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The Department of Entomology
The Robert H. Smith Faculty of Agriculture, Food and Environment
The Hebrew University of Jerusalem
P.O Box 12, Rehovot 76100, ISRAEL

Tel: 08-9489223 
Fax: 08-9366768
Email: orlytal@savion.huji.ac.il

Publications

2019
Meccariello, A. ; Salvemini, M. ; Primo, P. ; Hall, B. ; Koskinioti, P. ; Dalíková, M. ; Gravina, A. ; Gucciardino, M. A. ; Forlenza, F. ; Gregoriou, M. - E. ; et al. Maleness-on-the-Y (MoY) orchestrates male sex determination in major agricultural fruit fly pests. Science 2019, 365, 1457-1460. Publisher's VersionAbstract
In insects, rapidly evolving primary sex-determining signals are transduced by a conserved regulatory module controlling sexual differentiation. In the agricultural pest Ceratitis capitata (Mediterranean fruit fly, or Medfly), we identified a Y-linked gene, Maleness-on-the-Y (MoY), encoding a small protein that is necessary and sufficient for male development. Silencing or disruption of MoY in XY embryos causes feminization, whereas overexpression of MoY in XX embryos induces masculinization. Crosses between transformed XY females and XX males give rise to males and females, indicating that a Ychromosome can be transmitted by XY females. MoY is Y-linked and functionally conserved in other species of the Tephritidae family, highlighting its potential to serve as a tool for developing more effective control strategies against these major agricultural insect pests. Copyright © 2019 The Authors.
Nash, A. ; Urdaneta, G. M. ; Beaghton, A. K. ; Hoermann, A. ; Papathanos, P. A. ; Christophides, G. K. ; Windbichler, N. Integral gene drives for population replacement. Biol Open 2019, 8.Abstract
A first generation of CRISPR-based gene drives has now been tested in the laboratory in a number of organisms, including malaria vector mosquitoes. Challenges for their use in the area-wide genetic control of vector-borne disease have been identified, including the development of target site resistance, their long-term efficacy in the field, their molecular complexity, and practical and legal limitations for field testing of both gene drive and coupled anti-pathogen traits. We have evaluated theoretically the concept of integral gene drive (IGD) as an alternative paradigm for population replacement. IGDs incorporate a minimal set of molecular components, including drive and anti-pathogen effector elements directly embedded within endogenous genes - an arrangement that in theory allows targeting functionally conserved coding sequences without disrupting their function. Autonomous and non-autonomous IGD strains could be generated, optimized, regulated and imported independently. We performed quantitative modeling comparing IGDs with classical replacement drives and show that selection for the function of the hijacked host gene can significantly reduce the establishment of resistant alleles in the population, while drive occurring at multiple genomic loci prolongs the duration of transmission blockage in the face of pre-existing target site variation. IGD thus has potential as a more durable and flexible population replacement strategy.
2018
Papathanos, P. A. ; Windbichler, N. Redkmer: An Assembly-Free Pipeline for the Identification of Abundant and Specific X-Chromosome Target Sequences for X-Shredding by CRISPR Endonucleases. The CRISPR journal 2018, 1 88 - 98. Publisher's VersionAbstract
CRISPR-based synthetic sex ratio distorters, which operate by shredding the X-chromosome during male meiosis, are promising tools for the area-wide control of harmful insect pest or disease vector species. X-shredders have been proposed as tools to suppress insect populations by biasing the sex ratio of the wild population toward males, thus reducing its natural reproductive potential. However, to build synthetic X-shredders based on CRISPR, the selection of gRNA targets, in the form of high-copy sequence repeats on the X chromosome of a given species, is difficult, since such repeats are not accurately resolved in genome assemblies and cannot be assigned to chromosomes with confidence. We have therefore developed the redkmer computational pipeline, designed to identify short and highly abundant sequence elements occurring uniquely on the X chromosome. Redkmer was designed to use as input minimally processed whole genome sequence data from males and females. We tested redkmer with short- and long-read whole genome sequence data of Anopheles gambiae, the major vector of human malaria, in which the X-shredding paradigm was originally developed. Redkmer established long reads as chromosomal proxies with excellent correlation to the genome assembly and used them to rank X-candidate kmers for their level of X-specificity and abundance. Among these, a high-confidence set of 25-mers was identified, many belonging to previously known X-chromosome repeats of Anopheles gambiae, including the ribosomal gene array and the selfish elements harbored within it. Data from a control strain, in which these repeats are shared with the Y chromosome, confirmed the elimination of these kmers during filtering. Finally, we show that redkmer output can be linked directly to gRNA selection and off-target prediction. In addition, the output of redkmer, including the prediction of chromosomal origin of single-molecule long reads and chromosome specific kmers, could also be used for the characterization of other biologically relevant sex chromosome sequences, a task that is frequently hampered by the repetitiveness of sex chromosome sequence content.
Waters, A. J. ; Capriotti, P. ; Gaboriau, D. C. A. ; Papathanos, P. A. ; Windbichler, N. Rationally-engineered reproductive barriers using CRISPR & CRISPRa: an evaluation of the synthetic species concept in Drosophila melanogaster. 2018, 8 13125. Publisher's VersionAbstract
The ability to erect rationally-engineered reproductive barriers in animal or plant species promises to enable a number of biotechnological applications such as the creation of genetic firewalls, the containment of gene drives or novel population replacement and suppression strategies for genetic control. However, to date no experimental data exist that explores this concept in a multicellular organism. Here we examine the requirements for building artificial reproductive barriers in the metazoan model Drosophila melanogaster by combining CRISPR-based genome editing and transcriptional transactivation (CRISPRa) of the same loci. We directed 13 single guide RNAs (sgRNAs) to the promoters of 7 evolutionary conserved genes and used 11 drivers to conduct a misactivation screen. We identify dominant-lethal activators of the eve locus and find that they disrupt development by strongly activating eve outside its native spatio-temporal context. We employ the same set of sgRNAs to isolate, by genome editing, protective INDELs that render these loci resistant to transactivation without interfering with target gene function. When these sets of genetic components are combined we find that complete synthetic lethality, a prerequisite for most applications, is achievable using this approach. However, our results suggest a steep trade-off between the level and scope of dCas9 expression, the degree of genetic isolation achievable and the resulting impact on fly fitness. The genetic engineering strategy we present here allows the creation of single or multiple reproductive barriers and could be applied to other multicellular organisms such as disease vectors or transgenic organisms of economic importance.
Papathanos, P. A. ; Bourtzis, K. ; Tripet, F. ; Bossin, H. ; Virginio, J. F. ; Capurro, M. L. ; Pedrosa, M. C. ; Guindo, A. ; Sylla, L. ; Coulibaly, M. B. ; et al. A perspective on the need and current status of efficient sex separation methods for mosquito genetic control. Parasit Vectors 2018, 11, 654.Abstract
Major efforts are currently underway to develop novel, complementary methods to combat mosquito-borne diseases. Mosquito genetic control strategies (GCSs) have become an increasingly important area of research on account of their species-specificity, track record in targeting agricultural insect pests, and their environmentally non-polluting nature. A number of programs targeting Aedes and Anopheles mosquitoes, vectors of human arboviruses and malaria respectively, are currently being developed or deployed in many parts of the world. Operationally implementing these technologies on a large scale however, beyond proof-of-concept pilot programs, is hampered by the absence of adequate sex separation methods. Sex separation eliminates females in the laboratory from male mosquitoes prior to release. Despite the need for sex separation for the control of mosquitoes, there have been limited efforts in recent years in developing systems that are fit-for-purpose. In this special issue of Parasites and Vectors we report on the progress of the global Coordinated Research Program on "Exploring genetic, molecular, mechanical and behavioural methods for sex separation in mosquitoes" that is led by the Insect Pest Control Subprogramme of the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture with the specific aim of building efficient sex separation systems for mosquito species. In an effort to overcome current barriers we briefly highlight what we believe are the three main reasons why progress has been so slow in developing appropriate sex separation systems: the availability of methods that are not scalable, the difficulty of building the ideal genetic systems and, finally, the lack of research efforts in this area.
2017
Papa, F. ; Windbichler, N. ; Waterhouse, R. M. ; Cagnetti, A. ; D'Amato, R. ; Persampieri, T. ; Lawniczak, M. K. N. ; Nolan, T. ; Papathanos, P. A. Rapid evolution of female-biased genes among four species of Anopheles malaria mosquitoes. Genome research 2017, 27, 1536 - 1548. Publisher's VersionAbstract
Understanding how phenotypic differences between males and females arise from the sex-biased expression of nearly identical genomes can reveal important insights into the biology and evolution of a species. Among Anopheles mosquito species, these phenotypic differences include vectorial capacity, as it is only females that blood feed and thus transmit human malaria. Here, we use RNA-seq data from multiple tissues of four vector species spanning the Anopheles phylogeny to explore the genomic and evolutionary properties of sex-biased genes. We find that, in these mosquitoes, in contrast to what has been found in many other organisms, female-biased genes are more rapidly evolving in sequence, expression, and genic turnover than male-biased genes. Our results suggest that this atypical pattern may be due to the combination of sex-specific life history challenges encountered by females, such as blood feeding. Furthermore, female propensity to mate only once in nature in male swarms likely diminishes sexual selection of post-reproductive traits related to sperm competition among males. We also develop a comparative framework to systematically explore tissue- and sex-specific splicing to document its conservation throughout the genus and identify a set of candidate genes for future functional analyses of sex-specific isoform usage. Finally, our data reveal that the deficit of male-biased genes on the X Chromosomes in Anopheles is a conserved feature in this genus and can be directly attributed to chromosome-wide transcriptional regulation that de-masculinizes the X in male reproductive tissues.
Bernardini, F. ; Galizi, R. ; Wunderlich, M. ; Taxiarchi, C. ; Kranjc, N. ; Kyrou, K. ; Hammond, A. ; Nolan, T. ; Lawniczak, M. N. K. ; Papathanos, P. A. ; et al. Cross-Species Y Chromosome Function Between Malaria Vectors of the Species Complex. Genetics 2017, 207, 729-740.Abstract
Y chromosome function, structure and evolution is poorly understood in many species, including the genus of mosquitoes-an emerging model system for studying speciation that also represents the major vectors of malaria. While the Anopheline Y had previously been implicated in male mating behavior, recent data from the complex suggests that, apart from the putative primary sex-determiner, no other genes are conserved on the Y. Studying the functional basis of the evolutionary divergence of the Y chromosome in the gambiae complex is complicated by complete F1 male hybrid sterility. Here, we used an F1 × F0 crossing scheme to overcome a severe bottleneck of male hybrid incompatibilities that enabled us to experimentally purify a genetically labeled Y chromosome in an background. Whole genome sequencing (WGS) confirmed that the Y retained its original sequence content in the genomic background. In contrast to comparable experiments in , we find that the presence of a heterospecific Y chromosome has no significant effect on the expression of genes, and transcriptional differences can be explained almost exclusively as a direct consequence of transcripts arising from sequence elements present on the Y chromosome itself. We find that Y hybrids show no obvious fertility defects, and no substantial reduction in male competitiveness. Our results demonstrate that, despite their radically different structure, Y chromosomes of these two species of the gambiae complex that diverged an estimated 1.85 MYA function interchangeably, thus indicating that the Y chromosome does not harbor loci contributing to hybrid incompatibility. Therefore, Y chromosome gene flow between members of the gambiae complex is possible even at their current level of divergence. Importantly, this also suggests that malaria control interventions based on sex-distorting Y drive would be transferable, whether intentionally or contingent, between the major malaria vector species.
2016
Hall, A. B. ; Papathanos, P. A. ; Sharma, A. ; Cheng, C. ; Akbari, O. S. ; Assour, L. ; Bergman, N. H. ; Cagnetti, A. ; Crisanti, A. ; Dottorini, T. ; et al. Radical remodeling of the Y chromosome in a recent radiation of malaria mosquitoes. Proceedings of the National Academy of Sciences 2016, 113, E2114–E2123. Publisher's VersionAbstract
Interest in male mosquitoes has been motivated by the potential to develop novel vector control strategies, exploiting the fact that males do not feed on blood or transmit diseases, such as malaria. However, genetic studies of male Anopheles mosquitoes have been impeded by the lack of molecular characterization of the Y chromosome. Here we show that the Anopheles gambiae Y chromosome contains a very small repertoire of genes, with massively amplified tandem arrays of a small number of satellites and transposable elements constituting the vast majority of the sequence. These genes and repeats evolve rapidly, bringing about remodeling of the Y, even among closely related species. Our study provides a long-awaited foundation for studying mosquito Y chromosome biology and evolution.Y chromosomes control essential male functions in many species, including sex determination and fertility. However, because of obstacles posed by repeat-rich heterochromatin, knowledge of Y chromosome sequences is limited to a handful of model organisms, constraining our understanding of Y biology across the tree of life. Here, we leverage long single-molecule sequencing to determine the content and structure of the nonrecombining Y chromosome of the primary African malaria mosquito, Anopheles gambiae. We find that the An. gambiae Y consists almost entirely of a few massively amplified, tandemly arrayed repeats, some of which can recombine with similar repeats on the X chromosome. Sex-specific genome resequencing in a recent species radiation, the An. gambiae complex, revealed rapid sequence turnover within An. gambiae and among species. Exploiting 52 sex-specific An. gambiae RNA-Seq datasets representing all developmental stages, we identified a small repertoire of Y-linked genes that lack X gametologs and are not Y-linked in any other species except An. gambiae, with the notable exception of YG2, a candidate male-determining gene. YG2 is the only gene conserved and exclusive to the Y in all species examined, yet sequence similarity to YG2 is not detectable in the genome of a more distant mosquito relative, suggesting rapid evolution of Y chromosome genes in this highly dynamic genus of malaria vectors. The extensive characterization of the An. gambiae Y provides a long-awaited foundation for studying male mosquito biology, and will inform novel mosquito control strategies based on the manipulation of Y chromosomes.
Galizi, R. ; Hammond, A. ; Kyrou, K. ; Taxiarchi, C. ; Bernardini, F. ; O’Loughlin, S. M. ; Papathanos, P. A. ; Nolan, T. ; Windbichler, N. ; Crisanti, A. A CRISPR-Cas9 sex-ratio distortion system for genetic control. 2016, 6 31139. Publisher's VersionAbstract
Genetic control aims to reduce the ability of insect pest populations to cause harm via the release of modified insects. One strategy is to bias the reproductive sex ratio towards males so that a population decreases in size or is eliminated altogether due to a lack of females. We have shown previously that sex ratio distortion can be generated synthetically in the main human malaria vector Anopheles gambiae, by selectively destroying the X-chromosome during spermatogenesis, through the activity of a naturally-occurring endonuclease that targets a repetitive rDNA sequence highly-conserved in a wide range of organisms. Here we describe a CRISPR-Cas9 sex distortion system that targets ribosomal sequences restricted to the member species of the Anopheles gambiae complex. Expression of Cas9 during spermatogenesis resulted in RNA-guided shredding of the X-chromosome during male meiosis and produced extreme male bias among progeny in the absence of any significant reduction in fertility. The flexibility of CRISPR-Cas9 combined with the availability of genomic data for a range of insects renders this strategy broadly applicable for the species-specific control of any pest or vector species with an XY sex-determination system by targeting sequences exclusive to the female sex chromosome.