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Bruce S Baker - One of the best experts on this subject based on the ideXlab platform.
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constraints on the evolution of a Doublesex target gene arising from Doublesex s pleiotropic deployment
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Shengzhan D Luo, Bruce S BakerAbstract:“Regulatory evolution,” that is, changes in a gene’s expression pattern through changes at its regulatory sequence, rather than changes at the coding sequence of the gene or changes of the upstream transcription factors, has been increasingly recognized as a pervasive evolution mechanism. Many somatic sexually dimorphic features of Drosophila melanogaster are the results of gene expression regulated by the Doublesex (dsx) gene, which encodes sex-specific transcription factors (DSXF in females and DSXM in males). Rapid changes in such sexually dimorphic features are likely a result of changes at the regulatory sequence of the target genes. We focused on the Flavin-containing monooxygenase-2 (Fmo-2) gene, a likely direct dsx target, to elucidate how sexually dimorphic expression and its evolution are brought about. We found that dsx is deployed to regulate the Fmo-2 transcription both in the midgut and in fat body cells of the spermatheca (a female-specific tissue), through a canonical DSX-binding site in the Fmo-2 regulatory sequence. In the melanogaster group, Fmo-2 transcription in the midgut has evolved rapidly, in contrast to the conserved spermathecal transcription. We identified two cis-regulatory modules (CRM-p and CRM-d) that direct sexually monomorphic or dimorphic Fmo-2 transcription, respectively, in the midguts of these species. Changes of Fmo-2 transcription in the midgut from sexually dimorphic to sexually monomorphic in some species are caused by the loss of CRM-d function, but not the loss of the canonical DSX-binding site. Thus, conferring transcriptional regulation on a CRM level allows the regulation to evolve rapidly in one tissue while evading evolutionary constraints posed by other tissues.
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central brain neurons expressing Doublesex regulate female receptivity in drosophila
Neuron, 2014Co-Authors: Chuan Zhou, Carmen C Robinett, Geoffrey W Meissner, Bruce S BakerAbstract:Summary Drosophila melanogaster females respond to male courtship by either rejecting the male or allowing copulation. The neural mechanisms underlying these female behaviors likely involve the integration of sensory information in the brain. Because Doublesex ( dsx ) controls other aspects of female differentiation, we asked whether dsx -expressing neurons mediate virgin female receptivity to courting males. Using intersectional techniques to manipulate the activities of defined subsets of dsx -expressing neurons, we found that activation of neurons in either the pCd or pC1 clusters promotes receptivity, while silencing these neurons makes females unreceptive. Furthermore, pCd and pC1 neurons physiologically respond to the male-specific pheromone cis -vaccenyl acetate (cVA), while pC1 neurons also respond to male courtship song. The pCd and pC1 neurons expressing dsx in females do not express transcripts from the fruitless ( fru ) P1 promoter. Thus, virgin female receptivity is controlled at least in part by neurons that are distinct from those governing male courtship.
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Doublesex functions early and late in gustatory sense organ development
PLOS ONE, 2012Co-Authors: Bruce S Baker, Carmen C Robinett, David J MellertAbstract:Somatic sexual dimorphisms outside of the nervous system in Drosophila melanogaster are largely controlled by the male- and female-specific Doublesex transcription factors (DSXM and DSXF, respectively). The DSX proteins must act at the right times and places in development to regulate the diverse array of genes that sculpt male and female characteristics across a variety of tissues. To explore how cellular and developmental contexts integrate with Doublesex (dsx) gene function, we focused on the sexually dimorphic number of gustatory sense organs (GSOs) in the foreleg. We show that DSXM and DSXF promote and repress GSO formation, respectively, and that their relative contribution to this dimorphism varies along the proximodistal axis of the foreleg. Our results suggest that the DSX proteins impact specification of the gustatory sensory organ precursors (SOPs). DSXF then acts later in the foreleg to regulate gustatory receptor neuron axon guidance. These results suggest that the foreleg provides a unique opportunity for examining the context-dependent functions of DSX.
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joint control of drosophila male courtship behavior by motion cues and activation of male specific p1 neurons
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Geoffrey W Meissner, Bruce S BakerAbstract:Sexual behaviors in animals are governed by inputs from multiple external sensory modalities. However, how these inputs are integrated to jointly control animal behavior is still poorly understood. Whereas visual information alone is not sufficient to induce courtship behavior in Drosophila melanogaster males, when a subset of male-specific fruitless (fru)- and Doublesex (dsx)-expressing neurons that respond to chemosensory cues (P1 neurons) were artificially activated via a temperature-sensitive cation channel (dTRPA1), males followed and extended their wing toward moving objects (even a moving piece of rubber band) intensively. When stationary, these objects were not courted. Our results indicate that motion input and activation of P1 neurons are individually necessary, and under our assay conditions, jointly sufficient to elicit early courtship behaviors, and provide insights into how courtship decisions are made via sensory integration.
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turning males on activation of male courtship behavior in drosophila melanogaster
PLOS ONE, 2011Co-Authors: Carmen C Robinett, Bruce S BakerAbstract:The innate sexual behaviors of Drosophila melanogaster males are an attractive system for elucidating how complex behavior patterns are generated. The potential for male sexual behavior in D. melanogaster is specified by the fruitless (fru) and Doublesex (dsx) sex regulatory genes. We used the temperature-sensitive activator dTRPA1 to probe the roles of fruM- and dsx-expressing neurons in male courtship behaviors. Almost all steps of courtship, from courtship song to ejaculation, can be induced at very high levels through activation of either all fruM or all dsx neurons in solitary males. Detailed characterizations reveal different roles for fruM and dsx in male courtship. Surprisingly, the system for mate discrimination still works well when all dsx neurons are activated, but is impaired when all fruM neurons are activated. Most strikingly, we provide evidence for a fruM-independent courtship pathway that is primarily vision dependent.
Artyom Kopp - One of the best experts on this subject based on the ideXlab platform.
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modular tissue specific regulation of Doublesex underpins sexually dimorphic development in drosophila
Development, 2019Co-Authors: Olga Barmina, Michelle N. Arbeitman, Gavin R Rice, David Michael Luecke, Artyom KoppAbstract:The ability of a single genome to produce distinct and often dramatically different male and female forms is one of the wonders of animal development. In Drosophila melanogaster, most sexually dimorphic traits are controlled by sex-specific isoforms of the Doublesex (dsx) transcription factor, and dsx expression is mostly limited to cells that give rise to sexually dimorphic traits. However, it is unknown how this mosaic of sexually dimorphic and monomorphic organs arises. Here, we characterize the cis-regulatory sequences that control dsx expression in the foreleg, which contains multiple types of sex-specific sensory organs. We find that separate modular enhancers are responsible for dsx expression in each sexually dimorphic organ. Expression of dsx in the sex comb is co-regulated by two enhancers with distinct spatial and temporal specificities that are separated by a genitalia-specific enhancer. The sex comb-specific enhancer from D. willistoni, a species that primitively lacks sex combs, is not active in the foreleg. Thus, the mosaic of sexually dimorphic and monomorphic organs depends on modular regulation of dsx transcription by dedicated cell type-specific enhancers.
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modular tissue specific regulation of Doublesex underpins sexually dimorphic development in drosophila
bioRxiv, 2019Co-Authors: Olga Barmina, Michelle N. Arbeitman, Gavin R Rice, David Michael Luecke, Artyom KoppAbstract:Abstract The ability of a single genome to produce distinct and often dramatically different male and female forms is one of the wonders of animal development. In most animals, sex-specific phenotypes are shaped by interactions between a sex determination pathway and spatial patterning gene networks. In Drosophila melanogaster, most sexually dimorphic traits are controlled by sex-specific isoforms of the Doublesex (dsx) transcription factor, and dsx expression is mostly limited to cells that give rise to sexually dimorphic traits. However, it is unknown how this mosaic of “sex-naive” and “sex-aware” tissues arises. Here, we characterize the cis-regulatory sequences that control dsx expression in the foreleg, which contains multiple types of sex-specific sensory organs. We find that separate modular enhancers are responsible for dsx expression in each sexually dimorphic organ. Expression of dsx in the sex comb is co-regulated by two enhancers with distinct spatial and temporal specificities that are separated by a genitalia-specific enhancer. Thus, the mosaic of sexually dimorphic and monomorphic organs depends on modular regulation of dsx transcription by dedicated cell type-specific enhancers. Summary Statement We identify the modular cis-regulatory elements that direct expression of Doublesex in sexually dimorphic structures in Drosophila legs and genitalia. This regulatory landscape provides insight into how cells obtain their sex-specific identity.
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dmrt genes in the development and evolution of sexual dimorphism
Trends in Genetics, 2012Co-Authors: Artyom KoppAbstract:Most animals are sexually dimorphic, but different taxa have different sex-specific traits. Despite major differences in the genetic control of sexual development among animal lineages, the Doublesex/mab-3 related (Dmrt) family of transcription factors has been shown to be involved in sex-specific differentiation in all animals that have been studied. In recent years the functions of Dmrt genes have been characterized in many animal groups, opening the way to a broad comparative perspective. This review focuses on the similarities and differences in the functions of Dmrt genes across the animal kingdom. I highlight a number of common themes in the sexual development of different taxa, discuss how Dmrt genes have acquired new roles during animal evolution, and show how they have contributed to the origin of novel sex-specific traits.
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Evolution of Sex-Specific Traits through Changes in HOX-Dependent Doublesex Expression
PLoS biology, 2011Co-Authors: Kohtaro Tanaka, Olga Barmina, Laura E. Sanders, Michelle N. Arbeitman, Artyom KoppAbstract:Almost every animal lineage is characterized by unique sex-specific traits, implying that such traits are gained and lost frequently in evolution. However, the genetic mechanisms responsible for these changes are not understood. In Drosophila, the activity of the sex determination pathway is restricted to sexually dimorphic tissues, suggesting that spatial regulation of this pathway may contribute to the evolution of sex-specific traits. We examine the regulation and function of Doublesex (dsx), the main transcriptional effector of the sex determination pathway, in the development and evolution of Drosophila sex combs. Sex combs are a recent evolutionary innovation and show dramatic diversity in the relatively few Drosophila species that have them. We show that dsx expression in the presumptive sex comb region is activated by the HOX gene Sex combs reduced (Scr), and that the male isoform of dsx up-regulates Scr so that both genes become expressed at high levels in this region in males but not in females. Precise spatial regulation of dsx is essential for defining sex comb position and morphology. Comparative analysis of Scr and dsx expression reveals a tight correlation between sex comb morphology and the expression patterns of both genes. In species that primitively lack sex combs, no dsx expression is observed in the homologous region, suggesting that the origin and diversification of this structure were linked to the gain of a new dsx expression domain. Two other, distantly related fly lineages that independently evolved novel male-specific structures show evolutionary gains of dsx expression in the corresponding tissues, where dsx may also be controlled by Scr. These findings suggest that changes in the spatial regulation of sex-determining genes are a key mechanism that enables the evolution of new sex-specific traits, contributing to some of the most dramatic examples of phenotypic diversification in nature.
Lucas Sánchez - One of the best experts on this subject based on the ideXlab platform.
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an unusual role for Doublesex in sex determination in the dipteran sciara
Genetics, 2015Co-Authors: María Fernanda Ruiz, Francesca Sarno, Jose M Eirinlopez, Mercedes Alvarez, Leonor Kremer, Jose Luis Barbero, Lucas SánchezAbstract:The gene Doublesex, which is placed at the bottom of the sex-determination gene cascade, plays the ultimate discriminatory role for sex determination in insects. In all insects where this gene has been characterized, the dsx premessenger RNA (pre-mRNA) follows a sex-specific splicing pattern, producing male- and female-specific mRNAs encoding the male-DSXM and female-DSXF proteins, which determine male and female development, respectively. This article reports the isolation and characterization of the gene Doublesex of dipteran Sciara insects. The Sciara Doublesex gene is constitutively transcribed during development and adult life of males and females. Sciara had no sex-specific Doublesex mRNAs but the same transcripts, produced by alternative splicing of its primary transcript, were present in both sexes, although their relative abundance is sex specific. However, only the female DSXF protein, but not the male DSXM protein, was produced at similar amounts in both sexes. An analysis of the expression of female and male Sciara DSX proteins in Drosophila showed that these proteins conserved female and male function, respectively, on the control of Drosophila yolk-protein genes. The molecular evolution of gene Doublesex of all insects where this gene has been characterized revealed that Sciara Doublesex displays a considerable degree of divergence in its molecular organization and its splicing pattern with respect to the rest of dipterans as suggested by its basal position within the Doublesex phylogeny. It is suggested that the Doublesex gene is involved in Sciara sex determination although it appears not to play the discriminatory role performed in other insects.
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the gene transformer 2 of anastrepha fruit flies diptera tephritidae and its evolution in insects
BMC Evolutionary Biology, 2010Co-Authors: Francesca Sarno, André L.p. Perondini, Denise Selivon, María Fernanda Ruiz, Jose M Eirinlopez, Lucas SánchezAbstract:Background In the tephritids Ceratitis, Bactrocera and Anastrepha, the gene transformer provides the memory device for sex determination via its auto-regulation; only in females is functional Tra protein produced. To date, the isolation and characterisation of the gene transformer-2 in the tephritids has only been undertaken in Ceratitis, and it has been shown that its function is required for the female-specific splicing of Doublesex and transformer pre-mRNA. It therefore participates in transformer auto-regulatory function. In this work, the characterisation of this gene in eleven tephritid species belonging to the less extensively analysed genus Anastrepha was undertaken in order to throw light on the evolution of transformer-2.
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Effect of the gene Doublesex of anastrepha on the somatic sexual development of Drosophila.
PLOS ONE, 2009Co-Authors: Mercedes Alvarez, María Fernanda Ruiz, Lucas SánchezAbstract:Background The gene Doublesex (dsx) is at the bottom of the sex determination genetic cascade and is transcribed in both sexes, but gives rise to two different proteins, DsxF and DsxM, which impose female and male sexual development respectively via the sex-specific regulation of the so-called sexual cyto-differentiation genes. The present manuscript addressed the question about the functional conservation of the tephritid Anastrepha DsxF and DsxM proteins to direct the sexual development in Drosophila (Drosophilidae). Methodology To express these proteins in Drosophila, the GAL4-UAS system was used. The effect of these proteins was monitored in the sexually dimorphic regions of the fly: the foreleg basitarsus, the 5th, 6th and 7th tergites, and the external terminalia. In addition, we analysed the effect of Anastrepha DsxF and DsxM proteins on the regulation of Drosophila yolk protein genes, which are expressed in the fat body of adult females under the control of dsx. Conclusions The Anastrepha DsxF and DsxM proteins transformed Doublesex intersexual Drosophila flies into females and males respectively, though this transformation was incomplete and the extent of their influence varied in the different sexually dimorphic regions of the adult fly. The Anastrepha DsxF and DsxM proteins also behaved as activators and repressors, respectively, of the Drosophila yolk protein genes, as do the DsxF and DsxM proteins of Drosophila itself. Finally, the Anastrepha DsxF and DsxM proteins were found to counteract the functions of Drosophila DsxM and DsxF respectively, reflecting the normal behaviour of the latter proteins towards one another. Collectively, these results indicate that the Anastrepha DsxF and DsxM proteins show conserved female and male sex-determination function respectively in Drosophila, though it appears that they cannot fully substitute the latter's own Dsx proteins. This incomplete function might be partly due to a reduced capacity of the Anastrepha Dsx proteins to completely control the Drosophila sexual cyto-differentiation genes, a consequence of the accumulation of divergence between these species resulting in the formation of different co-adapted complexes between the Dsx proteins and their target genes.
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the gene transformer of anastrepha fruit flies diptera tephritidae and its evolution in insects
PLOS ONE, 2007Co-Authors: María Fernanda Ruiz, André L.p. Perondini, Denise Selivon, Andreina Milano, Marco Salvemini, Jose M Eirinlopez, Catello Polito, Giuseppe Saccone, Lucas SánchezAbstract:Background: In the tephritids Ceratitis, Bactrocera and Anastrepha, the gene transformer provides the memory device for sex determination via its auto-regulation; only in females is functional Tra protein produced. To date, the isolation and characterisation of the gene transformer-2 in the tephritids has only been undertaken in Ceratitis, and it has been shown that its function is required for the female-specific splicing of Doublesex and transformer pre-mRNA. It therefore participates in transformer auto-regulatory function. In this work, the characterisation of this gene in eleven tephritid species belonging to the less extensively analysed genus Anastrepha was undertaken in order to throw light on the evolution of transformer-2. Results: The gene transformer-2 produces a protein of 249 amino acids in both sexes, which shows the features of the SR protein family. No significant partially spliced mRNA isoform specific to the male germ line was detected, unlike in Drosophila. It is transcribed in both sexes during development and in adult life, in both the soma and germ line. The injection of Anastrepha transformer-2 dsRNA into Anastrepha embryos caused a change in the splicing pattern of the endogenous transformer and Doublesex pre-mRNA of XX females from the female to the male mode. Consequently, these XX females were transformed into pseudomales. The comparison of the eleven Anastrepha Transformer-2 proteins among themselves, and with the Transformer-2 proteins of other insects, suggests the existence of negative selection acting at the protein level to maintain Transformer-2 structural features. Conclusions: These results indicate that transformer-2 is required for sex determination in Anastrepha through its participation in the female-specific splicing of transformer and Doublesex pre-mRNAs. It is therefore needed for the auto-regulation of the gene transformer. Thus, the transformer/transfomer-2 > Doublesex elements at the bottom of the cascade, and their relationships, probably represent the ancestral state (which still exists in the Tephritidae, Calliphoridae and Muscidae lineages) of the extant cascade found in the Drosophilidae lineage (in which tra is just another component of the sex determination gene cascade regulated by Sex-lethal). In the phylogenetic lineage that gave rise to the drosophilids, evolution co-opted for Sex-lethal, modified it, and converted it into the key gene controlling sex determination.
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the gene Doublesex of the fruit fly anastrepha obliqua diptera tephritidae
Genetics, 2005Co-Authors: Fernanda M Ruiz, André L.p. Perondini, Denise Selivon, Rominy Novaes Stefani, Rodrigo O Mascarenhas, Lucas SánchezAbstract:The gene Doublesex of Anastrepha obliqua is composed of four instead of the usual six exons. It is transcribed in both sexes and its primary transcript undergoes sex-specific splicing, producing female DsxF and male DsxM proteins, which have in common the amino-terminal region but which differ at the carboxyl-terminal region.
Yongping Huang - One of the best experts on this subject based on the ideXlab platform.
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mutation of Doublesex in hyphantria cunea results in sex specific sterility
Pest Management Science, 2020Co-Authors: Qun Liu, Huihui Liu, Yaohui Wang, Xien Chen, Zhen Zhang, Yongping Huang, Hui ChenAbstract:Background The gene Doublesex (dsx) plays pivotal roles in sex determination and controls sexually dimorphic development in certain insects. Importantly, it also displays a potential candidate target for pest management due to its sex-specific splicing. Therefore, we used CRISPR/Cas9-mediated gene disruption to investigate the function of dsx in Hyphantria cunea, an invasive forest pest. Result In the present study, we identified the dsx gene from H. cunea which showed a sex-biased expression pattern that was different from other lepidopteran insects. Referring to sex-specific functional analyses in Bombyx mori, we performed a site-specific knockout of the Hcdsx gene by using a CRISPR/Cas9 system, which induced severe abnormalities in external genitalia and some incomplete sex reversal phenotypes, which in turn led to reduced sex-specific fecundity. An alternative splicing pattern of Hcdsx was altered by CRISPR/Cas9-induced mutation, and alterations in splicing affected expression of downstream genes encoding pheromone binding protein 1, vg1 and vg2 (encoding vitellogenin), which contributed to the sex-specific sterility phenotypes in the Hcdsx mutants. Conclusion The Hcdsx gene plays important roles in sexual differentiation in H. cunea. Disruption of Hcdsx induced sex-specific sterility, demonstrating a potential application in control of this pest. © 2019 Society of Chemical Industry.
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identification and functional characterization of Doublesex gene in the testis of spodoptera litura
Insect Science, 2019Co-Authors: Liang Wen, Yongping Huang, Sichun Zheng, Qili Feng, Lin LiuAbstract:Fusion of the testis occurs in most Lepidoptera insects, including Spodoptera litura, an important polyphagous pest. Testicular fusion in S. litura is advantageous for male reproduction, and the molecular mechanism of fusion remains unknown. Doublesex influences the formation of genitalia, the behavior of courtship, and sexually dimorphic traits in fruit-fly and silkworm, and is essential for sexual differentiation. However, its purpose in the testis of S. litura remains unknown. The Doublesex gene of S. litura (Sldsx) has male-specific SldsxM and female-specific SldsxF isoforms, and exhibits a higher expression level in the male testis. At the testicular fusion stage (L6D6), Sldsx attained the highest expression compared to the pre-fusion and post-fusion periods. Moreover, Sldsx had a higher expression in the peritoneal sheaths of testis than that of germ cells in the follicle. CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Cas9) was applied to S. litura to determine the role of Sldsx. A mixture of single guide RNA messenger RNA and Cas9 protein (300 ng/μL each) was injected into eggs within 2 h following oviposition. CRISPR/Cas9 successfully induced genomic mutagenesis of Sldsx at G0 generation. The mutant males had smaller testis surrounded by less tracheae. Moreover, the mutant males had abnormal external genitalia and could not finish mating with wild-type females. Additionally, testes were fused for almost all mutant males. The results showed that Sldsx was not related to testicular fusion, and is required for both testis development and the formation and function of external genitalia in S. litura. The main roles of Doublesex on the male are similar to other insects.
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sexually dimorphic traits in the silkworm bombyx mori are regulated by Doublesex
Insect Biochemistry and Molecular Biology, 2017Co-Authors: Shuai Zhan, Shuqing Chen, Baosheng Zeng, Anthony A James, Anjiang Tan, Yongping HuangAbstract:The DM domain genes, Doublesex (dsx) in insects, or their structural homologs, male abnormal 3 (mab-3) in nematodes and Dmrt1 (Doublesex and mab-3-related transcription factor 1) in mammals, are downstream regulators of the sex determination pathway that control sexually dimorphic development. Despite the functional importance of dsx and its potential applications in sterile insect technologies (SITs), the mechanisms by which it controls sexually dimorphic traits and the subsequent developmental gene networks in insects are poorly understood. Phylogenetic analyses indicate that insect dsx genes have sex-specific alternative splicing isoforms, whereas other taxa do not. We exploited genome editing and transgenesis technologies to induce mutations in either the male-specific isoform (dsxM) or common region (dsxC) of dsx in the somatic tissues of the lepidopteran model insect Bombyx mori. Disruptions of gene function produced either male-specific sexually-dimorphic defects or intersexual phenotypes; these results differ from those observed in other insects, including Drosophila melanogaster. Our data provide insights into the divergence of the insect sex determination pathways related to the most conserved downstream component dsx.
Giuseppe Saccone - One of the best experts on this subject based on the ideXlab platform.
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genomic organization and splicing evolution of the Doublesex gene a drosophila regulator of sexual differentiation in the dengue and yellow fever mosquito aedes aegypti
BMC Evolutionary Biology, 2011Co-Authors: Marco Salvemini, Lino C Polito, Andreina Milano, U Mauro, Fabrizio Lombardo, Vincenzo Zazzaro, Bruno Arca, Giuseppe SacconeAbstract:Background In the model system Drosophila melanogaster, Doublesex (dsx) is the double-switch gene at the bottom of the somatic sex determination cascade that determines the differentiation of sexually dimorphic traits. Homologues of dsx are functionally conserved in various dipteran species, including the malaria vector Anopheles gambiae. They show a striking conservation of sex-specific regulation, based on alternative splicing, and of the encoded sex-specific proteins, which are transcriptional regulators of downstream terminal genes that influence sexual differentiation of cells, tissues and organs.
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masculinization of xx drosophila transgenic flies expressing the ceratitis capitata Doublesexm isoform
The International Journal of Developmental Biology, 2008Co-Authors: Giuseppe Saccone, Attilio Pane, Marco Salvemini, Lino C PolitoAbstract:The Doublesex (DSX) transcription factor regulates somatic sexual differentiation in Drosophila melanogaster. Female and male isoforms (DSXF and DSXM) are produced due to sex-specific RNA splicing. Here we show that in the distantly related dipteran Ceratitis capitata, the DSXM male-specific isoform is conserved and able to induce masculinization of both somatic and germline tissues when ectopically expressed in XX Drosophila transgenic individuals.
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the gene transformer of anastrepha fruit flies diptera tephritidae and its evolution in insects
PLOS ONE, 2007Co-Authors: María Fernanda Ruiz, André L.p. Perondini, Denise Selivon, Andreina Milano, Marco Salvemini, Jose M Eirinlopez, Catello Polito, Giuseppe Saccone, Lucas SánchezAbstract:Background: In the tephritids Ceratitis, Bactrocera and Anastrepha, the gene transformer provides the memory device for sex determination via its auto-regulation; only in females is functional Tra protein produced. To date, the isolation and characterisation of the gene transformer-2 in the tephritids has only been undertaken in Ceratitis, and it has been shown that its function is required for the female-specific splicing of Doublesex and transformer pre-mRNA. It therefore participates in transformer auto-regulatory function. In this work, the characterisation of this gene in eleven tephritid species belonging to the less extensively analysed genus Anastrepha was undertaken in order to throw light on the evolution of transformer-2. Results: The gene transformer-2 produces a protein of 249 amino acids in both sexes, which shows the features of the SR protein family. No significant partially spliced mRNA isoform specific to the male germ line was detected, unlike in Drosophila. It is transcribed in both sexes during development and in adult life, in both the soma and germ line. The injection of Anastrepha transformer-2 dsRNA into Anastrepha embryos caused a change in the splicing pattern of the endogenous transformer and Doublesex pre-mRNA of XX females from the female to the male mode. Consequently, these XX females were transformed into pseudomales. The comparison of the eleven Anastrepha Transformer-2 proteins among themselves, and with the Transformer-2 proteins of other insects, suggests the existence of negative selection acting at the protein level to maintain Transformer-2 structural features. Conclusions: These results indicate that transformer-2 is required for sex determination in Anastrepha through its participation in the female-specific splicing of transformer and Doublesex pre-mRNAs. It is therefore needed for the auto-regulation of the gene transformer. Thus, the transformer/transfomer-2 > Doublesex elements at the bottom of the cascade, and their relationships, probably represent the ancestral state (which still exists in the Tephritidae, Calliphoridae and Muscidae lineages) of the extant cascade found in the Drosophilidae lineage (in which tra is just another component of the sex determination gene cascade regulated by Sex-lethal). In the phylogenetic lineage that gave rise to the drosophilids, evolution co-opted for Sex-lethal, modified it, and converted it into the key gene controlling sex determination.