Periodic Reporting for period 1 - mayFLYeye (The evolution of new organs during insects’ conquest of the sky)
Période du rapport: 2023-02-01 au 2025-07-31
The history of life on Earth has been defined by key game-changing events. In animals, this has often been driven by the appearance of novel organs, conferring new capabilities to explore new niches and adaptive landscapes, completely changing the subsequent evolution of certain lineages.
This project focus on one of the most fascinating and revolutionary events in animal history: insect’s conquest of the skies. First, by addressing the origin of the morphological change responsible for this revolution: the origin of wings. Second, by studying one of the multiple cascading effects set off by this initial event that continued shaping flying insects’ anatomy and boosted their diversification: the turbanate eyes (Turbeyes) of mayflies, a sex-specific extra set of eyes that males use to find mates during flight. We are using a unique experimental platform, the mayfly Cloeon dipterum, a key group to study insect evolution, as a new experimentally tractable model.
We are studying the genetic basis and key factors underlying the origin of these two organs, with a multidisciplinary view combining single cell -omics, developmental and functional approaches and, in the case of new eyes, their impact in physiology and diversification. By comparing these two evolutionary processes, we are unveiling key common and specific events in the evolution of the gene regulatory networks behind the origin of these two new organs.
This project focus on one of the most fascinating and revolutionary events in animal history: insect’s conquest of the skies. First, by addressing the origin of the morphological change responsible for this revolution: the origin of wings. Second, by studying one of the multiple cascading effects set off by this initial event that continued shaping flying insects’ anatomy and boosted their diversification: the turbanate eyes (Turbeyes) of mayflies, a sex-specific extra set of eyes that males use to find mates during flight. We are using a unique experimental platform, the mayfly Cloeon dipterum, a key group to study insect evolution, as a new experimentally tractable model.
We are studying the genetic basis and key factors underlying the origin of these two organs, with a multidisciplinary view combining single cell -omics, developmental and functional approaches and, in the case of new eyes, their impact in physiology and diversification. By comparing these two evolutionary processes, we are unveiling key common and specific events in the evolution of the gene regulatory networks behind the origin of these two new organs.
AIM1.1 Several hypotheses have been developed since the end of the 19th century to explain the origin of insect wings and whether wings and abdominal gills are serial homologues. Thus, the first question is to elucidate the Gene Regulatory Networks (GRNs) responsible for the appearance and development of gills and wings and to analyse the similarities between them. We have generated single cell (sc)-RNA-seq datasets from 6 developmental stages using ACME and SPLiT-seq methodologies (Rosselló, Tandonnet & Almudi, 2025. https://doi.org/10.1101/2025.02.04.635005(s’ouvre dans une nouvelle fenêtre)). We already identified wing and gills related clusters. We are also characterising these cell populations in embryos through HCR hybridizations for marker genes. In addition, we generated ATAC-seq datasets for the same developmental stgs (Pallarès-Albanell, Ortega-Flores et al., 2024, https://doi.org/10.1242/dev.203017(s’ouvre dans une nouvelle fenêtre)) that we will combine with the sc-RNA-seq data to build the GRNs responsible for the ontogeny of these organs.
AIM1.2 To reconstruct the ancestral wing GRN we have started a series of strategic collaborations which allowed us to obtain wing/tergum primordia from different insects.
AIM1.3 Although we are still selecting the list of candidates to test as players in wing appearance and development, we successfully established CRISPR/Cas9 in C. dipterum and the first KO lines are already growing in the lab.
AIM2.1 To identify the GRN responsible for the TurbEye we generated sc-RNA-seq and ATAC-seq datasets for different timepoints of TurbEye development.
AIM2.2 We have already characterised the expression pattern of the 10 opsins present in the genome of C. dipterum
AIM2.3 We collected specimens from the wild (Barcelona area and Brazil) from sixteen different species to identify Opsin genes and reconstruct phylogenetic trees.
AIM1.2 To reconstruct the ancestral wing GRN we have started a series of strategic collaborations which allowed us to obtain wing/tergum primordia from different insects.
AIM1.3 Although we are still selecting the list of candidates to test as players in wing appearance and development, we successfully established CRISPR/Cas9 in C. dipterum and the first KO lines are already growing in the lab.
AIM2.1 To identify the GRN responsible for the TurbEye we generated sc-RNA-seq and ATAC-seq datasets for different timepoints of TurbEye development.
AIM2.2 We have already characterised the expression pattern of the 10 opsins present in the genome of C. dipterum
AIM2.3 We collected specimens from the wild (Barcelona area and Brazil) from sixteen different species to identify Opsin genes and reconstruct phylogenetic trees.
- We have developed a new bioinformatic pipeline, BarQC (Rosselló, Tandonnet & Almudi, bioRxiv 2025) to pre-process data from SPLiT -seq experiments and asses the quality of them. With this new software, filtering becomes more robust, losing significant less reads due to mistakes in the adaptor and barcode sequences.
- We have implemented ATAC-seq approaches in C. dipterum (Pallares-Albanell, Ortega-Flores et al., Dev 2024), which are allowing us to identify regulatory regions in the genome of mayflies.
- We have also set up CRISPR/Cas9 genome editing in mayflies adapting DIPA-CRISPR methodology to C. dipterum. This new technique will permit us to functionally test candidate genes identified in all aims of the project.
In terms of scientific output, we have published so far:
1) Joan Pallares-Albanell*, Laia Ortega-Flores*, Tot Senar-Serra, Antoni Ruiz, Josep F Abril, Maria Rossello#, Isabel Almudi#. (2024). Gene regulatory dynamics during the development of a paleopteran insect, the mayfly Cloeon dipterum Development dev.203017. https://doi.org/10.1242/dev.203017(s’ouvre dans une nouvelle fenêtre)
2) Maria Rossello#, Sophie Tandonnet#, Isabel Almudi. BarQC: Quality Control and Preprocessing for SPLiT-Seq Data. (2025) bioRxiv 2025.02.04.635005. https://doi.org/10.1101/2025.02.04.635005(s’ouvre dans une nouvelle fenêtre)
3) Tòt Senar & Isabel Almudi#. El ojo de los insectos: una perspectiva evolutiva. eVOLUCIóN March, 2024 (in Spanish)
The insights we discovered in Pallares-Albanell, Ortega-Flores et al work are breakthroughs, as attested by its publication in the prestigious journal Development. First, we found a clear "transition point" in terms of gene regulation with two differential phases of early and late accessible chromatin during mayfly embryogenesis. This significant division between early and late embryogenesis had been previously suggested using gene expression data, but this is the first time it has been characterised using chromatin accessibility assays. Moreover, due to the key phylogenetic position of mayflies within the insect tree, these results are an unvaluable resource for the scientific community studying insect evolution and insect genomics.
- We have implemented ATAC-seq approaches in C. dipterum (Pallares-Albanell, Ortega-Flores et al., Dev 2024), which are allowing us to identify regulatory regions in the genome of mayflies.
- We have also set up CRISPR/Cas9 genome editing in mayflies adapting DIPA-CRISPR methodology to C. dipterum. This new technique will permit us to functionally test candidate genes identified in all aims of the project.
In terms of scientific output, we have published so far:
1) Joan Pallares-Albanell*, Laia Ortega-Flores*, Tot Senar-Serra, Antoni Ruiz, Josep F Abril, Maria Rossello#, Isabel Almudi#. (2024). Gene regulatory dynamics during the development of a paleopteran insect, the mayfly Cloeon dipterum Development dev.203017. https://doi.org/10.1242/dev.203017(s’ouvre dans une nouvelle fenêtre)
2) Maria Rossello#, Sophie Tandonnet#, Isabel Almudi. BarQC: Quality Control and Preprocessing for SPLiT-Seq Data. (2025) bioRxiv 2025.02.04.635005. https://doi.org/10.1101/2025.02.04.635005(s’ouvre dans une nouvelle fenêtre)
3) Tòt Senar & Isabel Almudi#. El ojo de los insectos: una perspectiva evolutiva. eVOLUCIóN March, 2024 (in Spanish)
The insights we discovered in Pallares-Albanell, Ortega-Flores et al work are breakthroughs, as attested by its publication in the prestigious journal Development. First, we found a clear "transition point" in terms of gene regulation with two differential phases of early and late accessible chromatin during mayfly embryogenesis. This significant division between early and late embryogenesis had been previously suggested using gene expression data, but this is the first time it has been characterised using chromatin accessibility assays. Moreover, due to the key phylogenetic position of mayflies within the insect tree, these results are an unvaluable resource for the scientific community studying insect evolution and insect genomics.