Title: Transcriptome profiling reveals sex‐specific gene expressions in pupal and adult stages of the mosquito Culex pipiens
Abstract Understanding the development process of male and female mosquitoes provides important basic information for sterile insect release programmes and is important for improving other vector control strategies. However, little is known about the molecular mechanisms that distinguish male from female‐specific developmental processes in this species. We used IlluminaRNA‐seq to identify sex‐specific genes during pupal and adult stages. One hundred and forty‐seven genes were expressed only in pupal males, 56 genes were expressed in adult males and another 82 genes were commonly expressed in both male samples. In addition, 26 genes were expressed only in the pupal females, 163 genes were found in the adult females and only one gene was expressed in both female samples. A further quantitative real‐time PCR validation of selected genes from the RNA sequencing (RNA‐seq) analysis confirmed upregulation of those genes in a sex‐specific manner, including: fibrinogen and fibronectin, a zinc finger protein, phospholipase A(2) and a serine protein for female pupae; venom allergen 3, a perlecan, testis‐specific serine/threonine‐protein kinase 1, testis‐specific serine/threonine‐protein kinase 6 and cytochrome c‐2 for male pupae; a salivary protein, D7 protein precursor, trypsin 7 precursor, D7 protein and nanos for female adults; and tetraspanin F139, cytosol aminopeptidase, testis‐specific serine/threonine‐protein kinase 1, a testis‐specific serine/threonine‐protein kinase 6 and a C‐type lectin for male adults. These findings provide insight into the development and physiology ofCulexmosquitoes, which will help in the development of more effective control methods for these disease vectors. more »« less
The transformer ( tra ) gene is essential for female development in many insect species, including the Australian sheep blow fly, Lucilia cuprina . Sex-specific tra RNA splicing is controlled by Sex lethal ( Sxl ) in Drosophila melanogaster but is auto-regulated in L . cuprina . Sxl also represses X chromosome dosage compensation in female D . melanogaster . We have developed conditional Lctra RNAi knockdown strains using the tet-off system. Four strains did not produce females on diet without tetracycline and could potentially be used for genetic control of L . cuprina . In one strain, which showed both maternal and zygotic tTA expression, most XX transformed males died at the pupal stage. RNAseq and qRT-PCR analyses of mid-stage pupae showed increased expression of X-linked genes in XX individuals. These results suggest that Lctra promotes somatic sexual differentiation and inhibits X chromosome dosage compensation in female L . cuprina . However, XX flies homozygous for a loss-of-function Lctra knockin mutation were fully transformed and showed high pupal eclosion. Two of five X-linked genes examined showed a significant increase in mRNA levels in XX males. The stronger phenotype in the RNAi knockdown strain could indicate that maternal Lctra expression may be essential for initiation of dosage compensation suppression in female embryos.
Zeh, Jeanne A.; Zawlodzki, Maya A.; Bonilla, Melvin M.; Su‐Keene, Eleanor J.; Padua, Michael V.; Zeh, David W.
(, Journal of Evolutionary Biology)
Abstract Maternal inheritance of mitochondria creates a sex‐specific selective sieve through which mitochondrial mutations harmful to males but not females accumulate and contribute to sexual differences in longevity and disease susceptibility. Because eggs and sperm are under disruptive selection, sperm are predicted to be particularly vulnerable to the genetic load generated by maternal inheritance, yet evidence for mitochondrial involvement in male fertility is limited and controversial. Here, we exploit the coexistence of two divergent mitochondrial haplogroups (A and B2) in a Neotropical arachnid to investigate the role of mitochondria in sperm competition. DNA profiling demonstrated that B2‐carrying males sired more than three times as many offspring in sperm competition experiments than A males, and this B2 competitive advantage cannot be explained by female mitochondrial haplogroup or male nuclear genetic background. RNA‐Seq of testicular tissues implicates differential expression of mitochondrial oxidative phosphorylation (OXPHOS) genes in the B2 competitive advantage, including a 22‐fold upregulation ofatp8in B2 males. Previous comparative genomic analyses have revealed functionally significant amino acid substitutions in differentially expressed genes, indicating that the mitochondrial haplogroups differ not only in expression but also in DNA sequence and protein functioning. However, mitochondrial haplogroup had no effect on sperm number or sperm viability, and, when females were mated to a single male, neither male haplogroup, female haplogroup nor the interaction between male/female haplogroup significantly affected female reproductive success. Our findings therefore suggest that mitochondrial effects on male reproduction may often go undetected in noncompetitive contexts and may prove more important in nature than is currently appreciated.
Abstract Vasoactive-intestinal peptide (Vip) is a pleiotropic peptide with a wide range of distribution and functions. Zebrafish possess 2 isoforms of Vip (a and b), in which Vipa is most homologous to the mammalian form. In female zebrafish, Vipa can stimulate LH secretion from the pituitary but is not essential for female reproduction, as vipa−/− females display normal reproduction. In contrast, we have found that vipa−/− males are severely subfertile and sex ratio of offspring is female-biased. By analyzing all aspects of male reproduction with wild-type (WT) males, we show that the testes of vipa−/− are underdeveloped and contain ∼70% less spermatids compared to WT counterparts. The sperm of vipa−/− males displayed reduced potency in terms of fertilization (by ∼80%) and motility span and duration (by ∼50%). In addition, vipa−/− male attraction to WT females was largely nonexistent, indicating decreased sexual motivation. We show that vipa mRNA and protein is present in Leydig cells and in developing germ cells in the testis of WT, raising the possibility that endogenous Vipa contributes to testicular function. Absence of Vipa in vipa−/− males resulted in downregulation of 3 key genes in the androgen synthesis chain in the testis, 3β-hsd, 17β-hsd1, and cyp11c1 (11β-hydrogenase), associated with a pronounced decrease in 11-ketotestosterone production and, in turn, compromised reproductive fitness. Altogether, this study establishes a crucial role for Vipa in the regulation of male reproduction in zebrafish, like in mammals, with the exception that Vipa is also expressed in zebrafish testis.
Meuti, Megan E; Siperstein, Alden; Wolkoff, Matthew
(, Cold Spring Harbor Protocols)
After overcoming the significant obstacle of getting adultCulexmosquitoes to reproduce and blood feed in the laboratory, maintaining a laboratory colony is much more achievable. However, great care and attention to detail are still required to ensure that the larvae have adequate food without being overwhelmed by bacterial growth. Additionally, achieving the appropriate densities of larvae and pupae is essential, as overcrowding delays development, prevents pupae from successfully emerging as adults, and/or reduces adult fecundity and alters sex ratios. Finally, adult mosquitoes should have constant access to H2O and nearly constant access to sugar sources to ensure that both males and females have adequate nutrition and can produce the maximum number of offspring. Here, we describe our methods for maintaining the Buckeye strain ofCulex pipiensand how other researchers might modify them to suit their specific needs.
Abstract 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, within vitroproduced 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 vitroproduced 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 asSOX21andPRDM14, and splicing factorsFMR1andHNRNPH2. 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 inDENND1B, DIS3L2, DOCK11, IL1RAPL2,andZRSR2Y,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. HighlightsMale embryos develop faster due to increased gene expression in metabolism pathwaysFemale embryos exhibit higher X-linked gene expression, suggesting X-dosage compensation plays a role in early developmentSex-biased alternative splicing events contribute to embryonic metabolism, autophagy, and transcriptional regulation in embryosSex-biased isoform diversity contributes to distinct developmental regulation in male and female embryosKey pluripotency factors (SOX21, PRDM14) and splicing regulators (FMR1, HNRNPH2) drive sex-specific gene expression
Martynova, T., Kamanda, P., and Sim, C. Transcriptome profiling reveals sex‐specific gene expressions in pupal and adult stages of the mosquito Culex pipiens. Insect Molecular Biology 31.1 Web. doi:10.1111/imb.12735.
Martynova, T., Kamanda, P., and Sim, C.
"Transcriptome profiling reveals sex‐specific gene expressions in pupal and adult stages of the mosquito Culex pipiens". Insect Molecular Biology 31 (1). Country unknown/Code not available: Wiley-Blackwell. https://doi.org/10.1111/imb.12735.https://par.nsf.gov/biblio/10306655.
@article{osti_10306655,
place = {Country unknown/Code not available},
title = {Transcriptome profiling reveals sex‐specific gene expressions in pupal and adult stages of the mosquito Culex pipiens},
url = {https://par.nsf.gov/biblio/10306655},
DOI = {10.1111/imb.12735},
abstractNote = {Abstract Understanding the development process of male and female mosquitoes provides important basic information for sterile insect release programmes and is important for improving other vector control strategies. However, little is known about the molecular mechanisms that distinguish male from female‐specific developmental processes in this species. We used IlluminaRNA‐seq to identify sex‐specific genes during pupal and adult stages. One hundred and forty‐seven genes were expressed only in pupal males, 56 genes were expressed in adult males and another 82 genes were commonly expressed in both male samples. In addition, 26 genes were expressed only in the pupal females, 163 genes were found in the adult females and only one gene was expressed in both female samples. A further quantitative real‐time PCR validation of selected genes from the RNA sequencing (RNA‐seq) analysis confirmed upregulation of those genes in a sex‐specific manner, including: fibrinogen and fibronectin, a zinc finger protein, phospholipase A(2) and a serine protein for female pupae; venom allergen 3, a perlecan, testis‐specific serine/threonine‐protein kinase 1, testis‐specific serine/threonine‐protein kinase 6 and cytochrome c‐2 for male pupae; a salivary protein, D7 protein precursor, trypsin 7 precursor, D7 protein and nanos for female adults; and tetraspanin F139, cytosol aminopeptidase, testis‐specific serine/threonine‐protein kinase 1, a testis‐specific serine/threonine‐protein kinase 6 and a C‐type lectin for male adults. These findings provide insight into the development and physiology ofCulexmosquitoes, which will help in the development of more effective control methods for these disease vectors.},
journal = {Insect Molecular Biology},
volume = {31},
number = {1},
publisher = {Wiley-Blackwell},
author = {Martynova, T. and Kamanda, P. and Sim, C.},
}
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