Genetic breakdown of a tet-off conditional lethality system for insect population control

ABSTRACT Genetically modified conditional lethal strains have been created to improve the control of insect pest populations damaging to human health and agriculture. However, understanding

the potential for the genetic breakdown of lethality systems by rare spontaneous mutations, or selection for inherent suppressors, is critical since field release studies are in progress.

This knowledge gap was addressed in a _Drosophila_ tetracycline-suppressible embryonic lethality system by analyzing the frequency and structure of primary-site spontaneous mutations and

second-site suppressors resulting in heritable survivors from 1.2 million zygotes. Here we report that F1 survivors due to primary-site deletions and indels occur at a 5.8 × 10−6 frequency,

while survival due to second-site maternal-effect suppressors occur at a ~10−5 frequency. Survivors due to inherent lethal effector suppressors could result in a resistant field population,

and we suggest that this risk may be mitigated by the use of dual redundant, albeit functionally unrelated, lethality systems. SIMILAR CONTENT BEING VIEWED BY OTHERS POPULATION SUPPRESSION

BY RELEASE OF INSECTS CARRYING A DOMINANT STERILE HOMING GENE DRIVE TARGETING _DOUBLESEX_ IN _DROSOPHILA_ Article Open access 14 September 2024 ENGINEERING MULTIPLE SPECIES-LIKE GENETIC

INCOMPATIBILITIES IN INSECTS Article Open access 08 September 2020 A TRANSGENIC FEMALE KILLING SYSTEM FOR THE GENETIC CONTROL OF _DROSOPHILA SUZUKII_ Article Open access 21 June 2021

INTRODUCTION Genetically modified (GM) conditional lethal insect strains have been developed to improve the sterile insect technique (SIT), with a focus on eliminating females during rearing

for sexing, and genetic sterilization of males due to inviable progeny1,2. Two systems achieve non-sex-specific and female-specific conditional lethality by using the _Escherichia coli_

tetracycline (Tet) resistance operon3 to suppress expression (Tet-off)4 of a lethal effector by feeding the antibiotic during rearing. One system, known as RIDL (release of insects carrying

a dominant lethal), uses toxic overexpression of the tet-transactivator (_tTA_) in a self-promoting driver/effector cassette to effect lethality in the absence of Tet5, while a Tet-off

embryonic lethality system (TELS) uses a developmentally regulated _tTA_ driver to promote the lethal effector expression of a pro-apoptotic cell death gene in embryos6. Both systems are

highly effective in small-scale experimental studies in several insect species7,8,9,10, and the RIDL system has advanced to field release studies for _Aedes aegypti_11,12. However, in

laboratory control studies for RIDL, survival in the medfly, _Ceratitis capitata_, and the yellow fever mosquito, _A. aegypti_, were reported at frequencies of 0.5% and 3.5%,

respectively7,9, while survival for the _Drosophila melanogaster_ TELS strain was approximately 0.01%6. These levels of initial F1 survival are likely due, primarily, to inherent “leakiness”

in the respective systems due to variable transgenic lethal effector expression or function, though heritable survival due to mutations in genetic components of the system have yet to be

reported. This is due, in part, to laboratory screening of lethality lines being limited to thousands of adults while spontaneous mutations in _Drosophila_, resulting in loss-of-function

alleles, have been reported in the range of 1–5 × 10−6 per locus per generation13. Thus, if mass-reared GM conditional lethal strains are released in the field at current levels of 106–108

insects/week for SIT14,15, there is a high likelihood that rare primary- or second-site mutations will occur resulting in lethal revertant survivors. This would result in the persistence of

GM insects in the field, and for some mutations (especially at second sites), revertants may be resistant to further control by the same or similar lethality system. Indeed, a recent report

of _A. aegypti_ populations in Brazil after the release of RIDL mosquitoes indicates introgression of RIDL strain genomic sequences in the surviving population16, which could be associated

with inheritance from non-mutant F1 escapers or with inherent resistance to the tTA lethal effector allowing hybrid survival. To experimentally assess the potential for breakdown of

Tet-suppressible lethality, we have tested the frequency and genetic basis of heritable lethal revertant survivors for a _D. melanogaster_ non-sex-specific TELS strain reared on Tet-free

diet. Despite not being a significant economically important species, its use as a genetic model provides relevant knowledge relating to the frequency and structure of spontaneous mutations,

and it is highly amenable to large-scale laboratory rearing. The binary driver/effector TELS system also provides the same primary _tTA_ target site for mutation that is necessary for

lethality in both the TELS and RIDL systems, thus allowing potential breakdown of both Tet-off conditional lethal systems to be evaluated within the same genomic context. In this respect,

after screening approximately 1.2 million zygotes on Tet-free diet, 20 heritable lethal revertant survivors were discovered, 7 of which are due to primary-site spontaneous mutations in the

driver/effector cassettes while the remaining 13 are due to second-site maternal effect suppressors. Primary-site survivors could still be controlled by the lethality system in the field,

though second-site lethal effector suppressors are likely to be resistant. We suggest that the risk of insects resistant to the primary lethality system survive and expand in a field

population may be mitigated by the concurrent use of a secondary redundant lethality system that is functionally independent. RESULTS EVALUATION OF A TET-OFF EMBRYO-SPECIFIC LETHALITY LINE

The TELS strain tested included the original _piggyBac_ lethal effector vector, pB{_3xP3-ECFP-5’HS4_>_5’HS4-TREp-hid__Ala5__-5’HS4_>_5’HS4_} (line M5.II) as described by Horn and

Wimmer6 (Fig. 1a). This vector is comprised of a phospho-mutated _hid_Ala5 pro-apoptotic cell death lethal effector gene17 linked to a tetracycline response element (TRE), surrounded by two

duplicate pairs of _5’ HS4_ chicken β-globin insulator sequences18, and the _3xP3-ECFP_ marker gene (Fig. 1b). The _piggyBac serendipity α_-regulated _tTA_ driver vector, pB{_PUb-DsRed.T3_,

_s1_-_tTA_} (line M18A), was modified from the original embryonic _tTA_ driver (_s2_-_tTA_)6,19 by use of the _s1_ promoter that has an additional 95 upstream promoter nucleotides (Fig. 1a)

and includes the _polyubiquitin_-regulated _DsRed.T3_ fluorescent marker (Fig. 1b). Both vectors were integrated into the second chromosome of independent _D. melanogaster white_- mutant

host strains, recombined for common linkage, and then inbred to create the double-homozygous driver/effector strain, DH-1. The applied use of TELS in the field would require the mass release

of double-homozygous P1 males reared on Tet-diet, with the expectation that their double-heterozygous F1 progeny would be inviable by the first larval instar in the absence of Tet. Thus F1

survivors would be considered to be putative lethal revertants due to spontaneous primary-site point mutations, deletions or insertions in the driver or effector cassettes, or second-site

modifiers or variations in the genome that would somehow suppress the otherwise functional TELS. However, fertile adult survivors expressing the DsRed driver (Red) and ECFP effector (Cyan)

cassette markers would require verification as true heritable lethal revertants from a backcross to _w_[m] that yields double-marked Red/Cyan F2 surviving progeny on Tet-free diet (Fig. 1b).

F1 SURVIVORS FROM LARGE-SCALE REARING Based on typical spontaneous mutation rates of 10−6 to 10−5 per locus in _Drosophila_13, we presumed that the recovery of a significant number of

heritable lethal revertants from the two loci TELS strain would require a minimal screen of ~106 F1 adults from large-scale rearing. This was achieved by establishing 6 control mating

groups, each having 100 homozygous DH-1 males crossed to 400 _w_[m] virgin females reared on permissive Tet-diet, with two successive transfers to fresh media, which yielded a total of

~15,000 F1 adult progeny from the three broods for each mating group (Table 1). The same crosses were then established for 80 experimental mating groups reared on non-permissive Tet-free

diet that would account, based on control matings, for the screening of approximately 1,200,000 double-heterozygous F1 progeny for adult survival. From the experimental matings, a total of

109 Red/Cyan (R/C) double-marked adults, for the driver and lethal effector vector cassettes (Fig. 1b), eclosed on Tet-free diet yielding an initial survival rate of 0.0091% (Table 1),

consistent with preliminary small-scale population survival rates. HERITABLE F2 LETHAL REVERTANTS IDENTIFIED FROM F1 SURVIVORS From the 109 F1 survivors, 88 remained viable allowing

backcross matings to _w_[m] to test for heritability of disruption or suppression of the lethality system (Table 1). Seventy-three F1 survivors were fertile, of which 20 resulted in R/C F2

progeny on Tet-free diet, indicating a heritable survival frequency of 18.3% relative to the initial number of survivors, and a survival frequency of 1.67 × 10−5 relative to the total number

of adults screened (Table 1). Seven of the 20 survivor lines exhibited an approximate 1:1 ratio of unmarked to double-heterozygous R/C marked F2 descendants on Tet-diet, with approximately

11% of the progeny expressing either the R or C marker (Table 2). Independent marker segregation was presumably due to recombination between the driver and lethal effector cassettes at their

respective chromosomal 2R loci of 56C8/9 and 53C4 (Supplementary Fig. 1), limited to the five F1 female lines due to the absence of male recombination in _D. melanogaster_20. These results

were consistent with complete disruption of the lethality system due to spontaneous primary-site genetic alterations in either, or both, the _tTA_ driver or lethal effector cassettes at a

frequency, relative to the population screened, of 5.8 × 10−6 events per generation. The significance of this frequency is supported by a power calculation21 to test for the optimal sample

size required for detecting primary-site genetic alterations based on the typical null mutation rate of 10−6. This resulted in a power of 0.93 at the 0.05 significance level for the screened

sample size of ~1,200,000 double-heterozygous F1 progeny (Supplementary Fig. 2). SECOND-SITE MATERNAL EFFECT LETHALITY SUPPRESSION LINES The 13 additional F2 R/C survivor lines were

heritable but exhibited significantly lower survival frequencies for R/C marked progeny relative to their unmarked siblings (Table 2), ranging from 1.4% to 32.4% survival, with 11 lines

being <11%. These variable R/C frequencies are more consistent with lethal suppression by second-site modifiers, either distantly or un-linked to the driver/effector cassettes, and

possibly the result of different suppressors. Notably, all of these lines originated from F1 females, although F2 survivors included both sexes, suggesting the possibility of maternal effect

heritability of lethal suppression. This was tested by backcrosses of R/C revertant males and females to _w_[m] flies in successive generations. For all 13 lines, F3 R/C survivors only

arose on Tet-free diet from maternal R/C F2 females, and not from R/C F2 males, while both F2 males and females gave rise to F3 R/C progeny on Tet-diet. For two lines (5-f1 and 14-f2) having

the highest levels of survival, the maternal effect was observed for 11 generations and confirmed by 10 to 12 individual backcross matings of F10 males and females to _w_[m] (Table 3).

MOLECULAR ANALYSIS OF LETHAL REVERTANT SURVIVOR LINES To determine the molecular integrity of the _tTA_ driver and lethal effector cassettes in the lethal revertant survivor lines, F2 R/C

adults for each line were inbred to homozygosity on Tet-free diet. PCR products from the _s1_-_tTA_ driver and _TREp_-_hid_Ala5 lethal effector cassettes from the revertant lines were

sequenced (Supplementary Table 2 and Supplementary Fig. 3) using adjacent and internal primers, though reliable PCR amplification of the highly repetitive _5’ HS4_ insulator sequences was

not possible. For some difficult sequences, more distant primers or TAIL-PCR were utilized. For the seven putative primary-site revertant lines, relatively short deletions were identified

within the _tTA_ and _hid_Ala5 genes, in addition to larger deletions and indels that included _TREp-hid_Ala5 and adjacent insulators (Fig. 2). Notably, mutations or alterations were not

evident in the driver or effector cassettes for any of the 13 putative modifier lines, consistent with second-site mutations or variations. For the primary-site mutations, two relatively

short spontaneous deletions were detected in lines 18-f1 and 45-f1. In line 18-f1, the _TREp-hid_Ala5 lethal effector sequence was unaltered, but a 27-bp deletion (nts 221–247 from the ATG)

and three adjacent nucleotide substitutions were identified in the _tTA_ driver coding sequence (Fig. 2 and Supplementary Fig. 4a). While the _tTA_ reading frame was maintained, we presume

the deleted and substituted peptides resulted in loss of tTA function. In line 45-f1, the _tTA_ driver was unaltered, but a 26-bp deletion (nts 27–52 from the ATG) occurred in the 1.2-kb

_hid_Ala5 lethal effector gene, resulting in an early frameshift that likely eliminated gene product function (Fig. 2 and Supplementary Fig. 4b). For the primary-site lines 21-f1 and 44-m1,

the _tTA_ driver sequence was unaltered, but a series of PCR primer pairs flanking or internal to the _5’HS4_5’HS4-TREp-hid__Ala5__-5’HS4_5’HS4_ lethal effector cassette failed to yield

amplicons, consistent with a large deletion or indel (see “Methods” and Supplementary Fig. 3b, c2–7). Control PCRs were positive for the unaltered presence of the _pB3’_ to _3xP3-ECFP_

sequence (AH1577–AH1636) in both lines (Supplementary Fig. 3b, c1) consistent with the expression of the marker gene. Internal PCR (AH1540–AH1652) indicated the presence of the effector

_pB5’_ sequence in both lines (Supplementary Fig. 3b, c8); however, PCR from _pB5’_ to adjacent 3’ genomic sequences were not positive (Supplementary Fig. 3b, c6) suggesting a transposition

or rearrangement of sequences. Partial insert sequences downstream from _ECFP-SV40_ were identified by TAIL-PCR, which included a 180-bp incomplete insert of unknown origin in line 21-f1

(Supplementary Fig. 5a), except for several short sequences identical to those found on the X and 2R chromosomes, the _3xP3_ promoter, and a _5’ HS4_ insulator. Line 44-m1 revealed a 540-bp

incomplete insert (Supplementary Fig. 5b) having high identity to telomeric HeT-A non-LTR retrotransposon sequences, with 87% identity to the X chromosome HeT element (ID: M81595.1)22

(Supplementary Fig. 6). Notably, retrotransposon insertions, among other mobile elements, have been associated with deletions that may provide the breakpoints for their insertion resulting

in spontaneous mutations in _Drosophila_ and other eukaryotes23,24. For the three remaining heritable survivor lines (9-f1, 62-f1, and 65-m2), PCR with primers in _ECFP_ and _pB_5’ and

_ECFP_-linked SV40 and _pB5’_ yielded 2.9 kb (Supplementary Fig. 3c7) and 2.6 kb (Supplementary Fig. 7a) products, respectively, that included partial _5’ HS4_ insulator sequences and the

absence of the internal _TREp_-_hid_Ala5 sequence. The actual sequence in this region was then inferred by _Afl_II and _Bgl_II digests of the 2.6-kb product that resulted in fragment sizes

consistent with a deletion of the lethal effector and two insulators (Supplementary Fig. 7a). If the two remaining insulator sequences were originally upstream and downstream of

_TREp_-_hid_Ala5, a parsimonious explanation for the deletion would be a single-strand inverted-loop meiotic pairing of the duplicate insulator tandem pairs having a single internal

recombination event. The resulting segregation products would thus consist of the two remaining insulators due to a circular DNA deletion of _5’HS4_TREphid__Ala5___5’HS4_ (Supplementary Fig.

 7b). DISCUSSION After nearly 20 years since Tet-off conditional lethality systems for pest insect population control were first reported5,6,25, a quantitative and structural assessment for

the spontaneous breakdown of the genetic components that comprise the _D. melanogaster_ Tet-off embryonic lethality system has now been described. After screening approximately 1.2 million

heterozygous TELS F1 adults on a restrictive Tet-free diet, 20 heritable revertant lethal survivors were recovered, yielding a frequency of 1.7 × 10−5 events per generation resulting in

genetic breakdown. However, primary-site mutations disrupting the _tTA_ driver or _hid_Ala5 lethal effector sequences were verified for seven of the survivor lines that included deletions

and indels of various length, including a retrotransposon-associated indel, resulting in a spontaneous primary-site mutation rate of approximately 5.8 × 10−6 events per generation. Given

that three of the lethal revertant lines were likely due to deletions resulting from _cis_-recombinations between the highly repetitive _5’ HS4_ chicken globin insulators, a more relevant

revertant frequency that includes only the remaining four mutant lines would be ~3.3 × 10−6 events per generation (Table 1). Since this mutation rate includes genetic alterations from both

primary-site targets, it more accurately reflects the overall rate of genetic breakdown for this specific lethality system, whereas the approximate spontaneous mutation rate per locus for

the 1.0-kb _tTA_ driver and 1.2-kb _hid_Ala5 lethal effector coding regions would be 0.83 × 10−6 and 2.5 × 10−6 events per locus, respectively. Various factors can affect the mutation rate

of specific loci, including the method of phenotypic screening and, indeed, our revertant lethal screens depended on detecting dual DsRed/ECFP marked survivors that would have missed

revertants due to deletions including critical sequences for either marker gene. With these caveats in mind, the observed spontaneous mutation rates for the driver and effector alleles

remain generally consistent with previous spontaneous mutation studies for a variety of alleles in _Drosophila_, having a mean spontaneous specific locus rate of 1–5 × 10−6 as estimated by

Ashburner13 and additional analyses24,26,27,28,29. The recovery of putative insulator recombinations also highlights an important consideration for the design of lethality systems since the

insulator pairs surrounding both driver and effector cassettes in the original TELS strain did improve lethal gene expressivity6. However, this was achieved at the cost of increasing the

primary-site mutation frequency by >40% (Table 1). Thus an important caveat for the use of the _5’ HS4_ insulator, and possibly other repetitive sequences, is the possibility for internal

_cis_-recombinations resulting in increased lethality strain breakdown due to deletions in a significant percentage of F1 progeny. The mutation recovered in this study most relevant to the

RIDL system, for which _A. aegypti_ RIDL lines have already been tested in open field release studies9,30, was the short internal deletion within the _tTA_ driver that acts as both driver

and lethal effector in the self-promoting RIDL system. Given the 0.83 × 10−6 mutation frequency of this locus from a single F1 TELS revertant, it is possible to expect a RIDL insect to

survive from at least every 1.2 million F1 embryos oviposited in the field. Not unexpectedly, the additional _hid_Ala5 effector cassette critical for TELS function, compared to RIDL,

provides an additional target for mutation and thus a higher frequency for disruption of lethality based on this study. In addition to primary-site mutations, this study revealed a class of

second-site maternal effect variations, found in 13 of the revertant lethality lines, that effectively suppress expression or function of the _hid_Ala5 pro-apoptotic lethal effector gene.

While it remains to be determined how ectopic _hid_Ala5 could be negatively influenced by a maternally derived factor, there is precedence for a maternally inherited mitochondrial role in

pro-apoptotic gene product function. In mammalian systems, cytochrome c is converted into holocytochrome c by a heme attachment within the mitochondrial intermembrane space that facilitates

pro-apoptotic caspase activation resulting in cell death31. While this specific function has not been observed in invertebrates, a mitochondrial role in _Drosophila_ cell death has been

inferred by the inhibition of apoptosis when pro-apoptotic protein localization within the mitochondria is blocked32. Although the observed maternal effect may be limited to the particular

_D. melanogaster w_[m] host strain tested, if ectopic pro-apoptosis is similarly subject to suppressor gene function due to variation in a small subset of an insect population, then the

potential for selection of these suppressors should be a concern for the use of pro-apoptotic lethal effectors. Indeed, the _hid_ gene has already been tested as a TELS conditional lethal

effector in four insect pest species8,10,33,34,35, some of which are in consideration for release. Another mechanism for maternal effect suppression, since the _sry-alpha_ promoter functions

transiently in early embryogenesis19, is a pre-zygotic maternal contribution of molecules that suppress ectopic _tTA_ or _hid_Ala5 expression or function at that developmental period. Since

the RIDL system relies on the toxic accumulation of tTA protein throughout development for lethality, neither of these types of maternal effect suppression may be relevant to RIDL, though

it is possible that tTA toxicity ultimately results in induced apoptosis. More broadly, however, is the realization that regardless of the lethal effector employed for population control, or

its mode of action, it may be subject to suppression by a pre-existing inherent variation in the targeted field population36 or a newly acquired modifier mutation in the mass-reared

lethality strain. In either case, such effects could have a significant impact on lethality system effectiveness and implications for its use in population control programs. Importantly,

this would require evaluation by contained large-scale preliminary testing as performed in this study but, ideally, using field population adults for P1 matings. Another important

consideration is that primary-site revertant survivors, due to _cis_-acting mutations, of both TELS and RIDL systems should still be susceptible to control by the same lethality system. In

contrast, a dominant-acting second-site suppressor of lethal effector expression or function, due to _trans_-acting modifiers, would more likely result in GM insects resistant to the

lethality system, and relatively small numbers of GM resistant survivors could be expected to rapidly re-populate an ecosystem where the susceptible population has been suppressed. Certain

factors will affect the rate of introgression of the resistant population and its ability to reach fixation. These include the number of suppressor alleles selected for and whether they are

recessive lethal, recessive or dominant fitness costs, maternal effect heritability, and the proportional population density of successive releases among other variables having positive or

negative effects37. All of the TELS suppressor lines were inbred to homozygosity (and share the same mitochondrial genome), and none exhibited recessive lethality or apparent semi-lethality

due to compromised fitness. Nevertheless, unless the negative costs to resistance are considerable, the eventual establishment of a lethality-resistant population may be expected from

programs that rely on continuous mass releases (especially preventative release programs38) in the absence of an efficient secondary control program. The potential for resistance to

bi-sexual and female-specific RIDL release programs, and its spread within a population, has been modeled taking into account many of the variables affecting resistance introgression for

this system37, but this remains theoretical in the absence of large-scale population tests and knowledge of the actual basis for tTA lethality. The importance of this knowledge for the RIDL

system, in particular, is heightened by recent studies showing significant introgression of genomic sequences from the _A. aegypti_ OX513A RIDL strain into the native Brazilian (Jacobina)

mosquito population 6 months to 2.5 years after the initiation of lethal strain releases16. Notably, sequences related to the lethality system were not reported, nor could they necessarily

be identified by the single-nucleotide polymorphism analysis performed, and the sequences identified could have simply been inherited from non-mutant escapers that comprised >70% of the

F1 TELS survivors in our tests. But until this is resolved, one explanation for at least some of the observed introgression is survival due to inherent resistance to the tTA lethal effector.

Alphey et al. suggest early and effective monitoring of release programs to detect resistance in time for remediation37, which is certainly important on a continuing basis, as it is for

most current SIT programs38. However, the additional use of preliminary large-scale contained population tests previous to a new release program would provide the advantage of assessing the

potential for resistance and a determination as to whether concurrent or sequential releases of a secondary control system, or other modifications, would be necessary. Considering the

possibility that resistance to current insect conditional lethality systems might occur, strategies to prevent or mitigate the survival of lethality-resistant individuals have been

considered. Two strategies are based on the potential for redundant, yet independent, dual lethality (or reproductive sterility) systems mitigating the survival of revertant individuals in a

mass release program39,40. Thus, based on the multiplicative law of probability [_P(AB)_ = _P(A)P(B)_], an approximate 10−6 lethal revertant frequency estimate for each system (_A_ and

_B_), as demonstrated here for a _Drosophila_ TELS strain, could result in a frequency as low as ~10−12 for both to fail concurrently in the same genome, presuming that the lethality systems

are functionally independent and do not differentially affect host physiology and fitness. In application, at a modest rate of release for current SIT programs (~107 males per week), use of

a single lethality system might result in ≥100 heritable revertant survivors per week assuming each male yields 10 F1 progeny, whereas the coincident breakdown of a dual lethality strain

might yield a single revertant survivor only after several decades. From these considerations, we would suggest that large-scale population screens for spontaneous mutations or pre-existing

genetic variations, resulting in the genetic breakdown of conditional lethal systems developed for mass field release, be a critical component in the risk assessment evaluation of these

programs. This could be achieved most efficiently in rearing facilities where mass-reared P1 males and females are mated under non-permissive conditions for survival, whereupon the frequency

and structure of mutations and suppressor/modifiers could be evaluated in surviving F1 progeny. Given that most current facilities for SIT mass-rearing generate a minimum of 107–108 insects

per week, a more significant and informative analysis could be achieved in insects of interest relative to large-scale laboratory studies with _Drosophila_. Importantly, host females for P1

matings should come from the targeted field populations to test for inherent suppressors or modifiers of the lethality system as a prelude to actual releases, though such evaluations may

prove to be less critical with the use of redundant lethality systems. This study has focused on a conditional lethal strain created by transposon-mediated transformation, yet most insect

genetic modifications should be subject to the same types of alteration, disruption, or suppression regardless of the methodology used to create them. However, the frequency of specific

types of mutations and suppressor/modifiers may vary with the genomic structure of the host species (e.g., genome size, chromatin structure, repetitive DNA content, etc.) and the specific

modification. This is especially relevant to the variety of manipulations envisioned by gene editing, and the development of gene drive (GD) systems in particular. Indeed, despite the great

potential for GD systems to modify or replace highly pestiferous insect strains in the field, it is well recognized that control or containment systems must be integrated into GD systems

before the field release of these self-propagating strains can be considered41,42. A variety of containment strategies proposed, and in some cases tested, include drives that are

self-limiting43 such as multi-component daisy-chained drives44, split drives45,46 and spatially restricted drives45 and small molecule-dependent drives47, among others. However, similar to

the lethality strain breakdown demonstrated here all of these strategies should be subject to mutation and modification and, potentially, at higher frequencies due to multi-generational GD

strategies and larger target sites for some. Thus it would seem imperative that similar large-scale studies for GD integrity be initiated for systems already created in _Drosophila_, and if

warranted, redundancy for GD containment be considered and tested for mitigation. Similarly, the desired systems for introgression should be equally subject to genetic breakdown for which

redundancy may also be required, at the risk of a GD population regaining its pestiferous function, or losing its beneficial function, and expanding in nature. In this respect, and in

general, care should be taken in the design of all genetic modifications for specific applications, in terms of understanding and testing the potential for mutation, suppression, and the

possible need for redundancy to maintain strain stability. METHODS INSECT STRAINS, REARING, AND SCREENING All _D. melanogaster_ transgenic lines, the white-eyed _w_[m] mutant line, and

large-scale matings were reared under standard laboratory conditions at 25 °C and 60% humidity on a 12-h light:12-h dark cycle48. Flies were anesthetized with CO2 for screening, fly

collections, and matings. Transgenic flies were screened by epifluorescence microscopy using a Leica MZFLIII fluorescence microscope and filter sets, TxRed for DsRed (ex: 560/40; em: 610 LP)

and CFP for ECFP (ex: 436/20; em: 480/40). The University of Florida Institutional Biosafety Committee provided oversight and regulatory approval for the creation and rearing of the GM _D.

melanogaster_ strains used in this study. TET-OFF _TTA_ DRIVER AND _HID_ _ALA5_ LETHAL EFFECTOR LINES The _D. melanogaster_ transgenic embryonic _tTA_ driver M18A strain was transformed with

the _piggyBac_ transgene vector, pBXL{_attP_PUbDsRed.T3_fa_s1-tTA_a_} (GenBank accession no.: MT453110). The vector was created by isolating the _s1-tTA_ driver sequence, as an _Asc_I

fragment, from pBac{_3xP3-EYFPafm; s1-tTA_}6 and inserting it into the _Asc_I site in pBXL{_attP_PUbDsRed_fa_}10. Unlike the _tTA_ driver vector used in the original embryonic lethality

strain6, the _serendipity α s1_ promoter has an additional 95 upstream nucleotides compared to the _s2_ promoter, the _5’ HS4_ insulator sequences that surround the _s2-tTA_ driver construct

are eliminated, an _attP_ recombination site is added for subsequent phiC31-mediated integrations, and a _polyubiquitin_-regulated _DsRed_ (_PUbDsRed_) whole-body marker replaces the

eye-specific _3xP3-EYFP_ marker. The vector was transformed into the _w_[m] strain by _piggyBac_-mediated germline transformation49 using a 500 ng/µl transgene vector plasmid and 200 ng/µl

_phspBac_ transposase helper plasmid mixture for preblastoderm injections. The transgenic M5.II lethal effector strain,

pBac{_3xP3-ECFPaf_5’HS_>_5’HS_>_TREp-hid_Ala>_5’HS_>_5’HS4_} was created and kindly provided by Horn and Wimmer6. This effector vector is marked with the eye-specific marker,

_3xP3-ECFP_, and carries a Tet-suppressible lethal effector construct having the phospho-mutated _hid_Ala5 pro-apoptotic cell death gene linked to a TRE. In the absence of tetracycline, the

tet-transactivator (_tTA-VP16_ originally isolated from the pTet-Off Vector plasmid; Takara BIO USA, Inc.) from the driver construct binds to the _tetO_ operator sequences within the TRE to

promote _hid_Ala5 transcription. Included within the vector construct is an _attP_ recombination site for subsequent phiC31-mediated integrations, and two tandem repeat 2.4 kb _5’ HS4_

chicken β-globin insulator repeat sequences flank both sides of the effector construct. VECTOR INSERTION SITE MAPPING The effector cassette in strain M5.II was previously mapped to

chromosome 2 but not to a specific locus6. Segregation patterns from the mating of the M18A and M5.II homozygous strains and subsequent inbreeding suggested that the effector cassette in

M18A also maps to chromosome 2. Therefore, the M18A and M5.II homozygous strains were inter-mated and double-heterozygous progeny were outcrossed to _w_[m] to select recombinant progeny

having the driver and effector cassettes linked on the same chromosome. Recombinants were inbred to create the double-homozygous driver/effector line, DH-1, that was mated to the _D.

melanogaster white_ mutant strain _w_[m] in large-scale rearing (Fig. 1). Both the effector and driver cassettes in DH-1 were mapped to specific loci by inverse PCR insertion-site sequencing

to one arm of each vector. Inverse PCR10 was performed by digestion of the DH-1 strain with _Ava_II or _Rsa_I and amplifying circularized fragment sequences with primer sets AH1537–AH1579

for the driver cassette 3’ flanking sequence and AH1566–AH1542 for the effector cassette 5’ flanking sequence (Supplementary Table 2). The flanking genomic sequences were subjected to a

BLASTn search of the _D. melanogaster_ genome assembly indicating an insertion of the driver cassette at the _Tab2_ gene (CG7417) and the effector cassette at the _Sema2b_ gene (CG33960),

both on chromosome 2R (Supplementary Fig. 1). However, while both transgenes inserted into a TTAA site (duplicated upon insertion) consistent with all _piggyBac_ insertions, the genomic

sequences immediately flanking both the 5’ and 3’ _piggyBac_ termini were not present in the _D. melanogaster_ NCBI database. The driver cassette 5’ flanking sequence and the effector

cassette 3’ flanking sequence were then re-confirmed by direct PCR using primer sets, AH1536–AH1585 and AH1543–AH1570 (Supplementary Table 2), to internal vector and genomic sequences and

were also confirmed by direct internal sequencing in the genomes of _w_[m] and another _white_ strain, _w_1118. A straightforward explanation for the anomalous sequences is that many _white_

strains have been maintained as inbred laboratory colonies for many years (at least 60 years for _w_1118) and may have been subjected to genetic drift from the wild-type strains used for

recent whole-genomic sequencing. TETRACYCLINE TESTS To generate an embryonic lethality strain exhibiting the lowest survival frequency on Tet-free diet and the highest level of survival on

the lowest Tet-diet concentration (considering potential fitness costs at high concentrations), F1 survival of progeny from small-scale matings of DH-1 males and _w_[m] females was first

tested on Tet-free diet and diet supplemented with tetracycline at concentrations of 10, 50, and 100 μg/ml (Supplementary Table 1). Eggs were collected on medium containing the corresponding

Tet concentration, with pupae and adult eclosion rates recorded. Since no significant difference was observed in eclosion rates between 10 and 50 μg/ml, a concentration of 20 μg/ml

tetracycline was used in media for large-scale rearing tests. LARGE-SCALE REARING TESTS For large-scale rearing tests, _w_[m] virgin females and double-homozygous DH-1 males were collected

<8 h after eclosion and reared separately for 2 days before P1 mating of 400 females and 100 males for brood I in 1-liter jars on 20 μg/ml Tet-diet for control tests and Tet-free media

for experimental tests. Brood I adults were transferred to fresh media after 4 days for brood II, with another transfer after 4 days for brood III, with brood III adults discarded after 6

days. Six control matings were performed to determine the mean total number of surviving adults from the three broods for each mating, which were counted individually. This resulted in a

mean number of 15,001 ± 389 standard error of the mean F1 adults per mating (Table 1). A total of 80 experimental matings (240 jars) were screened for pupal survivors that were collected and

placed in individual vials with Tet-free media and monitored for adult eclosion. F1 adults were counted and screened for the fluorescent markers, and survivors double-marked for the driver

(DsRed) and effector (ECFP) cassettes were backcrossed individually to _w_[m] adults on Tet-free diet for 3–5 days. F1 female survivor matings were then transferred to Tet-diet for 4–5 days,

while three _w_[m] females mated to F1 male survivors were left on Tet-free diet and three females were transferred to Tet-diet. Rearing on Tet-diet ensured that a line from fertile F1

adults was maintained and provided a control for the expected percentage of Red/Cyan F2 progeny. If F2 R/C progeny survived on Tet-diet, but not on Tet-free diet, then the F1 individual was

considered to be a non-heritable survivor and the line was discarded, but if the F2 R/C progeny survived on Tet-free diet, it was considered to be a putative heritable lethal revertant

survivor. F2 R/C males and females were inbred to create double-homozygous lines by single sib-pair matings with selection of the strongest fluorescing progeny (presumed to be homozygous)

for matings in successive generations until all progeny were strongly fluorescent, typically by the third generation. Homozygosity was then verified by backcrosses to _w_[m] that resulted in

100% heterozygous progeny. HERITABLE LETHAL REVERTANT SURVIVOR LINES Double-heterozygous F2 progeny, expressing both DsRed and ECFP (R/C) fluorescent markers, from backcrossed F1 survivors

reared on Tet-free diet, were considered to be lethal revertants due to a heritable mutation or variant modifier allele. Primary-site mutations in either the driver or effector sequences,

causing breakdown of the lethality system, were expected in approximately one-half of the F2 progeny with the remaining progeny consisting of unmarked _w_[m] flies and single-marked progeny

resulting from recombination between the markers from F1 female survivors (since recombination does not occur in _D. melanogaster_ males). Second-site modifiers were expected to have a

dominant-acting function and would also be expected to suppress lethality in one-half of the double-heterozygous F2 progeny only if the alleles had relatively close second chromosome linkage

to the driver/effector cassettes and were fully expressed. Lower frequencies of F2 survival would be expected if second-site modifiers were unlinked or distantly linked to the D/E cassettes

or were variably expressed possibly due to independent suppressor alleles. F1 and successive generation backcrosses on Tet-diet provided a control for the expected frequencies of marked and

unmarked F2 progeny. Heterozygous lethal revertant survivor lines were maintained by continuing backcrosses to _w_[m] on Tet-diet, in addition to inbred homozygous lines maintained on

Tet-free diet for molecular analysis. Putative second-site survivor lines, based on low F2 survival frequencies, were tested for putative maternal effect modifiers by three replicate single

pair backcrosses of F2 double-heterozygous males and females to _w_[m] reared on Tet-free diet for 3 days and then Tet-diet for 3 days. Adult progeny on each diet were screened for markers,

with the ratio of double-marked to unmarked F3 descendants compared between the two diets. F3 double-heterozygous survivors arising only from F2 females, and not from F2 males, was

considered to be consistent with a second-site maternal effect modifier that was verified by continued backcrosses in successive generations and verifying that the sequence integrity of the

driver and effector cassettes was unaltered (Table 2 and Supplementary Fig. 3a, b). For two putative maternal effect lines, 5f1 and 14f2, exhibiting relatively higher frequencies of

survival, backcrosses were continued for 10 generations at which time 10–12 replicate single pair backcrosses of both F10 double-heterozygous males and females was performed (Table 3).

MOLECULAR ANALYSIS OF LETHAL REVERTANT SURVIVOR LINES The _s1-tTA_ driver and _TREp-hid_Ala5 lethal effector cassette sequences were isolated by PCR for all heritable homozygous survivor

lines and sequenced to identify genetic alterations that would eliminate or diminish lethal effector expression. Primer pairs AH1601–AH1631 and AH1554–AH1582 were used to amplify the driver

cassette, while the primer pairs AH1616–AH1562 and AH1610–AH1581 were used to amplify the effector cassette (Supplementary Table 2 and Supplementary Fig. 3). Another primer pair,

AH1566–AH1615, that amplified the effector cassette and the flanking _5_′ _HS4_ insulator tandem repeat sequences was used if the effector cassette could not be amplified with the more

proximal primers. Owing to the highly repetitive _5_′ _HS4_ sequence, the use of primers within the sequence or PCR through the sequence with external primers was not always reliable. PCR

cycling conditions were: 30 s at 98 °C; 5 cycles of 10 s at 98 °C, 30 s at 58 °C, 2 min at 72 °C; 30 cycles of 10 s at 98 °C, 30 s at 55 °C, 2 min at 72 °C; and a final extension for 2 min

at 72 °C. If primer pairs flanking the driver or effector cassettes failed to generate amplicons, possibly due to large indel aberrations, TAIL-PCR was performed according to Liu and

Whittier50 using specific primers (SPs) SP1 (AH1557), SP2 (AH1635), SP3 (AH1638), and an arbitrary degenerate primer (AH1117). All PCR reactions and PCR product digestions resulting in the

same profile or sequence were replicated a minimum of two or more times. REPORTING SUMMARY Further information on research design is available in the Nature Research Reporting Summary linked

to this article. DATA AVAILABILITY The data and materials that support the findings of this study are available from the corresponding author upon reasonable request. The _piggyBac_ driver

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Article  CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We thank Dr. Carsten Horn and Dr. Ernst Wimmer for sharing the M5.II lethal effector strain and the _s1-tTA_ driver

construct plasmid, and Dr. Rodney Nagoshi and Dr. Daniel Hahn for comments on the manuscript. This project was supported by the USDA-Biotechnology Risk Assessment Program competitive grant

no. 2015-33522-24094 from the USDA National Institute of Food and Agriculture (to A.M.H.) and the Emmy Noether Program SCHE 1833-1/1 of the German Research Foundation (to M.F.S.). This study

benefited from discussions at meetings for the Coordinated Research Project, “Comparing Rearing Efficiency and Competitiveness of Sterile Male Strains Produced by Genetic, Transgenic or

Symbiont-based Technologies,” funded by the International Atomic Energy Agency (IAEA). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * State Key Lab for Conservation and Utilization of

Subtropical Agro-Biology Resources, Guangxi University, 100 Daxuedong Road, 530005, Nanning, Guangxi, China Yang Zhao * Center for Medical, Agricultural and Veterinary Entomology, USDA/ARS,

1700 SW 23rd Drive, Gainesville, FL, 32608, USA Yang Zhao & Alfred M. Handler * Department of Insect Biotechnology in Plant Protection, Justus-Liebig University Gießen, Winchesterstr. 2,

35394, Gießen, Germany Marc F. Schetelig Authors * Yang Zhao View author publications You can also search for this author inPubMed Google Scholar * Marc F. Schetelig View author

publications You can also search for this author inPubMed Google Scholar * Alfred M. Handler View author publications You can also search for this author inPubMed Google Scholar

CONTRIBUTIONS Y.Z. and A.M.H. designed experiments and analyzed data; Y.Z. and M.F.S. performed research; and all authors wrote the paper. CORRESPONDING AUTHOR Correspondence to Alfred M.

Handler. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PEER REVIEW INFORMATION _Nature Communications_ thanks Bruce Hay and the

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Handler, A.M. Genetic breakdown of a Tet-off conditional lethality system for insect population control. _Nat Commun_ 11, 3095 (2020). https://doi.org/10.1038/s41467-020-16807-3 Download

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