A novel gene REPTOR2 activates the autophagic degradation of wing disc in pea aphid
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eLife assessment
The aims and hypothesis of the study, which addresses the genetic basis of an iconic example of developmental plasticity, are clear, and the experiments are well conducted. The authors propose that a novel gene that arose through gene duplication, REPTOR2, stimulates autophagy to generate wingless aphid morphs. The implication of a novel gene in wing autophagy for the generation of wingless aphids is novel and interesting, but the link between TOR and REPTOR2 requires further support.
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Abstract
Wing dimorphism in insects is an evolutionarily adaptive trait to maximize insect fitness under various environments, by which the population could be balanced between dispersing and reproduction. Most studies concern the regulatory mechanisms underlying the stimulation of wing morph in aphids, but relatively little research addresses the molecular basis of wing loss. Here, we found that, while developing normally in winged-destined pea aphids, the wing disc in wingless-destined aphids degenerated 30-hr postbirth and that this degeneration was due to autophagy rather than apoptosis. Activation of autophagy in first instar nymphs reduced the proportion of winged aphids, and suppression of autophagy increased the proportion. REPTOR2 , associated with TOR signaling pathway, was identified by RNA-seq as a differentially expressed gene between the two morphs with higher expression in the thorax of wingless-destined aphids. Further genetic analysis indicated that REPTOR2 could be a novel gene derived from a gene duplication event that occurred exclusively in pea aphids on autosome A1 but translocated to the sex chromosome. Knockdown of REPTOR2 reduced autophagy in the wing disc and increased the proportion of winged aphids. In agreement with REPTOR’s canonical negative regulatory role of TOR on autophagy, winged-destined aphids had higher TOR expression in the wing disc. Suppression of TOR activated autophagy of the wing disc and decreased the proportion of winged aphids, and vice versa. Co-suppression of TOR and REPTOR2 showed that ds REPTOR2 could mask the positive effect of ds TOR on autophagy, suggesting that REPTOR2 acted as a key regulator downstream of TOR in the signaling pathway. These results revealed that the TOR signaling pathway suppressed autophagic degradation of the wing disc in pea aphids by negatively regulating the expression of REPTOR2 .
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Author Response
Reviewer #2 (Public Review):
Weaknesses: The authors do not make a direct link between TOR and REPTOR2 signalling. This seems important since REPTOR2 is a novel gene that arose from the duplication of REPTOR.
We have added several experiments to strengthen the connection between TOR and REPTOR2, and determined the effect of co-silencing of TOR and REPTOR2 on autophagy and proportion of the winged morph. Please see the details below in your comments point 3.
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eLife assessment
The aims and hypothesis of the study, which addresses the genetic basis of an iconic example of developmental plasticity, are clear, and the experiments are well conducted. The authors propose that a novel gene that arose through gene duplication, REPTOR2, stimulates autophagy to generate wingless aphid morphs. The implication of a novel gene in wing autophagy for the generation of wingless aphids is novel and interesting, but the link between TOR and REPTOR2 requires further support.
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Reviewer #1 (Public Review):
This paper elucidates the developmental-genetic mechanisms that generate the winged and wingless form (morph) of female pea aphids (Acyrthosiphon pisum). Pea aphids reproduce parthenogenetically generating genetically identical offspring, and so the difference between the winged and wingless morphs is environmentally induced (referred to as a polyphenism). Previous studies have shown that the crowding of mothers is sufficient to induce the winged phenotype in the offspring. The authors develop a technique so that they can reliably generate wing-destined (WD) or wingless-destined (WLD) offspring. This allowed them to examine the early development of WD and WLD offspring during the 1st nymphal stage, before wing development can be observed externally, in the 3rd nymphal stage. They find that the wing primordia …
Reviewer #1 (Public Review):
This paper elucidates the developmental-genetic mechanisms that generate the winged and wingless form (morph) of female pea aphids (Acyrthosiphon pisum). Pea aphids reproduce parthenogenetically generating genetically identical offspring, and so the difference between the winged and wingless morphs is environmentally induced (referred to as a polyphenism). Previous studies have shown that the crowding of mothers is sufficient to induce the winged phenotype in the offspring. The authors develop a technique so that they can reliably generate wing-destined (WD) or wingless-destined (WLD) offspring. This allowed them to examine the early development of WD and WLD offspring during the 1st nymphal stage, before wing development can be observed externally, in the 3rd nymphal stage. They find that the wing primordia are apparent in both WD and WLD 1st instars immediately after birth, but that the primordia degrades 30-36h after birth in WLD nymphs. They then demonstrate that this degeneration is due to autophagy rather than apoptosis, evident through the increased expression of autophagy-related genes in WLD nymphs, but not pro-apoptotic genes. The authors next ask what is responsible for inducing autophagy in the WLD nymphs and so transcriptomics to look for genes that are differentially up- or down-regulated in 1st insar WLD versus WD nymphs. One gene, REPTOR2, is markedly down-regulated in WLD versus WD nymphs. REPTOR is an established target of TOR-signaling, which is in turn an established regulator of autophagy. The authors, therefore, focus on REPTOR2 and show that it has arisen through gene duplication of REPTOR in the A. pisum lineages, that it is differentially upregulated in the thorax of WLD versus WD nymphs, and that knock-down of REPTOR2 both reduces levels of the autophagic protein ATG8 in the wing primordia of 1st instar nymph and increases the proportion of winged offspring. Finally, the authors demonstrate that TOR, which canonically represses RAPTOR, negatively regulates autophagy of the wing primordia and positively regulates the generation of the winged morph.
The strength of this paper is that it cleanly implicates a novel gene, REPTOR2, that has arisen through gene duplication, in the generation of alternative morphs in a polyphenism. The paper also provides compelling evidence that the degeneration of the wing primordia in wingless aphids is through autophagy rather than apoptosis. Further, the paper provides another example of how signaling pathways known to be involved in the generation of reaction norms (continuous phenotypic responses to environmental variation) are also implicated in the generation of polyphenisms (discrete phenotypic responses to environmental variation). The paper uses a reliable and reproducible technique to generate wing and wingless forms of aphids upon which developmental studies can be conducted. The results of the paper are straightforward and convincing. A weakness of the paper is that, while it implicates both RAPTOR2 and TOR-signaling in the generation of winged and wingless morphs, it does not provide a causal link between the two. REPTOR (Repressed by TOR) is known to be a regulator of TOR-signaling in Drosophila, activating transcriptional stress response upon TOR inhibition, and the authors argue that REPTOR2 serves to exert a negative effect of TOR signaling on autophagy initiation in wingless aphids. Nevertheless, their data do not unambiguously show this. Specifically, they do not demonstrate that REPTOR2 is downstream of TOR in the signaling pathway that regulates winglessness.
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Reviewer #2 (Public Review):
The developmental mechanisms underlying insect polyphenisms are understood for only a few species. Previous studies in pea aphids and planthoppers have shown that insulin signalling is important for differences in wing morphs across environmental conditions. In the pea aphid, mothers that are crowded produce a high proportion of offspring with wings, while mothers housed alone produce offspring without wings. The authors emphasise that in the pea aphid wing loss is a novel trait, and work to identify the developmental processes that lead to wing loss. They find that wing loss is induced by autophagy of the wing disc in the first instar nymphs. Using a transcriptomics approach, they identify a candidate gene, REPTOR2, whose expression is enriched in nymphs destined to become wingless. REPTOR2 is a novel gene …
Reviewer #2 (Public Review):
The developmental mechanisms underlying insect polyphenisms are understood for only a few species. Previous studies in pea aphids and planthoppers have shown that insulin signalling is important for differences in wing morphs across environmental conditions. In the pea aphid, mothers that are crowded produce a high proportion of offspring with wings, while mothers housed alone produce offspring without wings. The authors emphasise that in the pea aphid wing loss is a novel trait, and work to identify the developmental processes that lead to wing loss. They find that wing loss is induced by autophagy of the wing disc in the first instar nymphs. Using a transcriptomics approach, they identify a candidate gene, REPTOR2, whose expression is enriched in nymphs destined to become wingless. REPTOR2 is a novel gene that has arisen from duplication in REPTOR. They further demonstrate that reducing REPTOR2 expression by RNAi increases the proportion of winged nymphs. Similarly, reducing the target of rapamycin signalling or feeding mothers on a low-protein diet decreased the proportion of winged nymphs. The authors conclude from these studies that in crowded mothers, high TOR signalling represses REPTOR2 activity leading to reduced autophagy in the wing discs.
Strengths:
1. The authors have outlined very clear hypotheses and aims, which makes the arguments in the text very easy to follow.
2. This study is very carefully conducted, and the authors use multiple lines of approach to validate their claims (eg confocal imaging of wing discs to examine ATG8 expression and TUNEL staining to differentiate between autophagy or apoptosis respectively, followed up by qPCR for autophagy and apoptosis genes).
3. Experiments are appropriately quantified and are backed by statistical tests.
4. The results lend excellent support to the author's claims.Weaknesses: The authors do not make a direct link between TOR and REPTOR2 signalling. This seems important since REPTOR2 is a novel gene that arose from the duplication of REPTOR.
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