Genetically engineered insects with sex-selection and genetic incompatibility enable population suppression
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Curated by eLife
Evaluation Summary:
This paper is of interest to entomologists caring for genetic pest control or molecular biologists following synthetic biology. The authors describe a fruit fly strain that combines constructs that establish both repressible female-lethality and genetic incompatibility based on CRISPR transactivation. They show that this strain has high penetrance for these two traits and that it can suppress wild-type flies when released into cycling cage populations. The paper is thus a neat technology demonstration for a genetic control strategy possibly applicable to other insects including pests or disease vectors.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Abstract
Engineered Genetic Incompatibility (EGI) is a method to create species-like barriers to sexual reproduction. It has applications in pest control that mimic Sterile Insect Technique when only EGI males are released. This can be facilitated by introducing conditional female-lethality to EGI strains to generate a sex-sorting incompatible male system (SSIMS). Here, we demonstrate a proof of concept by combining tetracycline-controlled female lethality constructs with a pyramus -targeting EGI line in the model insect Drosophila melanogaster . We show that both functions (incompatibility and sex-sorting) are robustly maintained in the SSIMS line and that this approach is effective for population suppression in cage experiments. Further we show that SSIMS males remain competitive with wild-type males for reproduction with wild-type females, including at the level of sperm competition.
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Author Response:
Reviewer #2:
Weaknesses:
The competition assay used in this study may not truly reflect the competitiveness of SSIMS males. The mating assay used 20 virgin WT females and 4 males (including both WT and SSIMS), resulting 5:1 sex ratio so the males are not really competing for females. A more competitive ratio (such as WT females: WT males: SSIMA males at 1:1:1) should be designed to address this. Also, the sperm competition assay mixed the mated WT females with SSIMS males for 12 days, allowing plenty of time for the females to remate with these males. Therefore, it's more like a sperm replacement assay rather than competition assay. The authors should either repeat it with a strict time control, or soften their statements for sperm competitiveness.
We have repeated the experiment at a 1:1:1 ratio as suggested. The …
Author Response:
Reviewer #2:
Weaknesses:
The competition assay used in this study may not truly reflect the competitiveness of SSIMS males. The mating assay used 20 virgin WT females and 4 males (including both WT and SSIMS), resulting 5:1 sex ratio so the males are not really competing for females. A more competitive ratio (such as WT females: WT males: SSIMA males at 1:1:1) should be designed to address this. Also, the sperm competition assay mixed the mated WT females with SSIMS males for 12 days, allowing plenty of time for the females to remate with these males. Therefore, it's more like a sperm replacement assay rather than competition assay. The authors should either repeat it with a strict time control, or soften their statements for sperm competitiveness.
We have repeated the experiment at a 1:1:1 ratio as suggested. The new results are reported in the revised Figure 3. It is not clear to us how the timing of the mating experiments differentiates sperm competition versus sperm displacement, but we agree that sperm displacement is a better term to describe what we did. We have repeated the sperm displacement experiment with strict time control based on several published literature precedents and describe the results in the revised manuscript.
Some necessary information or statistics are not shown or mis-presented. For example, the alternative splicing diagram in Figure 1c likely was taken from the original transformer gene, but here it's the tTA gene so the male intron should be removed since it's not in the construct;
We have revised text in the manuscript to clarify some of these points. First of all, the male intron is still in the construct, even though we fused the intron to the tTA gene. The alternative splicing between males and females is caused by use of alternative 5' splice sites, which means the intron that is spliced out in males is just a smaller section of the intron that is spliced out in females. Use of an alternative 5' splice site in males means that a protein-coding sequence with multiple stop codons is incorporated to the mature mRNA. We do not support the precise splicing mechanism with empirical data in this paper, but this has been done in a number of previous publications (https://doi.org/10.1016/j.ibmb.2014.06.001; https://doi.org/10.1371/journal.pone.0056303).
Because the construct works as predicted (100% female lethality in the absence of tetracycline), and we did not change the genetic design in a way that would impact the mechanism of female lethality, we think there is little reason to believe that the splicing is occurring in a different way.
the panels of Figure 2 were not consistent to the legend and confusing; the statistics for different tetracycline concentration tests were not shown in Figure 2 or text to answer their hypothesis "(to) optimize rearing of SSIMS stock, …..we titrated Tet in the food";
We re-wrote the text describing Figure 2 to make the results more clear. We clarified in the legend that the symbol *** signifies p<0.0001 (we were not trying to imply that all experiments had this level of significance, only the ones marked with the *** symbol in the figure). We removed the word ‘optimize’ from the main text. Optimization was not the true aim of the experiment, and as the review points out, we did not statistically determine an optimal concentration of Tet. Our main goal was to show a dose- dependent response in the number of females surviving on Tet-free medium, which the data supports and which does not require statistical support.
Figure 3b shows 5-8 day old females were used but in the text it's 5-6 day, and it didn't mention the duration of the first crossing and time lag until the second crossing which are critical in such experiments; the conclusion and statistics for Figure 3c among tests with mixed males should also be mentioned.
We have corrected the figure (now Figure 3c) to indicate that the females were 5-6 days old. The first mating was for 5-6 days and there was no lag time between being co-housed with different males. We have performed multiple new experiments in revision that have been added to Figure 3. We have revised the discussion of these new experiments (and how they relate to the originally performed experiments) in the revised submission.
The discussion is largely towards the merits of SSIMS but missing some key points that might decide how it can be translated into applications or transferred to other species. First, the actual basis for tTA lethality that employed in this study is still unknown which is subject to suppression by a pre-existing inherent variation in the targeted field population. The very phenomenon may also be true for any gene-overexpression-based lethality including EGI lines generated here. Second, the complete penetrance observed from the relatively small sample size here can be hardly used to predict field or mass-rearing condition. Previous study showed that mutations in such lethal construct could occur at a one out of 10,000 frequency, and typical SIT program release millions of sterile insects every week. Third, while the authors claimed SSIMS is "one of the most complex engineered systems in insects", they also proposed that "the genetic design is likely to be portable to other species" without mention any potential obstacles along the way. Therefore, efforts should be made to give full picture of SSIMS including rain and sunshine.
We have added discussion of possible failure modes for this genetic biocontrol approach to the discussion section. We have also added text to discuss how the complexity of SSIMS is a potential obstacle to its translation to non-model organisms.
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Evaluation Summary:
This paper is of interest to entomologists caring for genetic pest control or molecular biologists following synthetic biology. The authors describe a fruit fly strain that combines constructs that establish both repressible female-lethality and genetic incompatibility based on CRISPR transactivation. They show that this strain has high penetrance for these two traits and that it can suppress wild-type flies when released into cycling cage populations. The paper is thus a neat technology demonstration for a genetic control strategy possibly applicable to other insects including pests or disease vectors.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their …
Evaluation Summary:
This paper is of interest to entomologists caring for genetic pest control or molecular biologists following synthetic biology. The authors describe a fruit fly strain that combines constructs that establish both repressible female-lethality and genetic incompatibility based on CRISPR transactivation. They show that this strain has high penetrance for these two traits and that it can suppress wild-type flies when released into cycling cage populations. The paper is thus a neat technology demonstration for a genetic control strategy possibly applicable to other insects including pests or disease vectors.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The authors describe a fruit fly strain that combines constructs that establish both repressible female-lethality and genetic incompatibility based on CRISPR transactivation. They show that this strain has high penetrance for these two traits and that it can suppress wild-type flies when released into cycling cage populations. The paper is thus a neat technology-demonstrator for a genetic control strategy possibly applicable to other insects such as pests or vectors.
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Reviewer #2 (Public Review):
Ambuj Upadhyay et al. developed a novel genetic control strategy named as sex-sorting incompatible male system (SSIMS) for insect pests and demonstrated it in the model insect Drosophila melanogaster. This study is based on the previous works from the same group which developed the genetic incompatibility based on the lethal overexpression of a target gene and conditional female lethality based on the Tetracycline(Tet)-off system. The authors successfully generated viable SSIMS flies that contain transgenes for both genetic incompatibility and female lethality, and used them for female lethality, male competitiveness and laboratory cage trials. The design of SSIMS system is highly complex but well executed by the authors, and the results from the cage trials were promising which suggest this could be an …
Reviewer #2 (Public Review):
Ambuj Upadhyay et al. developed a novel genetic control strategy named as sex-sorting incompatible male system (SSIMS) for insect pests and demonstrated it in the model insect Drosophila melanogaster. This study is based on the previous works from the same group which developed the genetic incompatibility based on the lethal overexpression of a target gene and conditional female lethality based on the Tetracycline(Tet)-off system. The authors successfully generated viable SSIMS flies that contain transgenes for both genetic incompatibility and female lethality, and used them for female lethality, male competitiveness and laboratory cage trials. The design of SSIMS system is highly complex but well executed by the authors, and the results from the cage trials were promising which suggest this could be an alternative method to species-specific pest control approaches such as insect sterile technique (SIT).
Strengths:
During the last seven decades, SIT has been used to battle insect pests worldwide. However, there are some factors may limit its application in certain species. Specifically, two major concerns are the fitness penalty that induced by radiation and inefficient control effect caused by bi-sex releasing. To compensate these two aspects, very often a large number of sterile insects are needed for releasing which considerably increased the running costs of SIT program. In some cases, bi-sex release may not be allowed since the harmful effects from released females cannot be tolerated. This study developed insect strains that could potentially address these two problems simultaneously: radiation is no longer needed due to the engineered genetic incompatibility (although the fitness of such insects needs to be further evaluated towards field application) and male-only releasing is possible due to the conditional female lethality. While female lethal strains have been generated in some insect species, this is one of the first studies that incorporating male sterility (mimicked by incompatibility) and female lethality into the same insect strain.
Due to the binary design for lethality, SSIMS created a redundant system that kill insects in two different mechanisms. This could be particular important to slow the resistance to such strain in mass-rearing or field scenarios.
The results from the cage trials supported some major claims of the authors. Specifically, releasing SSIMS caused collapse of wildtype (WT) population, and SSIMS females died out after the releasing stopped, suggesting that SSIMS can be an effective but also self-limited strategy which might be favoured by regulation.
Weaknesses:
The competition assay used in this study may not truly reflect the competitiveness of SSIMS males. The mating assay used 20 virgin WT females and 4 males (including both WT and SSIMS), resulting 5:1 sex ratio so the males are not really competing for females. A more competitive ratio (such as WT females: WT males: SSIMA males at 1:1:1) should be designed to address this. Also, the sperm competition assay mixed the mated WT females with SSIMS males for 12 days, allowing plenty of time for the females to remate with these males. Therefore, it's more like a sperm replacement assay rather than competition assay. The authors should either repeat it with a strict time control, or soften their statements for sperm competitiveness.
Some necessary information or statistics are not shown or mis-presented. For example, the alternative splicing diagram in Figure 1c likely was taken from the original transformer gene, but here it's the tTA gene so the male intron should be removed since it's not in the construct; the panels of Figure 2 were not consistent to the legend and confusing; the statistics for different tetracycline concentration tests were not shown in Figure 2 or text to answer their hypothesis "(to) optimize rearing of SSIMS stock, .....we titrated Tet in the food"; Figure 3b shows 5-8 day old females were used but in the text it's 5-6 day, and it didn't mention the duration of the first crossing and time lag until the second crossing which are critical in such experiments; the conclusion and statistics for Figure 3c among tests with mixed males should also be mentioned.
The discussion is largely towards the merits of SSIMS but missing some key points that might decide how it can be translated into applications or transferred to other species. First, the actual basis for tTA lethality that employed in this study is still unknown which is subject to suppression by a pre-existing inherent variation in the targeted field population. The very phenomenon may also be true for any gene-overexpression-based lethality including EGI lines generated here. Second, the complete penetrance observed from the relatively small sample size here can be hardly used to predict field or mass-rearing condition. Previous study showed that mutations in such lethal construct could occur at a one out of 10,000 frequency, and typical SIT program release millions of sterile insects every week. Third, while the authors claimed SSIMS is "one of the most complex engineered systems in insects", they also proposed that "the genetic design is likely to be portable to other species" without mention any potential obstacles along the way. Therefore, efforts should be made to give full picture of SSIMS including rain and sunshine.
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