Source: VIRGINIA POLYTECHNIC INSTITUTE submitted to
FUNCTION OF ANOPHELES MOSQUITO Y CHROMOSOME GENES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
Reporting Frequency
Annual
Accession No.
0233193
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Apr 1, 2013
Project End Date
Mar 31, 2018
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Project Director
Tu, Z.
Recipient Organization
VIRGINIA POLYTECHNIC INSTITUTE
(N/A)
BLACKSBURG,VA 24061
Performing Department
Biochemistry
Non Technical Summary
Anopheles mosquitoes are the primary vectors of malaria, which is one of the most deadly and costly infectious diseases in human history. Y chromosome is known to be involved in sex determination, spermatogenesis, and sexual behavior in different species. The long term objective of this study is to develop novel strategies to control mosquitoes and mosquito-borne diseases based on a better understanding of Anopheles Y chromosome genes. Specifically, we will use transgenic methods to investigate the function of a Y chromosome gene we discovered and named GUY1 in Anopheles stephensi, the Asian malaria mosquito. We will test the effect of GUY1 expression or the lack of GUY1 expression on sexual development in Anopheles stephensi. We will also uncover Y chromosome genes from Anopheles gambiae, the African malaria mosquito, to infer evolution of the Anopheles Y chromosome.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7223110104020%
7223110105030%
7223110108030%
7223110113020%
Goals / Objectives
Anopheles mosquitoes are the primary vectors of malaria, one of the most deadly and costly diseases in human history. Current control strategies are becoming less effective as insecticide- and drug-resistance increases. Novel approaches to malaria control are urgently needed. Only female mosquitoes feed on blood and transmit disease pathogens. Anopheles mosquitoes use the XX/XY sex-determination system. There is evidence that a dominant male-determining factor(s) on the Y chromosome initiates sexual differentiation in an Anopheles species. Y chromosome also has known functions in spermatogenesis and mating behaviors in other insects. Despite their importance, Y genes remain largely unexplored outside of a few model species, mostly due to the repetitive nature of the Y chromosome and the difficulty to clone repeat-rich heterochromatic sequences. Our long-term objective, which is to illuminate the genetic basis of sexual dimorphism in anopheline mosquitoes and develop relevant new control strategies. We have identified a few Y genes including GUY1 in An. stephensi. We have preliminary indication that GUY1 is involved in sexual differentiation. Although multiple new avenues can be explored, we choose to vigorously test the hypothesis that GUY1 is involved in sexual differentiation in the early embryo. The embryonic effect on sexual differentiation may manifest in later life stages. We will pursue the following specific aims: 1) Investigate the effect of ectopic GUY1 expression; 2) Investigate the effect of GUY1 knockdown; and 3) Discover and characterize Y-chromosome genes in An. gambiae.
Project Methods
We will generate and characterize transgenic mosquito lines that either ectopically express GUY1 or have GUY1 knocked down. We will generate transgenic An. stephensi mosquito lines using a piggyBac transposon. Knockdown will be achieved by artificial microRNA or shRNA targeting GUY1. To determine the impact of ectopic GUY1 expression or knockdown, we will perform both targeted and non-biased analysis. Specific analyses include 1) General observation: Transgenic individuals can be identified in larvae stage and we will monitor any abnormality associated with the transgenics from larvae to adult. 2) Sex-related phenotypic changes: manipulation of GUY1 expression may lead to sex-bias in the transgenics, morphological abnormalities in sexual dimorphic tissues (e.g., testis, ovary, and antenna), changes in fertility, and changes in mating behavior. We will monitor all these as we characterize the transgenic lines. 3) Effect on sex-specific splicing of doublesex (dsx): dsx is a highly conserved gene critical to sexual development and its sex-specific splicing has been characterized in mosquitoes. We will focus on the major isoforms in each sex. 4) RNA-seq of early embryos from one transgenic line: We will sex 96 embryos from each time point and pool the RNAs for RNA-seq. Sequence analysis will be performed as we described in Biedler et al. (2012). The genome assembly is available for An. gambiae (Holt et al., 2002) and we have just produced >20x coverage An. gambiae illumina reads for males and females, separately. We have also generated An. gambiae RNA-seq data from eight samples from embryonic time points to adult males and females (not shown). Therefore we will couple the chromosome quotient approach and RNA-seq and RT-PCR to uncover Y genes in An. gambiae. Given our success for An. stephensi, we do not expect any problems.

Progress 04/01/13 to 03/31/18

Outputs
Target Audience:Scientists, students, educators, and the general public who are interested in insect sex-determination. Scientists and the general public who are interested in developing genetic methods to control mosquito-borne infectious diseases. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?1) Publications in scientific journals are provided. 2) Dr. Tu has been giving presentations at international conferences and as invited seminar speakers 3) Dr. Tu has also given guest lectures to undergraduate and graduate students on this research. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? At the beginning of the 5 year project, we discovered four Y genes in An. stephensi (Criscione et al., 2013; Hall et al., 2013). Among these, Guy1 is the best candidate for the M factor because it encodes a small protein with predicted DNA-binding properties, its transcription starts at the very onset of embryonic development, and it does not require any other factor on the Y-chromosome for transcription initiation (Criscione et al., Insect molecular biology, 2013). During the last 4-5 years, we made significant progress towards understanding the function of Guy1 as a male determining factor, by demonstrating that the GUY1 protein confers 100% female-specific lethality and Guy1 transgenic males are reproductively more competitive than their non-transgenic siblings under laboratory conditions (Criscione et al., eLife, 2016) . During this reporting period, we further demonstrated the mechanism underlying the female-specific lethality, namely the mis-regulation of dosage compensation that results from the ectopic expression of Guy1 in XX females (Qi et al., eLife, under review). During this project period, we have also identified Y chromosome genes in the African malaria mosquito Anopheles gambiae and showed that the Y chromosomes underwent rapid remodeling within the past two million years (Hall et al., BMC genomics, 2014; Hall et al., PNAS, 2016). Using single molecule long read sequencing, we are improving the Y chromosome assembly of A. stephensi and A. gambiae, which will enable further evolutionary and functionary analysis of this important sex chromosome in mosquitoes.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Wu Y., Hu W., Bielder J., Chen X.G. and and Tu Z. 2018 Pure early zygotic genes in the Asian malaria mosquito Anopheles stephensi. Parasites & Vectors


Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Scientists, students, educators, and the general public who are interested in insect sex-determination. Scientists and general public who are interested in developing genetic methods to control mosquito-borne infectious diseases. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?1) Students and other personnel served as mentors to train undergraduate students, graduate rotation students and under-represented students. 2) Trainedundergraduate students, graduate rotation students and under-represented students. 3) Students and other personnel presented their research at lab group, departmental and international meetings. How have the results been disseminated to communities of interest?1) Publicationin scientific journal. 2) Dr. Tu and students gave presentations at international conferences and at invited seminar speakers. 3) New genomic data, once carefully verified and analyzed, are being submitted to SRA for data sharing. 4) Dr. Tu has given guest lectures to undergraduate students and the general public. What do you plan to do during the next reporting period to accomplish the goals?We will follow the research design described in the proposal to achieve our objectives. Key activities in the next year will include: 1) Investigate the mechanism of Guy1 function using the newly obtained tagged-guy1 transgenic mosquitoes byperforming Chip-Seqto systematically identify Guy1 targets; 2) make new tagged-Guy2 transgenic lines to help identify Guy2 targets; 3)obtain new antibodies using the newly purified Guy1 and Guy2 recombinant proteins; 4) make additionaltet-off transgenic lines to overcome potential position effects of the transgene insertions.

Impacts
What was accomplished under these goals? During this reporting period, we have completed RNA-seq of transgenic lines to directly assess the involvement of Guy1 and another related gene, Guy2, on dosage compensation. We have performed analyses to show that both genes are involved in regulation of dosage compensation. We have now improved protein expression of both Guy1 and Guy2 and made new transgenic lines that have improved affinity tags to the Guy1 transgene. The new lines showed the same complete female lethal phenotype as previous lines. Therefore, we are in a better position to study the mechanism of Guy1 function. Another significant result in this reporting period is that we were able to expand our discovery and analysis of Y chromosomal genes to a small number of Anopheles species, which will allow us to study the evolution of Y genes and potentially apply what we learned to help control additional Anopheles vectors of malaria parasites.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Jiang X, Hall AB, Biedler JK, and Tu Z. 2017. Single Molecule RNA Sequencing uncovers trans-splicing and improves annotations in Anopheles stephensi. Insect Mol Biol. DOI: 10.1111/imb.12294


Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Scientists, students, educators, and the general public who are interested in insect sex-determination. Scientists and general public who are interested in developing genetic methods to control mosquito-borne infectious diseases. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Students and other personnel published research articles. How have the results been disseminated to communities of interest?1) Publications in scientific journals and book chapters. 2) Dr. Tu has been giving presentations at international conferences and as invited seminar speakers. 3) Dr. Tu has also been engaging media outlets to disseminate information. 4) New genomic data, once carefully verified and analyzed, are submitted to SRA for data sharing. 5) Dr. Tu has also given guest lectures to undergraduate students and general public. What do you plan to do during the next reporting period to accomplish the goals?There is no change to our specific aims. We will follow the research design described in the proposal to achieve our objectives. Key activities in the next year will include: 1) continue RNA-seq of Guy1 transgenic lines; 2) obtain new Guy1 antibody; 3) perform Chip-Seq of Guy1 pull-down to systematically identify Guy1 targets; and 4) evaluate the new tet-off transgenic lines.

Impacts
What was accomplished under these goals? We have previously shown that a second Y-chromosome gene, sYG2 also conferred female-lethality. It is likely that Guy1 and sYG2 are components of a common signaling pathway. During this reporting period, we are performing RNA-seq of transgenic lines to directly assess the involvement of Guy1 and sYG2 on dosage compensation. These results indicate that sYG2 expression in females up-regulates X-linked genes, consistent with our hypothesis that sYG2 regulate dosage compensation by conferring hyper-expression of genes on the X chromosome. These results directly address the hypothesis of Aim 1 and it is the first direct evidence linking either Guy1 or sYG2 to dosage compensation and female lethality. We have also repeated the chromatin pull-down assay with additional controls using tagged Guy1 transgenic lines. We have now clearly shown that the Guy1 protein binds the sYG2 promoter specifically. This result is significant as it directly showed that Guy1 is a DNA-binding protein and it is thus very likely that Guy1 is a transcription factor that activates sYG2 and possibly other target genes. Another significant result in this reporting period is that we were able to purify the recombinant Guy1 protein in bacteria. We are in the process of making new antibodies using this recombinant protein. During this reporting period, we have also characterized Y chromosome genes and Y chromosome structure of Anopheles gambiae. These results provided the foundation for future comparative studies of Y genes in different Anopheles species.

Publications

  • Type: Book Chapters Status: Published Year Published: 2016 Citation: Biedler JK, and Tu Z. 2016. Sex determination in mosquitoes. In (Editor: Raikhel, A.) Advances in Insect Physiology, Elsevier.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Criscione, F. Qi, Y., and Tu, Z. 2016. GUY1 confers complete female lethality and is a strong candidate for a male-determining factor in Anopheles stephensi. eLife, http://dx.doi.org/10.7554/eLife.19281.
  • Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Jiang X, Hall AB, Biedler JK, and Tu Z. 2016. Single Molecule RNA Sequencing uncovers trans-splicing and improves annotations in Anopheles stephensi. Insect Mol Biol. Accepted.


Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Scientists, students, educators, and the general public who are interested in insect sex-determination. Scientists and the general public who are interested in developing genetic methods to control mosquito-borne infectious diseases. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One graduate student was trained. How have the results been disseminated to communities of interest?The results were published in scientific journals and book chapters. Dr. Tu has been invited to discuss his results in scientific conferences and seminars. What do you plan to do during the next reporting period to accomplish the goals?1) Investigate the involvement of Guy1 in dosage compensation using embryonic RNAseq data 2) Verify the candidate target genes of Guy1 by reporter assays and promoter analysis 3) Using these composite transgenic lines, we now can knockout Guy1 and study the impact of such knockout.

Impacts
What was accomplished under these goals? In the past year, we have made significant advances that will enable us to complete the first two specific aims. First, after a number of optimizations, we were able to generate Illumina RNAseq libraries from single-embryo. We obtained single-embryo RNA-seq data and are in the process of obtaining more for statistical analyses. Such analyses will allow us to determine the impact of transgenic Guy1 expression at the molecular level (Aim 1). Second, we have generated a number of Guy1 transgenic lines including lines that express Guy1 from a composite gene (Guy1 open reading frame mixed with untranslated regions and promoters from other Anopheles stephensi genes). Thus, instead of knocking down Guy1 by RNAi as originally proposed (Aim 2), we can now use CRISPR/cas9 to knockout the native Guy1 without affecting the transgenic Guy1. This will mitigate potential sex-specific lethality associated with Guy1 knockout because male lethality will not occur until the transgene on the autosome and the mutated Y segregate.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Jiang X, Biedler JK, Qi Y, Hall AB, and Tu Z. 2015. Complete dosage compensation in Anopheles stephensi and the evolution of sex-biased genes in mosquitoes. Genome Biol Evol. 7, 1914-24.
  • Type: Book Chapters Status: Accepted Year Published: 2015 Citation: Biedler, J.K.H., A.B.; Jiang, X.; Tu, Z. (2015) Exploring the sex-determination pathway for control of mosquito-borne infectious diseases. (Adelman, Z.N., ed), Elsevier, in press


Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Scientists, students, educators, and the general public who are interested in insect sex-determination. Scientists and the general public who are interested in developing genetic methods to control mosquito-borne infectious diseases. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? One postdoc was trained during the reporting period. How have the results been disseminated to communities of interest? The genome sequencing results were published. Dr. Tu was invited to scientific conferences and seminars to discuss the results. What do you plan to do during the next reporting period to accomplish the goals? 1) Continue working on the mechanism of the female lethality. Analysis the sequences of the transgenic and wild type embryos to identify genes that are affected by Guy1 expression. 2) Investiagte the target of Guy1 regulation by identifying the binding sites of the Guy1 protein in the early embryos. 3) Use CRISPR/cas9 to knockut Guy1 to investigate the impact of loss of Guy1.

Impacts
What was accomplished under these goals? We have recently uncovered four Y genes in An. stephensi (Criscione et al., 2013; Hall et al., 2013). Among these, Guy1 is the best candidate for the M factor because it encodes a small protein with predicted DNA-binding properties, its transcription starts at the very onset of embryonic development, and it does not require any other factor on the Y-chromosome for transcription initiation (Criscione et al., 2013). Multiple Guy1-expression transgenic lines were generated, which produced 6520 transgenic males and 0 transgenic females over multiple generations. A point mutation at the Guy1 initiation codon and a premature stop codon prior to the predicted DNA-binding domain abolished the male bias, indicating that the GUY1 protein is the cause of the male-only phenotype. Genotyping analysis showed that such a phenotype resulted from 100% lethality of XX individuals. Guy1 transgenic males were reproductively more competitive than their non-transgenic siblings under laboratory conditions.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Jiang, X., Peery, A., Hall, A.B., Sharakhov, I.V., and Tu, Z. 2014. Genome analysis of a major urban malaria mosquito Anopheles stephensi. Geome Biol., 15, 459.


Progress 04/01/13 to 09/30/13

Outputs
Target Audience: Scientists, students, educators, and the general public who are interested in insect sex-determination. Scientists and general public who are interested in developing genetic methods to control mosquito-borne infectious diseases. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Students participated in a mini-symposium on Vector-borne infectious diseases. Students and other personnel published research articles. How have the results been disseminated to communities of interest? Publications in peer-reviewed scientific journals: Hall, A.B., Qi , Y., Timoshevskiy, V. Sharakhova, M.V., Sharakhov, I.V. and Tu, Z. 2013. Six novel Y chromosome genes in Anopheles mosquitoes discovered by independently sequencing males and females. BMC Genomics, 14, 273 (13 print pages). Criscione, Z. Qi, Y., Saunders, T.R., Hall, B. and Tu, Z. 2013. A unique Y gene in the Asian malaria mosquito Anopheles stephensi encodes a small lysine-rich protein and is transcribed at the onset of embryonic development. Insect Mol Biol., 22, 433-441. What do you plan to do during the next reporting period to accomplish the goals? 1. We hypothesize that Guy1 is involved in both sex-determination and dosage compensation, similar to the Drosophila sex-lethal gene. To further determine the molecular mechanism of Guy1 function, we will perform RNA-seq or targeted analysis of doublesex transcripts on genotyped embryos (Crisione et al., 2013). We will also compare transcription of genes in the X with genes in the autosome. We have also demonstrated the feasibility of using Tet-off and Gal4/UAS to confer conditional gene expression in A. stephensi embryos. We will develop additional Guy1 expression lines based on gal4/uas conditional control. 2. Because the recent success of TALEN-based gene knockout in mosquitoes, which was described as an alternative in our proposal, we have now adopted the TALEN approach to achieve Guy1 knockout instead of the shRNA-based Guy1 knockdown. We have made three TALEN constructs that specifically target Guy1 and will perform transgenic experiments. We are also exploring the CRISPR-based method to knockout Guy1.

Impacts
What was accomplished under these goals? During this reporting period, we further characterized Guy1 and three additional Y genes in Anopheles stephensi and the results have now been published (Crisione et al., 2013; Hall et al., 2013). We then showed that embryonic expression of the Guy1 gene introduced dramatic male bias and obtained Guy1-expressing transgenic A. stephensi strains that produced all male or nearly all male transgenics. This is potentially exciting because these results indicate that Guy1 is possibly the first male-determining factor found on an insect Y chromosome. We are investigating the molecular mechanism of Guy1 function. We have also demonstrated the feasibility of using Tet-off and Gal4/UAS to confer conditional gene expression in A. stephensi embryos, which will facilitate the production of stable homozygous Guy1 transgenic lines that may improve sex separation and mosquito control methods. We are in the process of producing Guy1 knockout lines using site-specific nucleases.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Hall, A.B., Qi , Y., Timoshevskiy, V. Sharakhova, M.V., Sharakhov, I.V. and Tu, Z. 2013. Six novel Y chromosome genes in Anopheles mosquitoes discovered by independently sequencing males and females. BMC Genomics, 14, 273 (13 print pages).
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Criscione, Z. Qi, Y., Saunders, T.R., Hall, B. and Tu, Z. 2013. A unique Y gene in the Asian malaria mosquito Anopheles stephensi encodes a small lysine-rich protein and is transcribed at the onset of embryonic development. Insect Mol Biol., 22, 433-441.