Insects rely on the innate immune system for defense against pathogens, some aspects of which are under hormonal control. Here we provide direct experimental evidence showing that the juvenile hormone-binding protein (mJHBP) of Aedes aegypti is required for the regulation of innate immune responses and the development of mosquito blood cells (hemocytes). Using an mJHBP-deficient mosquito line generated by means of CRISPR-Cas9 gene editing technology we uncovered a mutant phenotype characterized by immunosuppression at the humoral and cellular levels, which profoundly affected susceptibility to bacterial infection. Bacteria-challenged mosquitoes exhibited significantly higher levels of septicemia and mortality relative to the wild type (WT) strain, delayed expression of antimicrobial peptides (AMPs), severe developmental dysregulation of embryonic and larval hemocytes (reduction in the total number of hemocytes) and increased differentiation of the granulocyte lineage. Interestingly, injection of recombinant wild type mJHBP protein into adult females three-days before infection was sufficient to restore normal immune function. Similarly, injection of mJHBP into fourth-instar larvae fully restored normal larval/pupal hemocyte populations in emerging adults. More importantly, the recovery of normal immuno-activation and hemocyte development requires the capability of mJHBP to bind JH III. These results strongly suggest that JH III functions in mosquito immunity and hemocyte development in a manner that is perhaps independent of canonical JH signaling, given the lack of developmental and reproductive abnormalities. Because of the prominent role of hemocytes as regulators of mosquito immunity, this novel discovery may have broader implications for the understanding of vector endocrinology, hemocyte development, vector competence and disease transmission.

Department of Biological Sciences and Biomolecular Science Institute Florida International University Miami United States of America

Department of Entomology and AgriLife Research Texas A and M University College Station United States of America

Institute of Parasitology Biology Centre CAS Ceske Budejovice Czech Republic

NIH NIAID Laboratory of Malaria and Vector Research 12735 Twinbrook Parkway Rockville MD United States of America

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Bartholomay LC, Michel K. Mosquito Immunobiology: The Intersection of Vector Health and Vector Competence. Annu Rev Entomol. 2018;63:145–67. Epub 2018/01/13. 10.1146/annurev-ento-010715-023530 . PubMed DOI

Rus F, Flatt T, Tong M, Aggarwal K, Okuda K, Kleino A, et al. Ecdysone triggered PGRP-LC expression controls Drosophila innate immunity. EMBO J. 2013;32(11):1626–38. Epub 2013/05/09. 10.1038/emboj.2013.100 PubMed DOI PMC

Zhang Z, Palli SR. Identification of a cis-regulatory element required for 20-hydroxyecdysone enhancement of antimicrobial peptide gene expression in Drosophila melanogaster. Insect Mol Biol. 2009;18(5):595–605. Epub 2009/09/17. 10.1111/j.1365-2583.2009.00901.x . PubMed DOI

Regan JC, Brandao AS, Leitao AB, Mantas Dias AR, Sucena E, Jacinto A, et al. Steroid hormone signaling is essential to regulate innate immune cells and fight bacterial infection in Drosophila. PLoS Pathog. 2013;9(10):e1003720 Epub 2013/11/10. 10.1371/journal.ppat.1003720 PubMed DOI PMC

Upton LM, Povelones M, Christophides GK. Anopheles gambiae blood feeding initiates an anticipatory defense response to Plasmodium berghei. J Innate Immun. 2015;7(1):74–86. Epub 2014/09/24. 10.1159/000365331 PubMed DOI PMC

Werling K, Shaw WR, Itoe MA, Westervelt KA, Marcenac P, Paton DG, et al. Steroid Hormone Function Controls Non-competitive Plasmodium Development in Anopheles. Cell. 2019;177(2):315–25 e14. Epub 2019/04/02. 10.1016/j.cell.2019.02.036 PubMed DOI PMC

Ahmed A, Martin D, Manetti AG, Han SJ, Lee WJ, Mathiopoulos KD, et al. Genomic structure and ecdysone regulation of the prophenoloxidase 1 gene in the malaria vector Anopheles gambiae. Proc Natl Acad Sci U S A. 1999;96(26):14795–800. Epub 1999/12/28. 10.1073/pnas.96.26.14795 PubMed DOI PMC

Flatt T, Heyland A, Rus F, Porpiglia E, Sherlock C, Yamamoto R, et al. Hormonal regulation of the humoral innate immune response in Drosophila melanogaster. J Exp Biol. 2008;211(Pt 16):2712–24. Epub 2008/08/12. 10.1242/jeb.014878 PubMed DOI PMC

Kim IH, Pham V, Jablonka W, Goodman WG, Ribeiro JMC, Andersen JF. A mosquito hemolymph odorant-binding protein family member specifically binds juvenile hormone. J Biol Chem. 2017;292(37):15329–39. Epub 2017/07/29. 10.1074/jbc.M117.802009 PubMed DOI PMC

Alvarenga PH, Francischetti IM, Calvo E, Sa-Nunes A, Ribeiro JM, Andersen JF. The function and three-dimensional structure of a thromboxane A2/cysteinyl leukotriene-binding protein from the saliva of a mosquito vector of the malaria parasite. PLoS Biol. 2010;8(11):e1000547 Epub 2010/12/15. 10.1371/journal.pbio.1000547 PubMed DOI PMC

Smykal V, Raikhel AS. Nutritional Control of Insect Reproduction. Curr Opin Insect Sci. 2015;11:31–8. Epub 2015/12/09. 10.1016/j.cois.2015.08.003 PubMed DOI PMC

Hernandez-Martinez S, Rivera-Perez C, Nouzova M, Noriega FG. Coordinated changes in JH biosynthesis and JH hemolymph titers in Aedes aegypti mosquitoes. J Insect Physiol. 2015;72:22–7. Epub 2014/12/03. 10.1016/j.jinsphys.2014.11.003 PubMed DOI PMC

Das D, Aradhya R, Ashoka D, Inamdar M. Macromolecular uptake in Drosophila pericardial cells requires rudhira function. Exp Cell Res. 2008;314(8):1804–10. Epub 2008/03/22. 10.1016/j.yexcr.2008.02.009 . PubMed DOI

Barletta ABF, Alves LR, Silva MCLN, Sim S, Dimopoulos G, Liechocki S, et al. Emerging role of lipid droplets in Aedes aegypti immune response against bacteria and Dengue virus. Sci Rep-Uk. 2016;6 ARTN 19928 10.1038/srep19928 WOS:000370340900001. PubMed DOI PMC

Bradburn MJ, Clark TG, Love SB, Altman DG. Survival analysis part II: multivariate data analysis—an introduction to concepts and methods. Br J Cancer. 2003;89(3):431–6. Epub 2003/07/31. 10.1038/sj.bjc.6601119 PubMed DOI PMC

Castillo J, Brown MR, Strand MR. Blood feeding and insulin-like peptide 3 stimulate proliferation of hemocytes in the mosquito Aedes aegypti. PLoS Pathog. 2011;7(10):e1002274 Epub 2011/10/15. 10.1371/journal.ppat.1002274 PubMed Central PMCID: PMC3188524. PubMed DOI PMC

Castillo JC, Ferreira ABB, Trisnadi N, Barillas-Mury C. Activation of mosquito complement antiplasmodial response requires cellular immunity. Sci Immunol. 2017;2(7). Epub 2017/07/25. 10.1126/sciimmunol.aal1505 PubMed DOI PMC

Hillyer JF, Schmidt SL, Christensen BM. Rapid phagocytosis and melanization of bacteria and Plasmodium sporozoites by hemocytes of the mosquito Aedes aegypti. J Parasitol. 2003;89(1):62–9. Epub 2003/03/28. 10.1645/0022-3395(2003)089[0062:RPAMOB]2.0.CO;2 . PubMed DOI

Hillyer JF, Schmidt SL, Christensen BM. The antibacterial innate immune response by the mosquito Aedes aegypti is mediated by hemocytes and independent of Gram type and pathogenicity. Microbes Infect. 2004;6(5):448–59. Epub 2004/04/28. 10.1016/j.micinf.2004.01.005 . PubMed DOI

Rodrigues J, Brayner FA, Alves LC, Dixit R, Barillas-Mury C. Hemocyte differentiation mediates innate immune memory in Anopheles gambiae mosquitoes. Science. 2010;329(5997):1353–5. Epub 2010/09/11. 10.1126/science.1190689 PubMed DOI PMC

Roy S, Saha TT, Zou Z, Raikhel AS. Regulatory Pathways Controlling Female Insect Reproduction. Annu Rev Entomol. 2018;63:489–511. Epub 2017/10/24. 10.1146/annurev-ento-020117-043258 . PubMed DOI

Zou Z, Saha TT, Roy S, Shin SW, Backman TW, Girke T, et al. Juvenile hormone and its receptor, methoprene-tolerant, control the dynamics of mosquito gene expression. Proc Natl Acad Sci U S A. 2013;110(24):E2173–81. Epub 2013/05/02. 10.1073/pnas.1305293110 PubMed DOI PMC

Kawaguchi R, Yu J, Honda J, Hu J, Whitelegge J, Ping P, et al. A membrane receptor for retinol binding protein mediates cellular uptake of vitamin A. Science. 2007;315(5813):820–5. Epub 2007/01/27. 10.1126/science.1136244 . PubMed DOI

Benoit JB, Vigneron A, Broderick NA, Wu Y, Sun JS, Carlson JR, et al. Symbiont-induced odorant binding proteins mediate insect host hematopoiesis. Elife. 2017;6 Epub 2017/01/13. 10.7554/eLife.19535 PubMed DOI PMC

Evans CJ, Hartenstein V, Banerjee U. Thicker than blood: conserved mechanisms in Drosophila and vertebrate hematopoiesis. Dev Cell. 2003;5(5):673–90. Epub 2003/11/07. 10.1016/s1534-5807(03)00335-6 . PubMed DOI

Hartenstein V. Blood cells and blood cell development in the animal kingdom. Annu Rev Cell Dev Biol. 2006;22:677–712. Epub 2006/07/11. 10.1146/annurev.cellbio.22.010605.093317 . PubMed DOI

Lebestky T, Chang T, Hartenstein V, Banerjee U. Specification of Drosophila hematopoietic lineage by conserved transcription factors. Science. 2000;288(5463):146–9. Epub 2001/02/07. 10.1126/science.288.5463.146 . PubMed DOI

Tepass U, Fessler LI, Aziz A, Hartenstein V. Embryonic origin of hemocytes and their relationship to cell death in Drosophila. Development. 1994;120(7):1829–37. Epub 1994/07/01. . PubMed

Jung SH, Evans CJ, Uemura C, Banerjee U. The Drosophila lymph gland as a developmental model of hematopoiesis. Development. 2005;132(11):2521–33. Epub 2005/04/29. 10.1242/dev.01837 . PubMed DOI

Rugendorff A, Younossi-Hartenstein A, Hartenstein V. Embryonic origin and differentiation of the Drosophila heart. Roux Arch Dev Biol. 1994;203(5):266–80. Epub 1994/03/01. 10.1007/BF00360522 . PubMed DOI

Holz A, Bossinger B, Strasser T, Janning W, Klapper R. The two origins of hemocytes in Drosophila. Development. 2003;130(20):4955–62. Epub 2003/08/22. 10.1242/dev.00702 . PubMed DOI

Robertson CW. The metamorphosis of Drosophila melanogaster, including an accurately timed account of the principal morphological changes. J Morphol. 1936;59:351–99.

Makhijani K, Alexander B, Tanaka T, Rulifson E, Bruckner K. The peripheral nervous system supports blood cell homing and survival in the Drosophila larva. Development. 2011;138(24):5379–91. Epub 2011/11/11. 10.1242/dev.067322 PubMed DOI PMC

Stofanko M, Kwon SY, Badenhorst P. A misexpression screen to identify regulators of Drosophila larval hemocyte development. Genetics. 2008;180(1):253–67. Epub 2008/09/02. 10.1534/genetics.108.089094 PubMed DOI PMC

Ghosh S, Singh A, Mandal S, Mandal L. Active hematopoietic hubs in Drosophila adults generate hemocytes and contribute to immune response. Dev Cell. 2015;33(4):478–88. Epub 2015/05/12. 10.1016/j.devcel.2015.03.014 PubMed DOI PMC

Basu S, Aryan A, Overcash JM, Samuel GH, Anderson MA, Dahlem TJ, et al. Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis in Aedes aegypti. Proc Natl Acad Sci U S A. 2015;112(13):4038–43. Epub 2015/03/17. 10.1073/pnas.1502370112 PubMed DOI PMC

Basu S, Aryan A, Haac ME, Myles KM, Adelman ZN. Methods for TALEN Evaluation, Use, and Mutation Detection in the Mosquito Aedes aegypti. Methods Mol Biol. 2016;1338:157–77. Epub 2015/10/08. 10.1007/978-1-4939-2932-0_13 PubMed DOI PMC

Nasci RS. Relationship of wing length to adult dry weight in several mosquito species (Diptera: Culicidae). J Med Entomol. 1990;27(4):716–9. Epub 1990/07/01. 10.1093/jmedent/27.4.716 . PubMed DOI

Ramirez CE, Nouzova M, Benigni P, Quirke JME, Noriega FG, Fernandez-Lima F. Fast, ultra-trace detection of juvenile hormone III from mosquitoes using mass spectrometry. Talanta. 2016;159:371–8. Epub 2016/07/31. 10.1016/j.talanta.2016.06.041 PubMed DOI PMC

Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9(7):676–82. Epub 2012/06/30. 10.1038/nmeth.2019 PubMed DOI PMC

Castillo JC, Robertson AE, Strand MR. Characterization of hemocytes from the mosquitoes Anopheles gambiae and Aedes aegypti. Insect Biochem Mol Biol. 2006;36(12):891–903. Epub 2006/11/14. 10.1016/j.ibmb.2006.08.010 PubMed DOI PMC

Rao X, Huang X, Zhou Z, Lin X. An improvement of the 2^(-delta delta CT) method for quantitative real-time polymerase chain reaction data analysis. Biostat Bioinforma Biomath. 2013;3(3):71–85. Epub 2013/08/01. PubMed PMC

Ramirez JL, Garver LS, Brayner FA, Alves LC, Rodrigues J, Molina-Cruz A, et al. The Role of Hemocytes in Anopheles gambiae Antiplasmodial Immunity. J Innate Immun. 2014;6(2):119–28. 10.1159/000353765 WOS:000331774900002. PubMed DOI PMC

Approaches and Tools to Study the Roles of Juvenile Hormones in Controlling Insect Biology