Antonia Monteiro | National University of Singapore (original) (raw)

Papers by Antonia Monteiro

BMC Developmental Biology, Nov 20, 2006

Background: Precise temporal and spatial regulation of transgene expression is a critical tool to... more Background: Precise temporal and spatial regulation of transgene expression is a critical tool to investigate gene function in developing organisms. The most commonly used technique to achieve tight control of transgene expression, however, requires the use of specific DNA enhancers that are difficult to characterize in non-model organisms. Here, we sought to eliminate the need for this type of sequence-based gene regulation and to open the field of functional genetics to a broader range of organisms. Results: We have developed a new laser mediated method to heat shock groups of cells that provides precise spatio-temporal control of gene expression without requiring knowledge of specific enhancer sequences. We tested our laser-system in a transgenic line of Bicyclus anynana butterflies containing the EGFP reporter gene attached to the heat sensitive hsp70 promoter of Drosophila melanogaster. Whole organismal heat shocks demonstrated that this Drosophila promoter can drive gene expression in butterflies, and the subsequent laser heat shocks showed that it was possible to activate cell-specific gene expression in very precise patterns on developing pupal wings. Conclusion: This laser-mediated gene expression system will enable functional genetic investigations, i.e., the ectopic expression of genes and their knock-down in targeted groups of cells in model and non-model organisms with little or no available regulatory data, as long as a compatible heat-shock promoter is used and the target tissue is accessible to a laser beam. This technique will also be useful in evolutionary developmental biology as it will enable the study of the evolution of gene function across a variety of organisms.

Evolution, Mar 20, 2014

Fixed, genetically determined, mate preferences for species whose adult phenotype varies with rea... more Fixed, genetically determined, mate preferences for species whose adult phenotype varies with rearing environment may be maladaptive, as the phenotype that is most fit in the parental environment may be absent in the offspring environment. Mate preference in species with polyphenisms (environmentally dependent alternative phenotypes) should therefore either not focus on polyphenic traits, be polyphenic themselves, or learned each generation. Here, we test these alternative hypotheses by first describing a female-limited seasonal polyphenism in a sexually dimorphic trait in the butterfly Bicyclus anynana, dorsal hindwing spot number (DHSN), and then testing whether male and female mate preferences for this trait exist, and whether they are seasonally polyphenic, or learned. Neither naïve males nor naïve females in either seasonal form exhibited mating preferences for DHSN. However, males, but not females, noticed DHSN variation and learned mate preferences for DHSN. These results suggest that individuals may accommodate environmentally dependent variation in morphological traits via learned mate preferences in each generation, and that learned mate preference plasticity can be sexually dimorphic.

Butterflies have evolved different colour patterns on their dorsal and ventral wing surfaces to s... more Butterflies have evolved different colour patterns on their dorsal and ventral wing surfaces to serve different signalling functions, yet the developmental mechanisms controlling surface-specific patterning are still unknown. Here, we mutate both copies of the transcription factor apterous in Bicyclus anynana butterflies using CRISPR/Cas9 and show that apterous A, expressed dorsally, functions both as a repressor and modifier of ventral wing colour patterns, as well as a promoter of dorsal sexual ornaments in males. We propose that the surface-specific diversification of wing patterns in butterflies proceeded via the co-option of apterous A or its downstream effectors into various gene regulatory networks involved in the differentiation of discrete wing traits. Further, interactions between apterous and sex-specific factors such as doublesex may have contributed to the origin of sexually dimorphic surface-specific patterns. Finally, we discuss the evolution of eyespot number diversity in the family Nymphalidae within the context of developmental constraints due to apterous regulation.

BioEssays, Jun 26, 2013

The differentiation of male and female characteristics in vertebrates and insects has long been t... more The differentiation of male and female characteristics in vertebrates and insects has long been thought to proceed via different mechanisms. Traditionally, vertebrate sexual development was thought to occur in two phases: a primary and a secondary phase, the primary phase involving the differentiation of the gonads, and the secondary phase involving the differentiation of other sexual traits via the influence of sex hormones secreted by the gonads. In contrast, insect sexual development was thought to depend exclusively on cell-autonomous expression of sexspecific genes. Recently, however, new evidence indicates that both vertebrates and insects rely on sex hormones as well as cell-autonomous mechanisms to develop sexual traits. Collectively, these new data challenge the traditional vertebrate definitions of primary and secondary sexual development, call for a redefinition of these terms, and indicate the need for research aimed at explaining the relative dependence on cell-autonomous versus hormonally guided sexual development in animals.

Journal of the Royal Society Interface, May 1, 2023

Optical transparency is rare in terrestrial organisms, and often originates through loss of pigme... more Optical transparency is rare in terrestrial organisms, and often originates through loss of pigmentation and reduction in scattering. The coloured wings of some butterflies and moths have repeatedly evolved transparency, offering examples of how they function optically and biologically. Because pigments are primarily localized in the scales that cover a colourless wing membrane, transparency has often evolved through the complete loss of scales or radical modification of their shape. Whereas bristle-like scales have been well documented in glasswing butterflies, other scale modifications resulting in transparency remain understudied. The butterfly Phanus vitreus achieves transparency while retaining its scales and exhibiting blue/cyan transparent zones. Here, we investigate the mechanism of wing transparency in P. vitreus by light microscopy, focused ion beam milling, microspectrophotometry and optical modelling. We show that transparency is achieved via loss of pigments and vertical orientation in normal paddle-like scales. These alterations are combined with an anti-reflective nipple array on portions of the wing membrane being more exposed to light. The blueish coloration of the P. vitreus transparent regions is due to the properties of the wing membrane, and local scale nanostructures. We show that scale retention in the transparent patches might be explained by these perpendicular scales having hydrophobic properties.

Mechanisms of Development, Jul 1, 2017

The cuticular skeleton of a butterfly wing scale cell serves both as a substrate for the depositi... more The cuticular skeleton of a butterfly wing scale cell serves both as a substrate for the deposition of pigments and as an exquisitely finely-sculpted material responsible for the production of structural colors. While cuticle rigidity and pigmentation depend to a large extent on the end products of a branched biochemical pathway-the melanin pathwaylittle is known whether genes in this pathway also play a role in the development of specific scale morphologies that might aid in the development of structural colors. Here we first show that male and female Bicyclus anynana butterflies display differences in scale size and scale morphology but no differences in scale color. Then we use CRISPR/Cas9 to show that knockout mutations in five genes that function in the melanin pathway, TH, DDC, yellow, ebony, and aaNAT, affect both the fine structure and the coloration of the wing scales. Most dramatically, mutations in yellow led to extra horizontal cuticular laminae on the surface of scales, whereas mutations in DDC led to taller and sheet-like vertical cuticular laminae throughout each scale. We identify some of the first genes affecting the development of scale morphology, and whose regulation and pleiotropic effects may be important in creating, as well as limiting, the diversity of structural as well as pigmentary colors observed in butterflies.

There are fewer eyespots on the forewings versus hindwings of nymphalids but the reasons for this... more There are fewer eyespots on the forewings versus hindwings of nymphalids but the reasons for this uneven distribution remain unclear. One possibility is that, in many butterflies, the hindwing covers part of the ventral forewing at rest and there are fewer forewing sectors to display eyespots (covered eyespots are not continuously visible and are less likely to be under positive selection). A second explanation is that having fewer forewing eyespots confers a selective advantage against predators. We analysed wing overlap at rest in 275 nymphalid species with eyespots and found that many have exposed forewing sectors without eyespots: i.e. wing overlap does not constrain the forewing from having the same number or more eyespots than the hindwing. We performed two predation experiments with mantids to compare the relative fitness of and attack damage patterns on two forms of Bicyclus anynana butterflies, both with seven hindwing eyespots, but with two (in wild-type) or four (in Spotty) ventral forewing eyespots. Spotty experienced more intense predation on the forewings, were shorter-lived and laid fewer eggs. These results suggest that predation pressure limits forewing eyespot number in B. anynana. This may occur if attacks on forewing eyespots have more detrimental consequences for flight than attacks on hindwing eyespots.

bioRxiv (Cold Spring Harbor Laboratory), May 27, 2021

A long-standing view in the field of evo-devo is that insect forewings develop without any Hox ge... more A long-standing view in the field of evo-devo is that insect forewings develop without any Hox gene input. The Hox gene Antennapedia (Antp), despite being expressed in the thoracic segments of insects, has no effect on wing development. This view has been obtained from studies in two main model species, Drosophila and Tribolium. Here, we show that partial loss of function of Antp resulted in reduced and malformed adult wings in Bombyx, Drosophila, and Tribolium. Antp mediates wing growth in Bombyx by directly regulating the ecdysteriod biosynthesis enzyme gene (shade) in the wing tissue, which leads to local production of the growth hormone 20E. In turn, 20E signaling also up-regulates Antp. Additional targets of Antp are wing cuticular protein genes CPG24, CPH28, and CPG9, essential for wing development. We propose, thus, that insect wing development occurs in an Antp-dependent manner.

Journal of insect science, Jul 1, 2017

Male investment towards reproduction is substantial in some species, and this leads to the evolut... more Male investment towards reproduction is substantial in some species, and this leads to the evolution of choosy males. Male choice is often directed towards female phenotypes that are good indicators of fecundity such as body size, age, or virgin status, and often acts in the same direction as fecundity selection. In insects, only a few examples exist where male choice is directed towards female ornaments as proxies Butler of female quality. Here, we use dry season males of the sex-role reversed butterfly species Bicyclus anynana to test for differences in male choosiness and investment towards females of varying attractiveness using ornamentmanipulations. Male reproductive investment in this species is in the form of a nuptial gift, a spermatophore, given to females upon mating. Males were placed in cages with either wild-type ornamented females or with nonornamented females (center of the dorsal forewing eyespots painted over), and time to mating, duration of mating, and longevity of males and females after a single mating were measured. Ornament manipulations consisted of blocking the UV-reflective scales in the center of the dorsal forewing eyespots of females, a known sexual ornament. Males displayed lower latency to mate and longer mating durations with ornamented females. The longer mating duration did not, however, translate in the transfer of a nuptial gift that increased female longevity or reduced male longevity. Instead, we propose that longer mating durations with ornamented females may represent increased mate guarding behavior or increased sperm transfer.

Development, 2020

We describe the expression pattern and function of candidate venation patterning genes in butterf... more We describe the expression pattern and function of candidate venation patterning genes in butterflies and propose how the simplified venation of flies might have evolved from a common ancestor to both flies and butterflies.

International Journal of Evolutionary Biology, Dec 5, 2013

Differences between sexes of the same species are widespread and are variable in nature. While it... more Differences between sexes of the same species are widespread and are variable in nature. While it is often assumed that males are more ornamented than females, in the nymphalid butterfly genus Bicyclus, females have, on average, more eyespot wing color patterns than males. Here we extend these studies by surveying eyespot pattern sexual dimorphism across the Nymphalidae family of butterflies. Eyespot presence or absence was scored from a total of 38 wing compartments for two males and two females of each of 450 nymphalid species belonging to 399 different genera. Differences in eyespot number between sexes of each species were tallied for each wing surface (e.g., dorsal and ventral) of forewings and hindwings. In roughly 44% of the species with eyespots, females had more eyespots than males, in 34%, males had more eyespots than females, and, in the remaining 22% of the species, there was monomorphism in eyespot number. Dorsal and forewing surfaces were less patterned, but proportionally more dimorphic, than ventral and hindwing surfaces, respectively. In addition, wing compartments that frequently displayed eyespots were among the least sexually dimorphic. This survey suggests that dimorphism arises predominantly in "hidden" or "private" surfaces of a butterfly's wing, as previously demonstrated for the genus Bicyclus.

Developmental Biology, Oct 1, 2014

In insects, forewings and hindwings usually have different shapes, sizes, and color patterns. A v... more In insects, forewings and hindwings usually have different shapes, sizes, and color patterns. A variety of RNAi experiments across insect species have shown that the hox gene Ultrabithorax (Ubx) is necessary to promote hindwing identity. However, it remains unclear whether Ubx is sufficient to confer hindwing fate to forewings across insects. Here, we address this question by over-expressing Ubx in the butterfly Bicyclus anynana using a heat-shock promoter. Ubx whole-body over-expression during embryonic and larvae development led to body plan changes in larvae but to mere quantitative changes to adult morphology, respectively. Embryonic heat-shocks led to fused segments, loss of thoracic and abdominal limbs, and transformation of head limbs to larger appendages. Larval heat-shocks led to reduced eyespot size in the expected homeotic direction, but neither additional eyespots nor wing shape changes were observed in forewings as expected of a homeotic transformation. Interestingly, Ubx was found to be expressed in a novel, non-characteristic domainin the hindwing eyespot centers. Furthermore, ectopic expression of Ubx on the pupal wing activated the eyespot-associated genes spalt and Distal-less, known to be directly repressed by Ubx in the fly's haltere and leg primordia, respectively, and led to the differentiation of black wing scales. These results suggest that Ubx has been co-opted into a novel eyespot gene regulatory network, and that it is capable of activating black pigmentation in butterflies.

Science Advances, Jul 28, 2023

Wnt signaling members are involved in the differentiation of cells associated with eyespot and ba... more Wnt signaling members are involved in the differentiation of cells associated with eyespot and band color patterns on the wings of butterflies, but the identity and spatio-temporal regulation of specific Wnt pathway members remains unclear. Here, we explore the localization and function of Armadillo/β-catenin dependent (canonical) and Armadillo/β-catenin independent (noncanonical) Wnt signaling in eyespot and band development in Bicyclus anynana by localizing Armadillo (Arm), the expression of all eight Wnt ligand and four frizzled receptor transcripts present in the genome of this species and testing the function of some of the ligands and receptors using CRISPR-Cas9. We show that distinct Wnt signaling pathways are essential for eyespot and band patterning in butterflies and are likely interacting to control their active domains.

Humana Press eBooks, Oct 24, 2013

Journal of Insect Behavior, 2018

Palatable animals sometimes mimic the color patterns of noxious animals to gain protection from p... more Palatable animals sometimes mimic the color patterns of noxious animals to gain protection from predators. This phenomenon, known as Batesian mimicry, is seen in many butterflies of the genus Papilio, and in some species the mimicry is limited to females. Although female-limited Batesian mimicry has been hypothesized to be caused by females preferring to mate with non-mimetic males, this hypothesis is rarely tested. In this study, we tested whether female mate choice is driving female-limited Batesian mimicry in Papilio polytes. Males have white spots on the dorsal forewings, which are absent in mimetic female sand in the toxic model, Pachliopta aristolochiae. Hence, we conducted mate choice experiments to examine whether these white spots are important to females. We offered females a choice of males with intact dorsal forewing white spots and males with artificially blackened dorsal forewings, resembling the model. Females did not show a preference for males with the white spots, suggesting that they are not being maintained by female mate choice. Future studies should investigate the presence of female mate choice on other parts of males' wings to further understand the role of female mate choice, as well as explore other factors driving female-limited mimicry in these butterflies. Keywords Female mate choice. Papilio polytes. sex-limited mimicry. Batesian mimicry Body coloration plays a key role in survival of prey animals, such as in reducing their likelihood of being eaten by predators. In some noxious prey animals, survival is tied to the display of bright and contrasting (i.e. aposematic) colors that warn potential predators that they are unpleasant or dangerous and therefore unprofitable prey (Poulton

Proceedings of The Royal Society B: Biological Sciences, Apr 6, 2022

The yellow gene regulates behavioural plasticity by repressing male courtship in Bicyclus anynana... more The yellow gene regulates behavioural plasticity by repressing male courtship in Bicyclus anynana butterflies.

bioRxiv (Cold Spring Harbor Laboratory), Sep 5, 2017

Eyespots on the wings of nymphalid butterflies represent colorful examples of the process of patt... more Eyespots on the wings of nymphalid butterflies represent colorful examples of the process of pattern formation, yet the developmental origins and the mechanisms behind eyespot differentiation are still not fully understood. Here we reexamine the function of Distal-less (Dll) in eyespot development, which is still unclear. We show that CRISPR-Cas9 induced exon 2 mutations in Bicyclus anynana leads to exon skipping and ectopic eyespots on the wing. Exon 3 mutations, however, lead to null/missense transcripts, missing eyespots, lighter wing coloration, loss of scales, and a variety of other phenotypes implicating Dll in the process of eyespot differentiation. Reaction-diffusion modeling enabled exploration of the function of Dll in eyespot formation, and accurately replicated a wide-range of mutant phenotypes. These results confirm that Dll is a required activator of eyespot development, scale growth and melanization and point to a new mechanism of alternative splicing to achieve Dll overexpression phenotypes. The genetic and developmental origins of the bullseye color patterns on the wings of nymphalid butterflies are still poorly understood. Eyespots originated once in ancestors of this butterfly lineage, around 90 million years ago 1-3 , to most likely function as targets for deflecting predators away from the butterfly's vulnerable body 1,4,5. Eyespots may have originated via the co-option of a network of pre-wired genes because several of the genes associated with eyespots gained their novel expression domain concurrently with the origin of eyespots 3. Some of these genes have since lost their expression in eyespots, without affecting eyespot development, suggesting that they did not play a functional role in eyespot development from the very beginning 3. Yet, one of the genes, Distal-less (Dll), has remained associated with eyespots in most nymphalid species examined so far, suggesting that it may have played a functional role in eyespot origins 3,6. The function of Dll in eyespot development was initially investigated in B. anynana using transgenic over-expression, RNAi, and ectopic expression tools 7. Overexpressing Dll in B. anynana led to the appearance of small additional eyespots on the wing as well as larger eyespots, whereas Dll downregulation produced smaller eyespots, strongly implicating Dll as an activator of eyespot development 7. However, a recent study using CRISPR-Cas9 to knockout Dll function in the painted lady butterfly, Vanessa cardui contradicted these findings. Zhang and Reed (2016) 8 found that using two guides to disrupt exon 2 in Dll led to the appearance not only of distally extended eyespots but also of ectopic eyespots developing in novel locations on the wing. These observations led to a conclusion that Dll represses eyespot development. In addition, these researchers also showed that targeting the same exon in another butterfly, Junonia coenia, produced darker wing pigmentation, whereas the exact same phenotype was obtained via ectopic expression of Dll in the wings of B. anynana 7 and in the wings of J. orythia, a close relative of J. coenia 9. One possibility for the discrepancies seen across species is that Dll has precisely opposite functions in the different butterfly species. Another possibility, which we believed more likely, is that the outcomes of genome editing may depend on the particular site that is targeted in the genome to disrupt the gene's function. In order to clarify the function of Dll in B. anynana, we separately targeted both exon 2 (using single guide RNAs Sg1 and Sg2) and exon 3 (using Sg3), within the homeobox (Fig. 1a). While screening. CC-BY-NC-ND 4.

Cell Reports, Jul 1, 2022