Biology subjects

Araujo, H. M.

Publications and source records attributed to Araujo, H. M..

6 recordsLinked to original sources

bioRxiv2026

Rp-vasa: a bona fide Primordial Germ Cell marker that drives embryonic expression in the Chagas disease vector Rhodnius prolixus

Rhodnius prolixus is an insect vector of the protozoan Trypanosoma cruzi, the causative agent of debilitating Chagas disease, which is transmitted to humans during blood feeding. Identifying germline markers is a critical step in advancing vector control and transgenic technologies of these medically important insects. Transmission of genetic traits to the next generation requires proper differentiation of the germline that gives rise to gametes. Germline precursors are established during early stages of development as the primordial germ cell (PGC) population. Among the genes required for this process, vasa homologues exert a conserved role in germline specification. Here, we characterize and validate the genomic structure of the R. prolixus Rp-vasa locus and assess its expression during early embryogenesis. We observe widespread Rp-vasa expression in preblastoderm embryos. Later, during the cellular blastoderm and at the beginning of gastrulation, Rp-vasa and Rp-piwi2 expression is restricted to PGCs, morphologically identifiable as a cluster of cells at the posterior of the embryo. We also report, for the first time, the use of R. prolixus regulatory sequences to drive the expression of exogenous genes. We identify the Rp-vasa regulatory region and show that these cis-regulatory sequences are sufficient to drive Cas9 and dsRed expression in the early embryo. Together, these findings demonstrate that Rp-vasa has great potential for use as a PGC marker and as a driver for gene expression in transgenic and gene editing approaches for Triatomine vectors.

developmental biology↗

bioRxiv2025

Sex-biased Transcriptome in in vitro Produced Bovine Early Embryos

BackgroundMorphologic sex differences between males and females typically emerge after the primordial germ cell migration and gonad formation, although sex is determined at fertilization based on chromosome composition. A key debated sexual difference is the embryonic developmental rate, with in vitro produced male embryos often developing faster. However, the molecular mechanisms driving early embryonic sex differences remain unclear. ResultsTo investigate the transcriptional sex difference during early development, in vitro produced bovine blastocysts were collected and sexed by PCR. A significant male-biased development was observed in expanded blastocysts. Ultra-low input RNA-seq analysis identified 837 DEGs, with 231 upregulated and 606 downregulated in males. Functional enrichment analysis revealed male-biased DEGs were associated with metabolic regulation, whereas female-biased DEGs were related to female gonad development, sex differentiation, inflammatory pathways, and TGF-beta signaling. Comparing X chromosome and autosome expression ratio, we found that female-biased DEGs contributed to the higher X-linked gene dosage, a phenomenon not observed in male embryos. Moreover, we identified the sex-biased transcription factors and RNA-bind proteins, including pluripotent factors such as SOX21 and PRDM14, and splicing factors FMR1 and HNRNPH2. Additionally, we revealed 1,555 significantly sex-biased differential alternative splicing (AS), predominantly skipped exons, mapped to 906 genes, with 59 overlapping with DEGs enriched in metabolic and autophagy pathways. By incorporating novel isoforms from long reads sequencing, we identified 1,151 sex-biased differentially expressed isoforms (DEIs) associated with 1,017 genes. Functional analysis showed that female-biased DEIs were involved in the negative regulation of transcriptional activity, while male-biased DEIs were related to energy metabolism. Furthermore, we identified sex-biased differential exon usage in DENND1B, DIS3L2, DOCK11, IL1RAPL2, and ZRSR2Y, indicating their sex-specific regulation in early embryo development. ConclusionThis study provided a comprehensive analysis of transcriptome differences between male and female bovine blastocysts, integrating sex-biased gene expression, alternative splicing, and isoform dynamics. Our findings indicate that enriched metabolism processes in male embryos may contribute to the faster developmental pace, providing insights into sex-specific regulatory mechanisms during early embryogenesis. Plain English summaryMale and female early embryos develop at different speeds, with male embryos often developing faster than female embryos. However, the reasons behind these early differences remain unclear. In this study, we examined gene activity in bovine embryos to uncover the biological factors regulating these early sex differences. We collected in vitro-produced bovine blastocysts, examined their sex, and confirmed that male embryos develop faster. By analyzing global gene activity, including alternative splicing, which allows one gene to code for multiple RNA isoforms and proteins, we found distinct gene expression profiles between male and female embryos. Male embryos showed higher activity in genes related to metabolism and cellular functions, while female embryos had increased activity in genes associated with female-specific gonad development and gene expression regulation. We also examined differences in how genes on the X chromosome were expressed. Female embryos had higher X-linked gene expression, which may contribute to sex-specific developmental regulation. Additionally, we identified sex-specific transcription factors and RNA-binding proteins that regulate early embryo development, some of which are known to control pluripotency and gene splicing. Overall, our study provides new insights into how gene activity shapes early sex differences, suggesting that enhanced metabolism in male embryos may be a key driver of their faster developmental rate. HighlightsO_LIMale embryos develop faster due to increased gene expression in metabolism pathways C_LIO_LIFemale embryos exhibit higher X-linked gene expression, suggesting X-dosage compensation plays a role in early development C_LIO_LISex-biased alternative splicing events contribute to embryonic metabolism, autophagy, and transcriptional regulation in embryos C_LIO_LISex-biased isoform diversity contributes to distinct developmental regulation in male and female embryos C_LIO_LIKey pluripotency factors (SOX21, PRDM14) and splicing regulators (FMR1, HNRNPH2) drive sex-specific gene expression C_LI

developmental biology↗

bioRxiv2023

Gene editing in the Chagas disease vector Rhodnius prolixus by Cas9-mediated ReMOT Control

Rhodnius prolixus is currently the model vector of choice for studying Chagas disease transmission, a debilitating disease caused by Trypanosoma cruzi parasites. Despite the broad use of double-stranded RNA interference for the knockdown of gene function in R. prolixus, transgenesis and gene editing protocols are still lacking. Here we tested Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) and direct parental injection of CRISPR (DIPA-CRISPR) for the maternal delivery of CRISPR/Cas9 elements to developing R. prolixus oocytes, and strategies for the identification of insertions/deletions (indels) in target loci of resulting gene-edited G0 nymphs. We demonstrate successful ReMOT Control-mediated gene editing of the eye color markers Rp-scarlet and Rp-white, and the cuticle color marker Rp-yellow, with highest effectiveness obtained using the ovary-targeting BtKV ligand. These results provide proof-of-concepts for generating somatic mutations in R. prolixus and potentially for generating germline edited lines in triatomines. Our studies also suggest that optimal strategies for recovery of mutations include performing multiple gRNA injections and the use of visible phenotypes such as those displayed in the Rp-scarlet, Rp-white and Rp-yellow loci for future Co-CRISPR experiments. These results will lay the foundation for gene editing protocols for triatomines and could lead to the development of novel control strategies for vectors of Chagas disease. Author SummaryRhodnius prolixus is an insect vector of the protozoan Trypanossoma cruzi, causative agent of debilitating Chagas disease. To fight the spread of the disease, it has been suggested that biological control of the insect should be attempted. Gene editing by the novel CRISPR methodology holds great promise in this sense, as it enables to target almost any gene in the genome for mutagenesis, thus allowing the control of insect physiology and reproduction. Here we have tested protocols for the delivery of CRISPR reagents as an attempt to enable genome editing of the vector. Our results show that maternal delivery of CRISPR by the ReMOT Control method is efficient for mutating eye and cuticle color genes in the resulting nymphs, generating edited animals with red eyes, white eyes or a yellow cuticle. This is the first report of gene editing in a vector of Chagas disease and should lay the basis to produce modified animals either unable to carry the T. cruzi parasite or to reproduce.

molecular biology↗

bioRxiv2020

Pigmentation loci as markers for genome editing in the Chagas disease vector Rhodnius prolixus

The kissing bug Rhodnius prolixus is a major vector for Chagas disease in the Americas, and also considered as the primary model for functional studies. Prospective transgenic approaches and genome editing strategies hold great promise for controlling insect populations as well as disease propagation. In this context, identifying visible genetic markers for transgenic methodologies is of paramount importance to advance the field. Here we have identified and analyzed the function of putative cuticle and eye color genes by investigating the effect of gene knockdown on fertility, viability, and the generation of visible phenotypes. Synthesis of the dark, yellow and tan pigments present in the cuticle of most insects depends on the function of key genes encoding enzymes in the tyrosine pathway. Knockdown of the R. prolixus yellow and aaNAT/pro orthologs produces striking alterations in cuticle color. Surprisingly, knockdown of ebony does not generate visible phenotypes. Since loss of ebony function results in a dark cuticle in several insect orders, we conclude that R. prolixus evolved alternative strategies for cuticle coloration, possibly including the loss of a pigmentation function for an entire branch of the tyrosine pathway. Knockdown of the scarlet and brown genes - encoding ABC transporters - alters cuticle and eye pigmentation, implying that the transport of pigment into proper organelles is an important process both for cuticle and eye coloration in this species. Therefore, this analysis identifies for the first time potential visible markers for transgenesis in a hemipteran vector for a debilitating human disease. Author SummaryThe hemipteran Rhodnius prolixus - also known as a kissing bug - is a main vector transmitting the parasite Trypanosoma cruzi, the causative agent of Chagas disease, a debilitating infection estimated to affect more than 6 million people in Central and South America. In order to limit disease spread, an important measure is insect vector control. However, kissing bugs - like other insects - develop resistance to insecticides. Alternative strategies based on transgenesis and the recently developed CRISPR- based genome edition hold great promise to control vector population or generate parasite-resistant insects. For these approaches to be feasible in R. prolixus, it is critical to identify visible phenotypic markers. Here we identify and describe several genes controlling cuticle and eye pigmentation that are well-suited putative landing sites for transformation strategies. Among these, loss-of-function mutations in the ABC transporter encoding scarlet and the tyrosine pathway enzyme encoding aaNAT/pro generate striking and easily visible phenotypes. Importantly, the knockdown of these genes does not affect insect viability and fertility under laboratory conditions. Our results suggest that R. prolixus has developed alternative strategies for cuticle coloration involving the loss of an entire branch of tanning loci, while the other branch producing cuticle patterns by generating non-pigmented areas has gained critical importance.

genetics↗

bioRxiv2020

A Reaction-Diffusion Network model predicts a dual role of Cactus/IκB to regulate Dorsal/NFκB nuclear translocation in Drosophila

Dorsal-ventral patterning of the Drosophila embryo depends on the NF{kappa}B superfamily transcription factor Dorsal (Dl). Toll receptor activation signals for degradation of the I{kappa}B inhibitor Cactus (Cact), leading to a ventral-to-dorsal nuclear Dl gradient. Cact is critical for Dl nuclear import, as it binds to and prevents Dl from entering the nuclei. Quantitative analysis of cact mutants revealed an additional Cact function to promote Dl nuclear translocation in ventral regions of the embryo. To investigate this dual Cact role, we developed a predictive model based on a reaction-diffusion regulatory network. This network considers non-uniform Toll activation as well as Toll-dependent Dl nuclear import and Cact degradation. In addition, it incorporates translational control of Cact levels by Dl, a Toll-independent pathway for Cact regulation and reversible nuclear-cytoplasmic Dl flow. Our model successfully reproduces wild-type data and emulates the Dl nuclear gradient in mutant dl and cact allelic combinations. Our results indicate that the dual role of Cact depends on targeting distinct Dl complexes along the dorsal-ventral axis: In the absence of Toll activation, free Dl-Cact trimers inhibit direct Dl nuclear entry; upon ventral-lateral Toll activation, Dl-Cact trimers are recruited into predominant signaling complexes and promote active Dl nuclear translocation. Simulations suggest that Toll-independent regulatory mechanisms that target Cact are fundamental to reproduce the full assortment of Cact effects. Considering the high evolutionary conservation of these pathways, our analysis should contribute to understand NF{kappa}B/c-Rel activation in other contexts such as in the vertebrate immune system and disease.

developmental biology↗

bioRxiv2019

Multiple roles of the polycistronic gene tarsaless/mille-pattes/polished-rice during embryogenesis of the kissing bug Rhodnius prolixus

Genes encoding small open-reading frames (smORFs) have been characterized as essential players of developmental processes. The smORF tarsaless/mille-pattes/polished-rice has been thoroughly investigated in holometabolous insects, such as the fruit fly Drosophila melanogaster and the red flour beetle Tribolium castaneum, while its function in hemimetabolous insects remains unknown. Thus, we analyzed the function of the tal/pri/mlpt ortholog in a hemimetabolous insect, the kissing bug Rhodnius prolixus (Rp). First, sequence analysis shows that Rp-tal/pri/mlpt polycistronic mRNA encodes two small peptides (11 to 14 amino acids) containing a LDPTG motif. Interestingly, a new hemipteran-specific conserved peptide of approximately 80 amino acids was also identified by in silico analysis. In silico docking analysis supports the high-affinity binding of the small LDPTG peptides to the transcription factor Shavenbaby. Rp-tal/pri/mlpt in situ hybridization and knockdown via RNA interference showed a conserved role of Rp-tal/pri/mlpt during embryogenesis, with a major role in the regulation of thoracic versus abdominal segmentation, leg development and head formation. Altogether, our study shows that tal/pri/mlpt segmentation role is conserved in the common ancestor of Paraneoptera and suggests that polycistronic genes might generate order specific smORFs.

developmental biology↗