Biology subjects

Monteiro, A.

Publications and source records attributed to Monteiro, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2022

Developmental exposure to a real-life environmental chemical mixture alters testicular transcription factor expression in neonatal and pre-pubertal rams, with morphological changes persisting into adulthood

Environmental chemical (EC) exposure may be impacting male reproductive health. The translationally relevant biosolids treated pasture (BTP) sheep model was used to investigate gestational low-level EC mixture exposure on the testis of F1 male offspring. Adult rams from ewes exposed to BTP 1 month before and throughout pregnancy had more seminiferous tubules with degeneration and depletion of elongating spermatids, indicating "recovery" from previously reported testicular dysgenesis syndrome-like phenotype in neonatal and pre-pubertal BTP lambs. Expression of transcription factors CREB1 (neonatal) and BCL11A and FOXP2 (pre-pubertal) were significantly higher in the BTP exposed testes, with no changes seen in the adults. Increased CREB1, which is crucial for testes development and regulation of steroidogenic enzymes, could be an adaptive response to gestational EC exposure to facilitate the phenotypic recovery. Overall, this demonstrates that testicular effects from gestational exposure to low-level mixtures of ECs can last into adulthood, potentially impacting fertility and fecundity.

pharmacology and toxicology↗

bioRxiv2022

The genetic basis of wing spots in Pieris canidia butterflies

Spots in pierid butterflies and eyespots in nymphalid butterflies are likely non-homologous wing colour pattern elements, yet they share a few features in common. Both develop black scales that depend on the function of the gene spalt, and both might have central signalling cells. This suggests that both pattern elements may be sharing common genetic circuitry. Hundreds of genes have already been associated with the development of nymphalid butterfly eyespot patterns, but the genetic basis of the simpler spot patterns on the wings of pierid butterflies has not been investigated. To facilitate studies of pierid wing patterns, we report a high-quality draft genome assembly for Pieris canidia, the Indian cabbage white. We then conducted transcriptomic analyses of pupal wing tissues sampled from the spot and non-spot regions of P. canidia at 3-6h post-pupation. A total of 1352 genes were differentially regulated between wing tissues with and without the black spot, including spalt, Kruppel-like factor 10, genes from the Toll, Notch, TGF-{beta}, and FGFR signalling pathways, and several genes involved in the melanin biosynthetic pathway. We identified 21 genes that are up-regulated in both pierid spots and nymphalid eyespots and propose that spots and eyespots share regulatory modules despite their likely independent origins.

evolutionary biology↗

bioRxiv2022

Lepidopteran prolegs are novel traits, not leg homologs

Lepidopteran larvae have both thoracic legs and abdominal prolegs, yet it is unclear whether these are serial homologs. We examined the role of three Hox genes in proleg development in Bicyclus anynana butterflies using CRISPR-Cas9 and discovered that under a partial segment abdominal-A (abd-A) knockout, both types of appendages can develop in the same segment, arguing for prolegs being a novel trait, not a leg serial homolog. We also discover that specificity protein (sp) genes do not co-localize with Distal-less (Dll) in prolegs, as they do in legs, and that the proleg gene-regulatory network (GRN) mostly resembles the head-horn GRN, another novel trait in the lepidoptera. We propose that larval prolegs evolved from the co-option of a partial limb GRN into novel embryonic coordinates in the abdomen. One-Sentence SummaryThe chubby legs of caterpillars are evolutionary innovations

evolutionary biology↗

bioRxiv2022

Detection and measurement of butterfly eyespot and spot patterns using convolutional neural networks

Butterflies are increasingly becoming model insects where basic questions surrounding the diversity of their color patterns are being investigated. Some of these color patterns consist of simple spots and eyespots. To accelerate the pace of research surrounding these discrete and circular pattern elements we developed two distinct convolutional neural networks (CNNs) for detection and measurement of butterfly spots and eyespots on digital images of butterfly wings. We tested the accuracy of the detection and of the area measurements using manual identifications and measurements. These methods were able to identify and distinguish marginal eyespots from spots, as well as distinguish these patterns from less symmetrical patches of color. In addition, the measurements of an eyespots central area and surrounding rings were highly accurate. These CNNs offer improvements of eyespot/spot detection and measurements relative to previous methods because it is not necessary to mathematically define the feature of interest. All that is needed is to point out the images that have those features to train the CNN. Author summaryWe developed two distinct convolutional neural networks (CNNs) for detection and measurement of butterfly spots and eyespots on digital images of butterfly wings. We tested the accuracy of the detection and of the area measurements using manual identifications and measurements. Our methods were able to identify and distinguish marginal eyespots from spots, as well as distinguish these patterns from less symmetrical patches of color. In addition, the measurements of an eyespots central area and surrounding rings were highly accurate.

systems biology↗

bioRxiv2022

Ultrabithorax modifies a regulatory network of genes essential for butterfly eyespot development in a wing sector-specific manner

Nymphalid butterfly species often have a different number of eyespots in forewings and hindwings but how the hindwing identity gene, Ultrabithorax (Ubx), drives this asymmetry, is not fully understood. It is also unclear why eyespot serial homologs originated initially only in hindwings. To address these questions, we examined a three-gene regulatory network (GRN) for eyespot development in the hindwings of Bicyclus anynana butterflies, and compared it to the same network previously described for forewings. We also examined how Ubx interacted with each of these three eyespot-essential genes. We found similar genetic interactions between the three genes in fore and hindwings, but we discovered three regulatory differences: Antennapedia (Antp) merely enhances spalt (sal) expression in the eyespot foci in hindwings but is not essential for sal activation, as in forewings; Ubx up- regulates Antp in all hindwing eyespot foci but represses Antp outside these wing regions and; Ubx is regulating sal expression in a wing-sector specific manner, i.e., it does not affect sal expression in the wing sectors that contain eyespots in both fore and hindwings, but it positively regulates sal expression in the sectors that have hindwing-specific eyespots. These results suggest that Ubx, or its downstream targets, might have paid a role in the origin of eyespots, restricted initially to hindwings, via the positive regulation of sal, an essential gene for eyespot development. We propose that Antp got co-opted into the eyespot GRN at a later stage by coming under Ubx regulation. This Hox gene redundancy, together with a novel positive feedback loop between sal and Antp, might have allowed Antp to functionally replace Ubx in forewings and lead to the origin of forewing eyespots. Outside the eyespot focal regions, we discovered that Ubx is up-regulated by Distal-less along the wing margin. We propose a model for how the regulatory connections between these genes might have evolved to produce wing- and sector-specific variation in eyespot number.

evolutionary biology↗

bioRxiv2022

Testicular Dysgenesis Syndrome-like morphology and gene expression, and activation of Hypoxia Inducible Factor 1 Alpha in juvenile lamb testes following developmental exposure to low-level environmental chemical mixture.

Current declines in male reproductive health may, in part, be driven by anthropogenic environmental chemical exposure. Using the biosolid treated pasture (BTP) sheep model, this study examined the effects of gestational exposure to a translationally relevant mixture of environmental chemicals. At 8 weeks of age, ram lambs gestationally exposed to BTP were lighter than control, and their testes contained fewer germ cells and had a greater proportion of Sertoli-cell-only seminiferous tubules. Effects of biosolid exposure on the testicular transcriptome correlated with human testicular dysgenesis syndrome (TDS) patient data. Common differentially expressed genes indicated changes in apoptotic and mTOR signalling, which concurs with previous gene expression data from neonatal BTP lambs. Gene expression data and immunohistochemistry indicates increased HIF1 activation and nuclear localisation in BTP exposed animals, known to disrupt testosterone synthesis. Together, these results provide a potential mechanism for the pathogenesis of this phenotype, and TDS in humans.

pharmacology and toxicology↗

bioRxiv2022

Butterfly wings exhibit spatial variation in chromatin accessibility

Butterfly wings exhibit a diversity of patterns which can vary between forewings and hindwings and spatially across the same wing. Regulation of morphological variation involves changes in how genes are expressed across different spatial scales which is driven by chromatin dynamics during development. How patterns of chromatin dynamics correspond to morphological variation remains unclear. Here we compared the chromatin landscape between forewings and hindwings and also across the proximal and distal regions of the hindwings in two butterfly species, Bicyclus anynana and Danaus plexippus. We found that the chromatin profile varied significantly between the different wing regions, however, there was no clear correspondence between the chromatin profile and the wing patterns. In some cases, wing regions with different phenotypes shared the same chromatin profile whereas those with a similar phenotype had a different profile. We also found that in the forewing, open chromatin regions (OCRs) were AT rich whereas those in the hindwing were GC rich. GC content of the OCRs also varied between the proximal and hindwing regions. These differences in GC content were also reflected in the transcription factor binding motifs that were differentially enriched between the wings and wing regions. Our results suggest that distinct wing patterns may result from the interaction of pioneer factors, including Hox genes, differentially opening chromatin in different wings and wing regions and cooperating with other transcriptions factors, that show preferences for specific GC content, to function either as activator or repressors of nearby genes.

genomics↗

bioRxiv2021

Antennapedia regulates metallic silver wing scale development and cell shape in Bicyclus anynana butterflies

Butterfly wing scale cells can develop very intricate cuticular nanostructures that interact with light to produce structural colors including silvery hues, but the genetic basis of such nanostructures is mostly unexplored. Here, we first identified that optix is necessary for metallic scale development in the butterfly Bicyclus anynana. We then characterized different subtypes of wildtype metallic silver scales and addressed the function of five genes - apterous A, Ultrabithorax, doublesex, Antennapedia, and optix - in the differentiation of silver scales, at a single-cell resolution, by leveraging crispants that exhibited either ectopic gains or losses of silver scales. Wildtype silver scales were generally rounded and had low amounts of pigmentation, exhibiting a common ultrastructural modification for metallic broadband reflectance, i.e., an undulatory air layer enclosed by an upper and lower lamina. Our results indicated that the varying air layer thickness was the important parameter of the bilaminate scale for producing a broadband reflectance across visible wavelengths. While a single lamina of the appropriate thickness could also produce broadband colors, the bilaminate structure is advantageous as it increases the overall reflectivity. Crispant brown scales differed from wildtype silver scales via the loss of the continuous upper lamina, increased lower lamina thickness, and increased pigmentation. The reverse was seen when brown scales became silver. We identified Antennapedia and optix as high-level regulators in the network differentiating different silver scale subtypes and determining overall cell shape in both sexes. In addition, Antp exhibits a novel, post-embryonic role in the determination of ridge and crossrib orientation.

developmental biology↗

bioRxiv2021

Reference genomes of 545 silkworms enable high-throughput exploring genotype-phenotype relationships

The silkworm Bombyx mori is a domestic insect for silk production and a lepidopteran model. The currently available genomes limit a full understanding of its genetic and phenotypic diversity. Here we assembled long-read genomes of 545 domestic and wild silkworms and constructed a high-resolution pan-genome dataset. We found that the silkworm population harbors extremely variable genomes containing 7,308 new gene families, 4,260 (22%) core gene families, and 3,432,266 non-redundant SVs. We deciphered a series of causal genes and variants associated with domestication, breeding, and ecological adaptation traits, and experimentally validated two of those genes using CRISPR-Cas9 or RNA interference. This unprecedented large-scale genomic resource allows for high-throughput screening of interesting traits for functional genomic research and breeding improvement of silkworms and may serve as a guideline for traits decoding in other species.

genomics↗

bioRxiv2021

Conserved roles of Distal-less and spalt in regulating butterfly melanic patterns

Two genes, Distal-less (Dll) and spalt (sal), are known to be involved in establishing nymphalid butterfly wing patterns. They function in several ways: in the differentiation of the eyespots central signaling cells, or foci; in the differentiation of the surrounding black disc; in overall scale melanisation (Dll); and in elaborating marginal patterns, such as parafocal elements. However, little is known about the functions of these genes in the development of wing patterns in other butterfly families. Here, we study the expression and function of Dll and sal in the development of spots and other melanic wing patterns of the Indian cabbage white, Pieris canidia, a pierid butterfly. In P. canidia, both Dll and Sal proteins are expressed in the scale-building cells at the wing tips, in chevron patterns along the pupal wing margins, and in areas of future scale melanisation. Additionally, Sal alone is expressed in the future black spots. CRISPR knockouts of Dll and sal showed that each gene is required for the development of melanic wing pattern elements, and repressing pteridine granule formation, in the areas where they are expressed. We conclude that both genes likely play ancestral roles in organising distal butterfly wing patterns, across pierid and nymphalid butterflies, but are unlikely to be differentiating signalling centers in pierids black spots. The genetic and developmental mechanisms that set up the location of spots and eyespots are likely distinct in each lineage.

evolutionary biology↗

bioRxiv2021

Developmental plasticity in male courtship in Bicyclus anynana butterflies is driven by hormone regulation of the yellow gene

The organizational role for hormones in the regulation of sexual behavior is currently poorly explored. Previous work showed that seasonal variation in levels of the steroid hormone 20-hydroxyecdysone (20E) during pupal development regulates plasticity in male courtship behavior in Bicyclus anynana butterflies. Wet season (WS) males, reared at high temperature, have high levels of 20-hydroxyecdysone (20E) during pupation and become active courters. Dry season (DS) males, reared at low temperatures, have lower levels of 20E and lower courtship rates. Rescue of WS courtship rates can be achieved via injection of 20E into DS male pupae, but it is still unknown whether 20E alters gene expression in the pupal brain, and if so, the identity of those targets. Using transcriptomics, qPCR, and behavioral assays with a transgenic knockout, we show that higher expression levels of the yellow gene in DS male pupal brains, relative to WS brains, represses courtship in DS males. Furthermore, injecting DS males with 20E downregulates yellow to WS levels 4 hours post-injection, revealing a hormone sensitive window that determines courtship behavior. These findings are in striking contrast to Drosophila, where yellow is required for active male courtship behavior. We conclude that 20E plays an organizational role during pupal brain development by regulating the expression of yellow, which is a repressor of the neural circuity for male courtship behavior in B. anynana. This work shows that similar to vertebrates, hormones can also play an organizational role in insect brains, leading to permanent changes in adult sexual behavior. Significance StatementBehavioral plasticity in adult insects is known to be regulated by hormones, which activate neural circuits in response to environmental cues. Here, we show that hormones can also regulate adult behavioral plasticity by altering gene expression during brain development, adjusting the insects behavior to predictable seasonal environmental variation. We show that seasonal changes in the hormone 20E alters expression of the yellow gene in the developing pupal brain of Bicyclus anynana butterflies, which leads to differences in male courtship behavior between the dry and wet seasonal forms. This work provides one of the first examples of the organizational role of hormones in altering gene expression and adult sexual behavior in the developing insect brain.

neuroscience↗

bioRxiv2021

The Hox gene Antennapedia regulates wing development through 20-hydroxyecdysone in insect

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.

developmental biology↗

bioRxiv2021

optix is involved in eyespot development via a possible positional information mechanism

Novel organismal traits might reuse ancestral gene-regulatory networks (GRNs) in their development, but data supporting this mechanism are still sparse. Here we show the reuse of an ancestral insect venation gene regulatory subnetwork patterning the sharp and distinct rings of color in butterfly eyespots. Using laser microdissection followed by RNA-Seq we first obtained transcriptional profiles of the anterior and posterior compartment of larval wings, and eyespot and adjacent control tissue in pupal wings of Bicyclus anynana butterflies. We identified key venation patterning genes such as Mothers against dpp 6 (Mad6), thickveins, Optix, spalt, optomotor-blind (omb), aristaless, cubitus interruptus, and patched differentially expressed (DE) across compartments, and a sub-set of these genes also DE across eyespot and non-eyespot samples. Fluorescent in-situ hybridization (HCR3.0) on the jointly DE genes Mad6, Optix, and spalt, as well as dpp showed clear eyespot-center, eyespot-rings, and compartment-specific expression. Knocking out dpp resulted in an individual with venation defects and loss of eyespots, whereas knockouts of Optix and spalt resulted in the loss of orange scales and black scales, respectively. Furthermore, using CRISPR-Cas9 followed by immunostainings, we showed that Spalt represses Optix in the central region of the eyespot, limiting Optix expression to a more peripheral ring, which parallels the regulatory interaction found in venation patterning in the anterior compartment of fly larval wings. These network similarities suggest that part of the venation GRN was co-opted to aid in the differentiation of the eyespot rings. One-sentence summaryWe showed the reuse of an ancestral insect wing venation GRN in patterning a novel complex trait in butterflies.

developmental biology↗

bioRxiv2021

Butterfly eyespots evolved via co-option of the antennal gene-regulatory network

Butterfly eyespots are beautiful novel traits with an unknown developmental origin. Here we show that eyespots likely originated via co-option of the antennal gene-regulatory network (GRN) to novel locations on the wing. Using comparative transcriptome analysis, we show that eyespots cluster with antennae relative to multiple other tissues. Furthermore, three genes essential for eyespot development (Distal-less (Dll), spalt (sal), and Antennapedia (Antp)) share similar regulatory connections as those observed in the antennal GRN. CRISPR knockout of cis-regulatory elements (CREs) for Dll and sal led to the loss of eyespots and antennae, and also legs and wings, demonstrating that these CREs are highly pleiotropic. We conclude that eyespots likely re-used the ancient antennal GRN, a network previously implicated also in the development of legs and wings.

evolutionary biology↗

bioRxiv2020

Molecular mechanism underlying venation patterning in butterflies

The mechanism of wing vein differentiation in Drosophila is a classic text-book example of pattern formation using a system of positional-information, yet very little is known about how this mechanism differs in species with a different number of veins and how insect venation patterns evolved. Here, we examine the expression patterns of genes previously implicated in vein differentiation in Drosophila in two butterfly species with more complex venation, the African squinting bush brown Bicyclus anynana and the Asian cabbage white, Pieris canidia. We also test the function of one of these genes, spalt (sal), with CRISPR-Cas9 in B. anynana. We identify both conserved as well as new domains of decapentaplegic (dpp), engrailed (en), invected (inv) and sal gene expression in B. anynana, and propose how the simplified venation in Drosophila might have evolved via loss of dpp and sal gene expression domains, silencing of vein inducing programs at Sal-expression boundaries, and changes in gene expression of vein maintenance genes. Summary statementThe paper describes new domains of venation patterning genes in butterflies and proposes how simplified venation in other insect lineages might have evolved.

developmental biology↗

bioRxiv2019

Hox genes are essential for the development of novel serial homologous eyespots on the wings of Bicyclus anynana butterflies

The eyespot patterns found on the wings of nymphalid butterflies are novel serial homologous traits that originated first in hindwings and subsequently in forewings, suggesting that eyespot development might be dependent on Hox genes. Hindwings differ from forewings in the expression of Ultrabithorax (Ubx), but the function of this Hox gene in eyespot development as well as that of another Hox gene Antennapedia (Antp), expressed specifically in eyespots centers on both wings, are still unclear. We used CRISPR-Cas9 to target both genes in Bicyclus anynana butterflies. We show that Antp is essential for eyespot development on the forewings and for the differentiation of white centers and larger eyespots on hindwings, whereas Ubx is essential for the development of at least some hindwing eyespots but also for repressing the size of other eyespots. Additionally, Antp is essential for the development of silver scales in male wings. In summary, Antp and Ubx, in addition to their conserved roles in modifying serial homologous traits along the anterior-posterior axis of animals, have acquired a novel role in promoting the development of a new set of serial homologs, the eyespot patterns, in both forewings (Antp) and hindwings (Antp and Ubx) of B. anynana butterflies. We propose that the peculiar pattern of eyespot origins on hindwings first, followed by forewings, could be due to an initial co-option of Ubx into eyespot development, followed by a later, partially redundant co-option of Antp into the same network.

evolutionary biology↗

bioRxiv2019

Doublesex mediates the development of sex-specific pheromone organs in Bicyclus butterflies via multiple mechanisms

The Bicyclus lineage of satyrid butterflies exhibits male-specific traits, the scent organ complex, used for chemical communication during courtship. This complex consists of tightly packed brush-like scales (hair-pencils) that rub against scent patches to disperse pheromones, but the evolution and molecular basis of the organs male-limited development remains unknown. Here, we examine the evolution of the number and location of the scent patches and hair-pencils within 53 species of Bicyclus butterflies, and the involvement of the sex determinant gene doublesex (dsx) in scent organ development in Bicyclus anynana using CRISPR/Cas9. We show that scent patches and hair-pencils arose via multiple, independent gains, in a correlated manner. Further, an initially non-sex-specific Dsx protein expression pattern in developing wing discs becomes male-specific and spatially refined to areas that develop the scent organ complex over the course of development. Functional perturbations of dsx show that this gene is required for male patch development whereas hair-pencils can develop in both sexes without Dsx input. Dsx in females is, instead, required to repress hair-pencils. These findings suggest that the patches and hair-pencils evolve as correlated composite organs that are sex-limited via the spatial regulation of dsx. Divergence in the function of dsx isoforms occurs in both sexes, where the male isoform promotes patch development in males and the female isoform represses hair-pencil development in females, both leading to the development of male-limited traits. Furthermore, evolution in number and location of patches, but not of hair-pencils, appears to be regulated by spatial regulation of dsx.

evolutionary biology↗

bioRxiv2019

Multiple loci control eyespot number variation on the hindwings of Bicyclus anynana butterflies

The underlying genetic changes that regulate the appearance and disappearance of repeated traits, or serial homologs, remain poorly understood. One hypothesis is that variation in genomic regions flanking master regulatory genes, also known as input-output genes, controls variation in trait number, making the locus of evolution almost predictable. Other hypotheses implicate genetic variation in up-stream or downstream loci of master control genes. Here, we use the butterfly Bicyclus anynana, a species which exhibits natural variation in eyespot number on the dorsal hindwing, to test these two hypotheses. We first estimated the heritability of dorsal hindwing eyespot number by breeding multiple butterfly families differing in eyespot number, and regressing eyespot number of offspring on mid-parent values. We then estimated the number and identity of independent genetic loci contributing to eyespot number variation by performing a genome-wide association study with restriction site-associated DNA Sequencing (RAD-seq) from multiple individuals varying in number of eyespots sampled across a freely breeding lab population. We found that dorsal hindwing eyespot number has a moderately high heritability of approximately 0.50. In addition, multiple loci near previously identified genes involved in eyespot development display high association with dorsal hindwing eyespot number, suggesting that homolog number variation is likely determined by regulatory changes at multiple loci that build the trait and not by variation at single master regulators or input-output genes.\n\nData accessibilityThe Bicyclus anynana PstI RAD-tag sequencing data is available via the Genbank BioProject PRJNA509697. Genotype VCF files will be made available through Figshare upon acceptance.

genetics↗