay.
Minor edits:
Use of articles and singular/plural should be ch
ecked carefully thoughout the mansucript (also
Figure legends!!)
We thank you for pointing this out, and have
subsequently had the entire manuscript
professionally edited for language.
Line 28: tra and dsx are
recessive lethal allele
s and not capitalized. Plea
se check this throughout
the mansucript, as this is changing agai
n and again. Also check the Figure legends.
This has been corrected.
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 D
obson (Mosquito Mate). Thus
publications by him should be
cited in this respect!
Thank you. This has been corrected.
Line 59: There is a sole focus
on mosquitoes. What about refere
nces to Tephritid fruit flies
(pupal colours) or Tse-Tse flies (hatch
time) in respect to sexing methods!
We added more comments to the sexing methods
used for Tephritid fruit flies (Medfly and
Mexfly) and Tsetse flies, and toned down discussi
on of mosquito sexing
methods especially in
the introduction. The Medfly sexi
ng method (the linkage of the
white pupae
+
temperature
sensitive lethal
loci) is the most advanced sexing me
thods and is used for high-throughput
production of Medfly males by culling females. S
till, it was established serendipitously with
classic genetic methods and could not be tran
sfered to other Tephritid fruit fly species.
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 co
rrect here! This one could be replaced by Mesa
et al. 2018: Comparison of classica
l 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
.
Thank you, we added both references, and removed ref. #30.
Lines 95 to 97. While their is resistance to loss
of function mutations, th
eir could be mutations
that interfere with sex-specifi
c splicing and thereby
disrupt the sexing. To
present this as
completely resistant to mutations
is wrong and should not be done!
This has been changed to be more specific. The
described system is resistant to loss-of-function
mutations, gain-of-function mutations can sti
ll disrupt sex-sorting,
though gain-of-function
mutations are much less frequent than loss-of-function ones.
Line 115 and elsewhere (e.g. line
272): piggyBac (in italics) Not cap
italized or any other way!
This has been corrected.
Lines 160 to 161: Splicing is not ne
cessary affecting expression! Thus
it is not the expression of
..... genes, but "the production of
....resistance can be limited...
This has been corrected.
Lines 229- : The assay used is not conclusive for
competition! See above! Thus either do a real
competitive experiment or remove this statements!
We removed the male competition assay.
Line 244: there is an "ove
r " missing: advantages over
This has been corrected.
Line 252: Yes LOFs will selected against, but mu
tations in the construct causing the loss of sex-
specific splicing could generate a problem. Might be
more rare indeed but should be mentioned!
This has been corrected.
Line 255: What are Y-autosome or X-autosomes?
(probably chrosmosomes
or translocations?)
Translocation. We removed
this unclear writing.
Line 281: NO, the Drosophila tra in
tron 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. tr
a intron yes it could be transferable (as shown in
the not cited Fu et al., 2007)
but not the D.m. tra!!!
We completely agree with the raised
point and updated the text accordingly.
Lines 287 to 289: The promotion of using insectic
ide resistance genes in such approaches is
ludicrous! Float the environment even more with th
ings we don't want to have there! No No No
!!!!
Please see our response above.
Lines 291 - : the positive selection al
so 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!
We agree that the advantage a
nd the selection depe
nds on the context, and we hope that
antibiotic concentratio
ns in the wild are not that high. That
said, of course these things should be
denoted and implemented in an environment specific manner. If the antibiotic concentrations are
used in areas for cattle or plant protection (which
they sometimes are) this may benefit or not the
strategy, depending on the goal whethe
r it is useful to ha
ve the engineered males have a fitness
advantage.
Lines 311- : Drop sentences to mating comp
etitivess or do the experiments correctly!
We excluded the male competition assay..
Line 328 Point 7: No!!! Floating the environmen
t with antibiotica resi
stance genes is NOT
sellable! PERIOD!
We agree and corrected the text accord
ingly (please see our response above)
Line 362: Where exactly were th
e introns integrated. Any thought
or specific consideration on
the exact sequence position as made in the no
n-cited Fu et al., 2007? Sequences outside the
intron might influence the effici
ency of sex-specific splicing!
Both introns were integrated right after the star
t codon (ATG) of an
tibiotic-resistance genes. We
submitted the sequences and map of every cons
truct to AddGene.org, after publication these
plasmid and sequence will be released. We used
the consensus sequences for Drosophila splice
junctions, to match the most frequent bases adja
cent to an intron sequenc
e (..TG[gt---ag]AT/C..).
Line 370: "Sp" is a dominant mutation!
This has been corrected.
Lines 410- : One and one male for 10 females is
not a competitive situation!! This is not a
competition experiment!
We do not use the word “competitiveness” in the current text.
Line 439: Only one line was deposited.
We deposited one homozygous lines w
ith the complete sex-sotter casse
tte, since it contains all
genetic parts and can be used to generate
males or females. Bloomington Drosophila Stock
Center prefers to keep Drosophila lines that are requested by multiple researchers and ‘retires’
unused stocks.
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)
This has been corrected.
Figure S1: This should become a main
Figure. This provides the clear data!
We added the data from this figur
e to the current figures 1 and 2.
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!
This is the survival percentage. The hetero
zygous not-yet-balanced
transgenic flies and
wt
flies
were raised on the food supplemente
d with puromycin or geneticin.
Line 652: 1 mg/mL NOT 0.1!!!!!
This has been corrected.