In search of the links between environmental signals and polyphenism
Dopamine pathway characterization during the reproductive mode switch in the pea aphid
Recommendation: posted 25 May 2022, validated 24 June 2022
Polyphenisms offer an opportunity to study the links between phenotype, development, and environment in a controlled genomic context (Simpson, Sword, & Lo, 2011). In organisms with short generation times, individuals living and developing in different seasons encounter different environmental conditions. Adaptive plasticity allows them to express different phenotypes in response to seasonal cues, such as temperature or photoperiod. Such phenotypes can be morphological variants, for instance displaying different wing patterns as seen in butterflies (Brakefield & Larsen, 1984; Nijhout, 1991; Windig, 1999), or physiological variants, characterized for instance by direct development vs winter diapause in temperate insects (Dalin & Nylin, 2012; Lindestad, Wheat, Nylin, & Gotthard, 2019; Shearer et al., 2016).
Many aphids display cyclical parthenogenesis, a remarkable seasonal polyphenism for reproductive mode (Tagu, Sabater-Muñoz, & Simon, 2005), also sometimes coupled with wing polyphenism (Braendle, Friebe, Caillaud, & Stern, 2005), which allows them to switch between parthenogenesis during spring and summer to sexual reproduction and the production of diapausing eggs before winter. In the pea aphid Acyrthosiphon pisum, photoperiod shortening results in the production, by parthenogenetic females, of embryos developing into the parthenogenetic mothers of sexual individuals. The link between parthenogenetic reproduction and sexual reproduction, therefore, occurs over two generations, changing from a parthenogenetic form producing parthenogenetic females (virginoparae), to a parthenogenetic form producing sexual offspring (sexuparae), and finally sexual forms producing overwintering eggs (Le Trionnaire et al., 2022).
The molecular basis for the transduction of the environmental signal into reproductive changes is still unknown, but the dopamine pathway is an interesting candidate. Form-specific expression of certain genes in the dopamine pathway occurs downstream of the perception of the seasonal cue, notably with a marked decrease in the heads of embryos reared under short-day conditions and destined to become sexuparae. Dopamine has multiple roles during development, with one mode of action in cuticle melanization and sclerotization, and a neurological role as a synaptic neurotransmitter. Both modes of action might be envisioned to contribute functionally to the perception and transduction of environmental signals.
In this study, Le Trionnaire and colleagues aim at clarifying this role in the pea aphid (Le Trionnaire et al., 2022). Using quantitative RT-PCR, RNA-seq, and in situ hybridization of RNA probes, they surveyed the timing and spatial patterns of expression of dopamine pathway genes during the development of different stages of embryo to larvae reared under long and short-day conditions, and destined to become virginoparae or sexuparae females, respectively. The genes involved in the synaptic release of dopamine generally did not show differences in expression between photoperiodic treatments. By contrast, pale and ddc, two genes acting upstream of dopamine production, generally tended to show a downregulation in sexuparare embryo, as well as genes involved in cuticle development and interacting with the dopamine pathway. The downregulation of dopamine pathway genes observed in the heads of sexuparare juveniles is already detectable at the embryonic stage, suggesting embryos might be sensing environmental cues leading them to differentiate into sexuparae females.
The way pale and ddc expression differences could influence environmental sensitivity is still unclear. The results suggest that a cuticle phenotype specifically in the heads of larvae could be explored, perhaps in the form of a reduction in cuticle sclerotization and melanization which might allow photoperiod shortening to be perceived and act on development. Although its causality might be either way, such a link would be exciting to investigate, yet the existence of cuticle differences between the two reproductive types is still a hypothesis to be tested. The lack of differences in the expression of synaptic release genes for dopamine might seem to indicate that the plastic response to photoperiod is not mediated via neurological roles. Yet, this does not rule out the role of decreasing levels of dopamine in mediating this response in the central nervous system of embryos, even if the genes regulating synaptic release are equally expressed.
To test for a direct role of ddc in regulating the reproductive fate of embryos, the authors have generated CrispR-Cas9 knockout mutants. Those mutants displayed egg cuticle melanization, but with lethal effects, precluding testing the effect of ddc at later stages in development. Gene manipulation becomes feasible in the pea aphid, opening up certain avenues for understanding the roles of other genes during development.
This study adds nicely to our understanding of the intricate changes in gene expression involved in polyphenism. But it also shows the complexity of deciphering the links between environmental perception and changes in physiology, which mobilise multiple interacting gene networks. In the era of manipulative genetics, this study also stresses the importance of understanding the traits and phenotypes affected by individual genes, which now seems essential to piece the puzzle together.
Braendle C, Friebe I, Caillaud MC, Stern DL (2005) Genetic variation for an aphid wing polyphenism is genetically linked to a naturally occurring wing polymorphism. Proceedings of the Royal Society B: Biological Sciences, 272, 657–664. https://doi.org/10.1098/rspb.2004.2995
Brakefield PM, Larsen TB (1984) The evolutionary significance of dry and wet season forms in some tropical butterflies. Biological Journal of the Linnean Society, 22, 1–12. https://doi.org/10.1111/j.1095-8312.1984.tb00795.x
Dalin P, Nylin S (2012) Host-plant quality adaptively affects the diapause threshold: evidence from leaf beetles in willow plantations. Ecological Entomology, 37, 490–499. https://doi.org/10.1111/j.1365-2311.2012.01387.x
Le Trionnaire G, Hudaverdian S, Richard G, Tanguy S, Gleonnec F, Prunier-Leterme N, Gauthier J-P, Tagu D (2022) Dopamine pathway characterization during the reproductive mode switch in the pea aphid. bioRxiv, 2020.03.10.984989, ver. 4 peer-reviewed and recommended by Peer Community in Zoology. https://doi.org/10.1101/2020.03.10.984989
Lindestad O, Wheat CW, Nylin S, Gotthard K (2019) Local adaptation of photoperiodic plasticity maintains life cycle variation within latitudes in a butterfly. Ecology, 100, e02550. https://doi.org/10.1002/ecy.2550
Nijhout HF (1991). The development and evolution of butterfly wing patterns. Washington, DC: Smithsonian Institution Press.
Shearer PW, West JD, Walton VM, Brown PH, Svetec N, Chiu JC (2016) Seasonal cues induce phenotypic plasticity of Drosophila suzukii to enhance winter survival. BMC Ecology, 16, 11. https://doi.org/10.1186/s12898-016-0070-3
Simpson SJ, Sword GA, Lo N (2011) Polyphenism in Insects. Current Biology, 21, R738–R749. https://doi.org/10.1016/j.cub.2011.06.006
Tagu D, Sabater-Muñoz B, Simon J-C (2005) Deciphering reproductive polyphenism in aphids. Invertebrate Reproduction & Development, 48, 71–80. https://doi.org/10.1080/07924259.2005.9652172
Windig JJ (1999) Trade-offs between melanization, development time and adult size in Inachis io and Araschnia levana (Lepidoptera: Nymphalidae)? Heredity, 82, 57–68. https://doi.org/10.1038/sj.hdy.6884510
Mathieu Joron (2022) In search of the links between environmental signals and polyphenism. Peer Community in Zoology, 100013. https://doi.org/10.24072/pci.zool.100013
The recommender in charge of the evaluation of the article and the reviewers declared that they have no conflict of interest (as defined in the code of conduct of PCI) with the authors or with the content of the article. The authors declared that they comply with the PCI rule of having no financial conflicts of interest in relation to the content of the article.
Evaluation round #2
DOI or URL of the preprint: https://doi.org/10.1101/2020.03.10.984989
Version of the preprint: v2
Author's Reply, 14 Mar 2022
Decision by Mathieu Joron, posted 13 Sep 2021
Dear Gael Le Trionnaire and Denis Tagu
Thank you for your revised version of the manuscript, and apologies for the delayed response. Many of the concerns expressed by the referees of the initial submission have been addressed adequately, most significantly the exclusion of inconclusive data on pharmacological experiments and the amplification data for the Crispr experiement.
To me the manuscript appears simplified and the aim clearer. I agree with you that this represents a significant amount of work and cannot be simply considered to be entirely built upon negative results. Although the amount or work does not necessarily indicate its value, the work performed is indeed informative with respect to the timing and spatial distribution of gene expression in the dopamine pathway. Showing that it is the cuticular and melanin synthesis components of the pathway that are differentially expressed in different photoperiodic conditions while the synaptic components are not is of interest. Therefore, I think there is value in recommending and publishing your revised work in PCI Zoology. However, other concerns are still outstanding and your manuscript is in my opinion still somewhat confusing and warrants further clarification before I can commit on a recommendation.
First, the manuscript is still very much presented as a dissection of the response to photoperiodic change and as the dopamine pathway as a main component of this response, yet your analyses do not truly test this. Your starting point is the observation of a downregulation of dopamin synthesis genes pale and ddc in sexuparae, which your paper documents further using RNAseq, qRTPCR and in situ staining in different tissue or stages. But this tells little about induction of this response following the change in photoperiod. Your response to Reviewer 2 states that “it is clear that our data indicate that dopamine is probably not directly involved in photoperiod signalling”, yet at least from my reading the manuscript does not acknowledges this so clearly. Therefore, I think you could be more explicit with respect to the true objectives of your study and avoid any ambiguity about the link with the photoperiodic cue.
Second, to me the CrispR experiment is not part of a dissection of the photoperiod-induced changes in expression of dopamine-pathway genes. I see that you made an important effort in addressing the concern of the referees regarding the efficiency of the Crispr editing. Yet the difficulty in maintaining stable lines because of the lethal effects of the transformation precludes using this experiment as a tool to assess the role of ddc in the regulation of the polyphenism (even though this was the initial aim). The apparent role of ddc in egg cuticle melanisation may be indicative of the general role of ddc in melanisation, but this was known already from many other organisms. So I am not sure exactly how this connects with the general purpose of the paper of investigating seasonal polyphenism. There may be a logical link, because an egg trait involved in surviving winter could be considered important, but this may be a long stretch. As it stands the position of the CrispR experiment adds more confusion than it contributes to the general message. So, you may consider keeping this section in, or decide to leave it out, or in supplementary material, but the value of this experiment in the context of deciphering the response to photoperiod must be clarified to make the entire manuscript a more integrated body of work. Perhaps it may be sufficient to improve the logic linking the different experiments, and to frame those as part of an effort to document the expression of dopamine-pathway genes in the context of a response to seasonal changes.
On a more precise note, I found it unclear whether the authors consider that the response in the embryos is due to embryos, or the mother, sensing the environmental cue. Could both play a role? This could perhaps be clarified to understand how the presence of “residual” maternal RNA on the early embryo relates to the response to photoperiod.
Regarding the spatial expression of pale and ddc in the central nervous system of the embryo, this is intriguing, because readers may wonder whether this could reflect expression associated with the downstream synaptic compartment of the signaling pathway. Since the genes involved in the synaptic compartment of the pathway are not downregulated with the change in photoperiod, there seems to be potential for an interesting discussion.
In short, I would be happy to recommend a manuscript with clarified link between all the different experiments, providing a more integrated analysis and discussion to improve our understanding of the response to seasonal cues. I thank you in advance for your efforts in this direction and am looking forward to reading a revised version.
Evaluation round #1
DOI or URL of the preprint: https://doi.org/10.1101/2020.03.10.984989
Author's Reply, 18 Jun 2021
Decision by Mathieu Joron, posted 17 Jul 2020
Dear Gael Le Trionnaire and Denis Tagu,
Your manuscript entitled “Dopamine pathway characterization during the reproductive mode switch in the pea aphid” was evaluated by three referees. As you will see from their reviews, two of them praised the quality of the expression analysis of dopamine pathway and cuticle formation genes, but all three raised concerns regarding the manipulative experiments either on the design itself or on the validity of the negative results.
I tend to agree with Referee 2 that the lack of response in the manipulative experiments is difficult to interpret confidently as a lack of role of the dopamine pathway on the switch in reproductive mode, because of possible inefficiency or toxic effects of the manipulation. Referee 1 has concerns regarding the novelty of the role of dopamine on cuticle melanisation and sclerotization, and although your work does not aim at establishing this role, it is unclear how you envision the possibility of a link between sclerotization and reproductive mode switch. Referee 3 also has concerns about the overall design of the study in order to understand the pathways involved in switching the reproductive mode following the perception of an environmental cue. My opinion is that your study adds exciting information regarding the involvement of the dopamine pathway in the construction of phenotypes that are associated with changes in reproductive mode and in egg diapause, but falls short of providing decisive information on a role of this pathway in causing the reproductive switch. It is not entirely clear why this pathway might not simply respond to the same cues as those causing the reproductive switch, but acting in parallel rather than as a necessary link to the switch itself.
Overall the concerns expressed are substantial and I think that your manuscript would require major improvements to lead to a recommendation. Following Referee 2, you may want to consider changing the focus of your manuscript in a way that gives more credit to the novelty and robustness of your results on the timing of expression of the genes in the context of comparing virginoparae and sexuparae. This would allow addressing the concerns of both Referees 2 and 3. Alternatively, you may want to address the concerns of Referees 1 and 2 directly by providing more data or information.
I would therefore be glad to consider a thoroughly revised version taking those suggestions into account, and explaining in details how you have dealt with the points raised by the reviewers.
Thank you for sending your work to PCI Entomology. I look forward to seeing a revision.
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