Biology subjects

Mazo-Vargas, A.

Publications and source records attributed to Mazo-Vargas, A..

10 recordsLinked to original sources

bioRxiv2026

Genetic circuit rewiring underlies a lineage-specific patterning machinery on butterfly wings

Phenotypes evolve through changes in gene regulatory networks (GRNs), but how changes in network topology contribute to phenotypic evolution is still poorly understood. Butterfly wing color patterns provide a tractable system to answer this question. Here, we compared the expression of four color pattern genes, WntA, pdm3, ivory, and Optix, in five nymphalid butterflies, and tested their regulatory connections through genetic perturbations in three species spanning 80 million years of divergence. We found broadly conserved 1) regulatory hierarchy linking early pattern instruction via WntA signaling to subsequent color readout via ivory and Optix, 2) positive autoregulation in WntA signaling, and 3) positive regulation from WntA signaling to pdm3. Notably, we discovered lineage-specific negative feedback from pdm3 to WntA signaling in Vanessa cardui, which closes a reciprocal WntA-pdm3 feedback loop that refines complex pattern elements. We show that developmental programs can evolve by rewiring internal interactions among pre-existing GRN nodes, emphasizing the importance of network topology in phenotypic evolution.

evolutionary biology↗

bioRxiv2026

ABCG transporter knockouts alter integument and eye pigmentation with gene-specific effects on viability in butterflies and moths

Insects possess remarkable diversity in pigmentation across life stages, generated from a limited number of pigment precursors. ABCG transporters play central roles in pigmentation by mediating the intracellular transport of these precursors, yet their functions outside a few model systems remain poorly characterized. Here, we investigate the roles of the ABCG transporter genes white (w), scarlet (st), and oily kinshiryu (ok) across five lepidopteran species representing both butterflies and moths. CRISPR-Cas9 knockouts reveal conserved, tissue-specific functions of ABCG transporters throughout development, affecting larval and pupal integuments as well as adult eyes, but not scale coloration. Notably, our data indicate that both ommochrome and uric acid deposition underlie the coloration and opacity of Lepidoptera larvae and pupae, with the relative contributions of these pathways differing across species and stages. We also reveal that the vivid orange coloration of Gulf fritillary caterpillars arises from the interplay of ommochromes and urate granules, providing a clear example of how these pathways combine to produce diverse integument pigmentation. Loss of white was deleterious in four species examined, whereas scarlet knockouts produced viable individuals with easily detectable pigmentation phenotypes, identifying scarlet mutant backgrounds as a practical genetic marker for functional studies. Together, these findings show that conserved ABCG transporter complexes are differentially deployed across tissues and life stages, providing a mechanistic basis for the evolutionary diversification of pigmentation in Lepidoptera and enabling expanded functional genomics and transgenesis in emerging model species. Research HighlightsCRISPR-Cas9 disruption of ABCG transporters across Lepidoptera reveals conserved, tissue-specific roles in pigmentation. Interactions between ommochrome and uric acid deposition generate stage-specific coloration in larval and pupal integument.

genetics↗

bioRxiv2024

Combinatorial Wnt Signaling induces Margin Color Elements in Swallowtail Butterfly Ground Plan.

The intricate wing patterns of butterflies are thought to derive from a morphological ground plan that anchors homology relationships between individual color pattern elements and serves as an archetype for comparative analysis. These patterns undergo modifications that drive the diverse morphologies observed in nature. While brush-footed butterflies (Nymphalidae) have been well studied, assigning homologies with other lepidopteran families remains challenging due to substantial divergence. Here, we focus on swallowtails (Papilionidae), an early-diverging butterfly lineage known for its outstanding diversity in wing shapes and patterns but lacking a developmental framework. Through qualitative and phylogenetic analyses, CRISPR perturbation assays, and in situ expression experiments, we investigate homologies between papilionid butterflies, offering phylogenetic and molecular characterization of the Papilionidae wing ground plan. Our results highlight the roles of WntA and Wnt6 in patterning papilionid signature wing elements, such as the glauca and the Submarginal spots. Notably, the nymphalids distinct Central Symmetry System is either reduced or absent in the family, with marginal systems expanding proximally. Our data illuminate a highly adaptable patterning system driven by Wnt signaling pathways in developing butterfly wings.

developmental biology↗

bioRxiv2024

A long non-coding RNA at the cortex locus controls adaptive colouration in butterflies

Evolutionary variation in the wing pigmentation of butterflies and moths offers striking examples of adaptation by crypsis and mimicry. The cortex locus has been independently mapped as the locus controlling colour polymorphisms in 14 lepidopteran species, suggesting it acts as a genomic hotspot for the diversification of wing patterns, but functional validation through protein-coding knockouts has proven difficult to obtain. Our study unveils the role of a novel long non-coding RNA (lncRNA) which we name ivory, transcribed from the cortex locus, in modulating colour patterning in butterflies. Strikingly, ivory expression prefigures most melanic patterns during pupal development, suggesting an early developmental role in specifying scale identity. To test this, we generated CRISPR mosaic knock-outs in five nymphalid butterfly species and show that ivory mutagenesis yields transformations of dark pigmented scales into white or light-coloured scales. Genotyping of Vanessa cardui germline mutants associates these phenotypes to small on-target deletions at the conserved first exon of ivory. In contrast, cortex germline mutant butterflies with confirmed null alleles lack any wing phenotype, and exclude a colour patterning role for this adjacent gene. Overall, these results show that a lncRNA acts as a master switch of colour pattern specification, and played key roles in the adaptive diversification of colour patterns in butterflies. Significance statementDeciphering the genetic underpinnings of adaptive variation is fundamental for a comprehensive understanding of evolutionary processes. Long non-coding RNAs (lncRNAs) represent an emerging category of genetic modulators within the genome, yet they have been overlooked as a source of phenotypic diversity. In this study, we unveil the pivotal role of a lncRNA in orchestrating colour transitions between dark and light patterns during butterfly wing development. Remarkably, this lncRNA gene is nested within the cortex locus, a genetic region known to control multiple cases of adaptive variation in butterflies and moths, including iconic examples of natural selection. These findings highlight the significant influence of lncRNAs in developmental regulation, and also underscore their potential as key genetic players in the evolutionary process itself.

evolutionary biology↗

bioRxiv2024

The ivory lncRNA regulates seasonal color patterns in buckeye butterflies

Long non-coding RNAs (lncRNAs) are transcribed elements increasingly recognized for their roles in regulating gene expression. Thus far, however, we have little understanding of how lncRNAs contribute to evolution and adaptation. Here we show that a conserved lncRNA, ivory, is an important color patterning gene in the buckeye butterfly Junonia coenia. ivory overlaps with cortex, a locus linked to multiple cases of crypsis and mimicry in Lepidoptera. Along with a companion paper by Livraghi et. al., we argue that ivory, not cortex, is the color pattern gene of interest at this locus. In J. coenia a cluster of cis-regulatory elements (CREs) in the first intron of ivory are genetically associated with natural variation in seasonal color pattern plasticity, and targeted deletions of these CREs phenocopy seasonal phenotypes. Deletions of different ivory CREs produce other distinct phenotypes as well, including loss of melanic eyespot rings, and positive and negative changes in overall wing pigmentation. We show that the color pattern transcription factors Spineless, Bric-a-brac, and Ftz-f1 bind to the ivory promoter during wing pattern development, suggesting that they directly regulate ivory. This case study demonstrates how cis-regulation of a single non-coding RNA can exert diverse and nuanced effects on the evolution and development of color patterns, including modulating seasonally plastic color patterns. SignificanceThe genomic locus hosting the cortex gene has been linked to numerous cases of color pattern adaptation in moths and butterflies, including crypsis, mimicry, and seasonal polyphenism. Here we show in buckeye butterflies that the actual color pattern gene at the cortex locus is an evolutionarily conserved long non-coding RNA (lncRNA), dubbed ivory, that overlaps with cortex. Compared with other wing pattern genes, ivory stands out because of the highly nuanced, quantitative changes in pigmentation that can be achieved by manipulating adjacent cis-regulatory sequences. This study highlights how lncRNAs can be important factors underlying morphological evolution, and emphasizes the importance of considering non-coding transcripts in comparative genomics.

genetics↗

bioRxiv2023

Cis-regulatory modes of Ultrabithorax inactivation in butterfly forewings

Hox gene clusters encode transcription factors that drive regional specialization during animal development: e.g. the Hox factor Ubx is expressed in the insect metathoracic (T3) wing appendages and differentiates them from T2 mesothoracic identities. Hox transcriptional regulation requires silencing activities that prevent spurious activation and regulatory crosstalks in the wrong tissues, but this has seldom been studied in insects other than Drosophila, which shows a derived Hox dislocation into two genomic clusters that disjoined Antennapedia (Antp) and Ultrabithorax (Ubx). Here we investigated how Ubx is restricted to the hindwing in butterflies, amidst a contiguous Hox cluster. By analysing Hi-C and ATAC-seq data in the butterfly Junonia coenia, we show that a Topologically Associated Domain (TAD) maintains a hindwing-enriched profile of chromatin opening around Ubx. This TAD is bordered by a Boundary Element (BE) that separates it from a region of joined wing activity around the Antp locus. CRISPR mutational perturbation of this BE releases ectopic Ubx expression in forewings, inducing homeotic clones with hindwing identities. Further mutational interrogation of two non-coding RNA encoding regions and one putative cis-regulatory module within the Ubx TAD cause rare homeotic transformations in both directions, indicating the presence of both activating and repressing chromatin features. We also describe a series of spontaneous forewing homeotic phenotypes obtained in Heliconius butterflies, and discuss their possible mutational basis. By leveraging the extensive wing specialization found in butterflies, our initial exploration of Ubx regulation demonstrates the existence of silencing and insulating sequences that prevent its spurious expression in forewings.

genomics↗

bioRxiv2023

Frizzled2 receives the WntA morphogen during butterfly wing pattern formation

Butterfly color patterns provide visible and biodiverse phenotypic readouts of the patterning processes that occur in a developing epithelium. While the secreted ligand WntA was shown to instruct the color pattern formation in butterflies, its modes of reception and signal transduction remain elusive. Butterfly genomes encode four homologues of the Frizzled-family of Wnt receptors. Here we show that CRISPR mosaic knock-outs of frizzled2 (fz2) phenocopy the color pattern effects of WntA loss-of-function in multiple nymphalids. While WntA mosaic clones result in intermediate patterns of reduced size, consistently with a morphogen function, fz2 clones are cell-autonomous. Shifts in pupal expression in WntA crispants show that WntA and fz2 are under positive and negative feedback, respectively. Fz1 is required for Wnt-independent planar cell polarity (PCP) in the wing epithelium. Fz3 and Fz4 show phenotypes consistent with Wnt competitive-antagonist functions in vein formation (Fz3 and Fz4), wing margin specification (Fz3), and color patterning in the Discalis and Marginal Band Systems (Fz4). Overall, these data show that the WntA/Frizzled2 morphogen-receptor pair forms a signaling axis that instructs butterfly color patterning, and shed light on the functional diversity of insect Frizzled receptors.

developmental biology↗

bioRxiv2022

Efficient hyperactive piggyBac transgenesis in Plodia pantry moths

While piggyBac transposon-based transgenesis is widely used in various emerging model organisms, its relatively low transposition rate in butterflies and moths has hindered its use for routine genetic transformation in Lepidoptera. Here, we tested the suitability of a codon-optimized hyperactive piggyBac transposase (hyPBase) in mRNA form to deliver and integrate transgenic cassettes into the genome of the pantry moth Plodia interpunctella. Co-injection of hyPBase mRNA with donor plasmids successfully integrated 1.5-4.4 kb expression cassettes driving the fluorescent markers EGFP, DsRed, or EYFP in eyes and glia with the 3xP3 promoter. Somatic integration and expression of the transgene in the G0 injected generation was detectable from 72-hr embryos and onward in larvae, pupae and adults carrying a recessive white-eyed mutation. Overall, 2.5% of injected eggs survived into transgene-bearing adults with mosaic fluorescence. Subsequent outcrossing of fluorescent G0 founders transmitted single-insertion copies of 3xP3::EGFP and 3xP3::EYFP and generated stable isogenic lines. Random in-crossing of a small cohort of G0 founders expressing 3xP3::DsRed yielded a stable transgenic line segregating for more than one transgene insertion site. We discuss how hyPBase can be used to generate stable transgenic resources in Plodia and other moths.

genetics↗

bioRxiv2022

Evolutionary dynamics of genome size and content during the adaptive radiation of Heliconiini butterflies

Heliconius butterflies, a speciose genus of Mullerian mimics, represent a classic example of an adaptive radiation that includes a range of derived dietary, life history, physiological and neural traits. However, key lineages within the genus, and across the broader Heliconiini tribe, lack genomic resources, limiting our understanding of how adaptive and neutral processes shaped genome evolution during their radiation. We have generated highly contiguous genome assemblies for nine new Heliconiini, 29 additional reference-assembled genomes, and improve 10 existing assemblies. Altogether, we provide a major new dataset of annotated genomes for a total of 63 species, including 58 species within the Heliconiini tribe. We use this extensive dataset to generate a robust and dated heliconiine phylogeny, describe major patterns of introgression, explore the evolution of genome architecture, and the genomic basis of key innovations in this enigmatic group, including an assessment of the evolution of putative regulatory regions at the Heliconius stem. Our work illustrates how the increased resolution provided by such dense genomic sampling improves our power to generate and test gene-phenotype hypotheses, and precisely characterize how genomes evolve.

evolutionary biology↗

bioRxiv2019

Multiple roles for laccase2 in butterfly wing pigmentation, scale development, and cuticle tanning

Lepidopteran wing scales play important roles in a number of functions including color patterning and thermoregulation. Despite the importance of wing scales, however, we still have a limited understanding of the genetic mechanisms that underlie scale patterning, development, and coloration. Here we explore the function of the phenoloxidase-encoding gene laccase2 in wing and scale development in the nymphalid butterfly Vanessa cardui. Somatic deletion mosaics of laccase2 generated by CRISPR/Cas9 genome editing presented several distinct mutant phenotypes. Consistent with work in other non-lepidopteran insect groups, we observed reductions in melanin pigmentation and defects in cuticle formation. We were also surprised, however, to see distinct effects on scale development including complete loss of wing scales. This work highlights laccase2 as a gene that plays multiple roles in wing and scale development and provides new insight into the evolution of lepidopteran wing coloration.

developmental biology↗