A genome-wide association study implicates the olfactory system in Drosophila melanogaster diapause-associated lifespan extension and fecundity

Reviewer #1 (Public Review):

Summary:

The paper begins with phenotyping the DGRP for post-diapause fecundity, which is used to map genes and variants associated with fecundity. There are overlaps with genes mapped in other studies and also functional enrichment of pathways including most surprisingly neuronal pathways. This somewhat explains the strong overlap with traits such as olfactory behaviors and circadian rhythm. The authors then go on to test genes by knocking them down effectively at 10 degrees. Two genes, Dip-gamma and sbb, are identified as significantly associated with post-diapause fecundity, and they also find the effects to be specific to neurons. They further show that the neurons in the antenna but not the arista are required for the effects of Dip-gamma and sbb. They show that removing the antenna has a diapause-specific lifespan-extending effect, which is quite interesting. Finally, ionotropic receptor neurons are shown to be required for the diapause-associated effects.

Strengths and Weaknesses:

Overall I find the experiments rigorously done and interpretations sound. I have no further suggestions except an ANOVA to estimate the heritability of the post-diapause fecundity trait, which is routinely done in the DGRP and offers a global parameter regarding how reliable phenotyping is. A minor point is I cannot find how many DGRP lines are used.

Reviewer #2 (Public Review):

Summary

In this study, Easwaran and Montell investigated the molecular, cellular, and genetic basis of adult reproductive diapause in Drosophila using the Drosophila Genetic Reference Panel (DGRP). Their GWAS revealed genes associated with variation in post-diapause fecundity across the DGRP and performed RNAi screens on these candidate genes. They also analyzed the functional implications of these genes, highlighting the role of genes involved in neural and germline development. In addition, in conjunction with other GWAS results, they noted the importance of the olfactory system within the nervous system, which was supported by genetic experiments. Overall, their solid research uncovered new aspects of adult diapause regulation and provided a useful reference for future studies in this field.

Strengths:

The authors used whole-genome sequenced DGRP to identify genes and regulatory mechanisms involved in adult diapause. The first Drosophila GWAS of diapause successfully uncovered many QTL underlying post-diapause fecundity variations across DGRP lines. Gene network analysis and comparative GWAS led them to reveal a key role for the olfactory system in diapause lifespan extension and post-diapause fecundity.

Weaknesses:

(1) I suspect that there may be variation in survivorship after long-term exposure to cold conditions (10ºC, 35 days), which could also be quantified and mapped using genome-wide association studies (GWAS). Since blocking Ir21a neuronal transmission prevented flies from exiting diapause, it is possible that natural genetic variation could have a similar effect, influencing the success rate of exiting diapause and post-diapause mortality. If there is variation in this trait, could it affect post-diapause fecundity? I am concerned that this could be a confounding factor in the analysis of post-diapause fecundity. However, I also believe that understanding phenotypic variation in this trait itself could be significant in regulating adult diapause.

(2) On p.10, the authors conclude that "Dip-𝛾 and sbb are required in neurons for successful diapause, consistent with the enrichment of this gene class in the diapause GWAS." While I acknowledge that the results support their neuronal functions, I remain unconvinced that these genes are required for "successful diapause". According to the RNAi scheme (Figure 4I), Dip-γ and sbb are downregulated only during the post-diapause period, but still show a significant effect, comparable to that seen in the nSyb Gal4 RNAi lines (Figure 4K). In addition, two other RNAi lines (SH330386, 80461) that did not show lethality did not affect post-diapause fecundity. Notably, RNAi (27049, KK104056) substantially reduced non-diapause fecundity, suggesting impairment of these genes affects fecundity in general regardless of diapause experience. Therefore, the reduced post-diapause fecundity observed may be a result of this broader effect on fecundity, particularly in a more "sensitized" state during the post-diapause period, rather than a direct regulation of adult diapause by these genes.

(3) The authors characterized 546 genetic variants and 291 genes associated with phenotypic variation across DGRP lines but did not prioritize them by significance. They did prioritize candidate genes with multiple associated variants (p.9 "Genes with multiple SNPs are good candidates for influencing diapause traits."), but this is not a valid argument, likely due to a misunderstanding of LD among variants in the same gene. A gene with one highly significantly associated variant may be more likely to be the causal gene in a QTL than a gene with many weakly associated variants in LD. I recommend taking significance into account in the analysis.

Reviewer #3 (Public Review):

Summary:

Drosophila melanogaster of North America overwinters in a state of reproductive diapause. The authors aimed to measure 'successful' D. melanogaster reproductive diapause and reveal loci that impact this quantitative trait. In practice, the authors quantified the number of eggs produced by a female after she exited 35 days of diapause. The authors claim that genes involved with olfaction in part contribute to some of the variation in this trait.

Strengths:

The work used the power platform of the fly DRGP/GWAS. The work tried to verify some of the candidate loci with targeted gene manipulations.

Weaknesses:

Some context is needed. Previous work from 2001 established that D. melanogaster reproductive diapause in the laboratory suspends adult aging but reduces post-diapause fecundity. The work from 2001 showed the extent fecundity is reduced is proportional to diapause duration. As well, the 2001 data showed short diapause periods used in the current submission reduce fecundity only in the first days following diapause termination; after this time fecundity is greater in the post-diapause females than in the non-diapause controls.

In this context, the submission fails to offer a meaningful concept for what constitutes 'successful diapause'. There is no biological rationale or relationship to the known patterns of post-diapause fecundity. The phenotype is biologically ambiguous.

I have a serious concern about the antenna-removal design. These flies were placed on cool/short days two weeks after surgery. Adults at this time will not enter diapause, which must be induced soon after eclosion. Two-week-old adults will respond to cool temperatures by 'slowing down', but they will continue to age on a time scale of day-degrees. This is why the control group shows age-dependent mortality, which would not be seen in truly diapaused adults. Loss of antennae increases the age-dependent mortality of these cold adults, but this result does not reflect an impact on diapause.

• Appraisal of whether the authors achieved their aims, and whether the results support their conclusions.

The work falls well short of its aim because the concept of 'successful diapause' is not biologically established. The paper studies post-diapause fecundity, and we don't know what that means. The loci identified in this analysis segregate for a minimally constructed phenotype. The results and conclusions are orthogonal.

• The likely impact of the work on the field, and the utility of the methods and data to the community.

The work will have little likely impact. Its phenotype and operational methods are weakly developed. It lacks insight based on the primary literature on post-diapause. The community of insect diapause investigators are not likely to use the data or conclusions to understand beneficial or pest insects, or the impact of a changing climate on how they over-winter.

Author response:

eLife assessment

This useful study shows how genetic variation is associated with fecundity following a period of reproductive diapause in female Drosophila. The work identifies the olfactory system as central to successful diapause with associated changes in longevity and fecundity. While the genetic screening and methods used are solid, the approach to assessing diapause is incomplete and could benefit from additional orthogonal experiments.

Response: We agree that, as with most studies, additional follow-up work will be informative.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

The paper begins with phenotyping the DGRP for post-diapause fecundity, which is used to map genes and variants associated with fecundity. There are overlaps with genes mapped in other studies and also functional enrichment of pathways including most surprisingly neuronal pathways. This somewhat explains the strong overlap with traits such as olfactory behaviors and circadian rhythm. The authors then go on to test genes by knocking them down effectively at 10 degrees. Two genes, Dip-gamma and sbb, are identified as significantly associated with post-diapause fecundity, and they also find the effects to be specific to neurons. They further show that the neurons in the antenna but not the arista are required for the effects of Dip-gamma and sbb. They show that removing the antenna has a diapause-specific lifespan-extending effect, which is quite interesting. Finally, ionotropic receptor neurons are shown to be required for the diapause-associated effects.

Strengths and Weaknesses:

Overall I find the experiments rigorously done and interpretations sound. I have no further suggestions except an ANOVA to estimate the heritability of the post-diapause fecundity trait, which is routinely done in the DGRP and offers a global parameter regarding how reliable phenotyping is. A minor point is I cannot find how many DGRP lines are used.

Response: Thank you for the suggestions. We screened 193 lines and we will add that information to the methods.

Additionally, we will add the heritability estimate of the post-diapause fecundity trait.

Reviewer #2 (Public Review):

Summary

In this study, Easwaran and Montell investigated the molecular, cellular, and genetic basis of adult reproductive diapause in Drosophila using the Drosophila Genetic Reference Panel (DGRP). Their GWAS revealed genes associated with variation in post-diapause fecundity across the DGRP and performed RNAi screens on these candidate genes. They also analyzed the functional implications of these genes, highlighting the role of genes involved in neural and germline development. In addition, in conjunction with other GWAS results, they noted the importance of the olfactory system within the nervous system, which was supported by genetic experiments. Overall, their solid research uncovered new aspects of adult diapause regulation and provided a useful reference for future studies in this field.

Strengths:

The authors used whole-genome sequenced DGRP to identify genes and regulatory mechanisms involved in adult diapause. The first Drosophila GWAS of diapause successfully uncovered many QTL underlying post-diapause fecundity variations across DGRP lines. Gene network analysis and comparative GWAS led them to reveal a key role for the olfactory system in diapause lifespan extension and post-diapause fecundity.

Weaknesses:

(1) I suspect that there may be variation in survivorship after long-term exposure to cold conditions (10ºC, 35 days), which could also be quantified and mapped using genome-wide association studies (GWAS). Since blocking Ir21a neuronal transmission prevented flies from exiting diapause, it is possible that natural genetic variation could have a similar effect, influencing the success rate of exiting diapause and post-diapause mortality. If there is variation in this trait, could it affect post-diapause fecundity? I am concerned that this could be a confounding factor in the analysis of post-diapause fecundity. However, I also believe that understanding phenotypic variation in this trait itself could be significant in regulating adult diapause.

Response: We agree that it is possible that the ability to endure cool temperatures per se may influence post-diapause fecundity. However, cool temperature is the essential diapause-inducing condition in Drosophila, so it is not obvious how to separate those effects experimentally, and we agree that phenotypic variation in the cool-sensitivity trait itself could be significant in regulating diapause.

(2) On p.10, the authors conclude that "Dip-𝛾 and sbb are required in neurons for successful diapause, consistent with the enrichment of this gene class in the diapause GWAS." While I acknowledge that the results support their neuronal functions, I remain unconvinced that these genes are required for "successful diapause". According to the RNAi scheme (Figure 4I), Dip-γ and sbb are downregulated only during the post-diapause period, but still show a significant effect, comparable to that seen in the nSyb Gal4 RNAi lines (Figure 4K).

Response: Our definition of successful diapause is the ability to produce viable adult progeny post-diapause, which requires that the flies enter, maintain, and exit diapause, alive and fertile. We will restate our conclusion to say that Dip-γ and sbb are required for post-diapause fecundity.

In addition, two other RNAi lines (SH330386, 80461) that did not show lethality did not affect post-diapause fecundity.

Response: We interpret those results to mean that those RNAi lines were not effective since Dip-γ and sbb are known to be essential.

Notably, RNAi (27049, KK104056) substantially reduced non-diapause fecundity, suggesting impairment of these genes affects fecundity in general regardless of diapause experience. Therefore, the reduced post-diapause fecundity observed may be a result of this broader effect on fecundity, particularly in a more "sensitized" state during the post-diapause period, rather than a direct regulation of adult diapause by these genes.

Response: Ubiquitous expression of RNAi lines #27049 or #KK104056 was lethal, so we included the tubGAL80ts repressor to prevent RNAi from taking effect during development. Flies had to be shifted to 30 °C to inactivate the repressor and thereby activate the RNAi. At 30 °C, fecundity of the controls (GFP RNAi lines #9331, KK60102) were also lower (average non-diapause fecundity = 12 and 19 respectively) and similar to #27049 or #KK104056. We also assessed the knockdown using Repo GAL4 and nSyb GAL4 and did not find a significant difference/decline in the non diapause fecundity for #27049 and #KK104056 as compared to a nonspecific RNAi control (#54037).

(3) The authors characterized 546 genetic variants and 291 genes associated with phenotypic variation across DGRP lines but did not prioritize them by significance. They did prioritize candidate genes with multiple associated variants (p.9 "Genes with multiple SNPs are good candidates for influencing diapause traits."), but this is not a valid argument, likely due to a misunderstanding of LD among variants in the same gene. A gene with one highly significantly associated variant may be more likely to be the causal gene in a QTL than a gene with many weakly associated variants in LD. I recommend taking significance into account in the analysis.

We agree with the reviewer, and in Supplemental Table S3 we list top-associated SNPs in order from the lowest (most significant) p-value. Most of the top-associated genes from this analysis were uncharacterized CG numbers for which there were insufficient tools available for validation purposes. Nevertheless, there is overlap amongst the highly significant genes by p-value and those with multiple SNPs. Amongst the top 15 genes with multiple associated SNPsCG18636 & CR15280 ranked 3rd by p-value, CG7759 ranked 4th, CG42732 ranked 10th, and Drip ranked 30th (all above the conservative Bonferroni threshold of 4.8e-8) while three Sbb-associated SNPs also appear in Table 3 above the standard e-5 threshold.

Reviewer #3 (Public Review):

Summary:

Drosophila melanogaster of North America overwinters in a state of reproductive diapause. The authors aimed to measure 'successful' D. melanogaster reproductive diapause and reveal loci that impact this quantitative trait. In practice, the authors quantified the number of eggs produced by a female after she exited 35 days of diapause. The authors claim that genes involved with olfaction in part contribute to some of the variation in this trait.

Strengths:

The work used the power platform of the fly DRGP/GWAS. The work tried to verify some of the candidate loci with targeted gene manipulations.

Weaknesses:

Some context is needed. Previous work from 2001 established that D. melanogaster reproductive diapause in the laboratory suspends adult aging but reduces post-diapause fecundity. The work from 2001 showed the extent fecundity is reduced is proportional to diapause duration. As well, the 2001 data showed short diapause periods used in the current submission reduce fecundity only in the first days following diapause termination; after this time fecundity is greater in the post-diapause females than in the non-diapause controls.

Response: The 2001 paper by Tatar et al. reports the number of eggs laid after 3, 6, or 9 weeks in diapause conditions. Thus the diapause conditions used in this study (35 days or 5 weeks) are neither short nor long, rather intermediate. Does the reviewer have a specific concern?

In this context, the submission fails to offer a meaningful concept for what constitutes 'successful diapause'. There is no biological rationale or relationship to the known patterns of post-diapause fecundity. The phenotype is biologically ambiguous.

Response: We have unambiguously defined successful diapause as the ability to produce viable adult progeny post-diapause. Other groups have measured % of flies that arrest ovarian development or % of post-diapause flies with mature eggs in the ovary, or # eggs laid post-diapause; however we suggest that # of viable adult progeny produced post-diapause is more meaningful than the other measurements from the point of view of perpetuating the species.

I have a serious concern about the antenna-removal design. These flies were placed on cool/short days two weeks after surgery. Adults at this time will not enter diapause, which must be induced soon after eclosion. Two-week-old adults will respond to cool temperatures by 'slowing down', but they will continue to age on a time scale of day-degrees. This is why the control group shows age-dependent mortality, which would not be seen in truly diapaused adults. Loss of antennae increases the age-dependent mortality of these cold adults, but this result does not reflect an impact on diapause.

Response: The reviewer has a point. We carried out the lifespan study under two different conditions: either by removing the antenna and moving the flies directly to 10 °C or by removing the antenna and allowing a “wound healing” period prior to moving the flies to 10 °C (out of concern that the flies might have died quickly because wound healing may be impaired at 10 °C). In both cases, lifespan was shortened. We will add a discussion of the technical limitations of this experiment.

• Appraisal of whether the authors achieved their aims, and whether the results support their conclusions.

The work falls well short of its aim because the concept of 'successful diapause' is not biologically established. The paper studies post-diapause fecundity, and we don't know what that means. The loci identified in this analysis segregate for a minimally constructed phenotype. The results and conclusions are orthogonal.

Response: It is unclear to us why the reviewer has such a negative opinion of measuring post-diapause fecundity, specifically the ability to produce viable progeny post-diapause. The value of this measurement seems obvious from the point of view of perpetuating the species.

• The likely impact of the work on the field, and the utility of the methods and data to the community.

The work will have little likely impact. Its phenotype and operational methods are weakly developed. It lacks insight based on the primary literature on post-diapause. The community of insect diapause investigators are not likely to use the data or conclusions to understand beneficial or pest insects, or the impact of a changing climate on how they over-winter.

Response: The reviewer has not explained why his/her opinion is so negative.