{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,28]],"date-time":"2025-11-28T12:33:11Z","timestamp":1764333191613,"version":"build-2065373602"},"reference-count":46,"publisher":"MDPI AG","issue":"14","license":[{"start":{"date-parts":[[2022,7,13]],"date-time":"2022-07-13T00:00:00Z","timestamp":1657670400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R01AI141532"],"award-info":[{"award-number":["R01AI141532"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Bloomberg Philanthropies and Johns Hopkins Malaria Institute","award":["R01AI141532"],"award-info":[{"award-number":["R01AI141532"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Cysteine-rich trypsin inhibitor-like domain (TIL)-harboring proteins are broadly distributed in nature but remain understudied in vector mosquitoes. Here we have explored the biology of a TIL domain-containing protein of the arbovirus vector Aedes aegypti, cysteine-rich venom protein 379 (CRVP379). CRVP379 was previously shown to be essential for dengue virus infection in Ae. aegypti mosquitoes. Gene expression analysis showed CRVP379 to be highly expressed in pupal stages, male testes, and female ovaries. CRVP379 expression is also increased in the ovaries at 48 h post-blood feeding. We used CRISPR-Cas9 genome editing to generate two mutant lines of CRVP379 with mutations inside or outside the TIL domain. Female mosquitoes from both mutant lines showed severe defects in their reproductive capability; mutant females also showed differences in their follicular cell morphology. However, the CRVP379 line with a mutation outside the TIL domain did not affect male reproductive performance, suggesting that some CRVP379 residues may have sexually dimorphic functions. In contrast to previous reports, we did not observe a noticeable difference in dengue virus infection between the wild-type and any of the mutant lines. The importance of CRVP379 in Ae. aegypti reproductive biology makes it an interesting candidate for the development of Ae. aegypti population control methods.<\/jats:p>","DOI":"10.3390\/ijms23147736","type":"journal-article","created":{"date-parts":[[2022,7,13]],"date-time":"2022-07-13T22:06:00Z","timestamp":1657749960000},"page":"7736","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Trypsin-like Inhibitor Domain (TIL)-Harboring Protein Is Essential for Aedes aegypti Reproduction"],"prefix":"10.3390","volume":"23","author":[{"given":"Chinmay Vijay","family":"Tikhe","sequence":"first","affiliation":[{"name":"W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, USA"},{"name":"Johns Hopkins Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4605-092X","authenticated-orcid":false,"given":"Victor","family":"Cardoso-Jaime","sequence":"additional","affiliation":[{"name":"W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, USA"},{"name":"Johns Hopkins Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8218-4688","authenticated-orcid":false,"given":"Shengzhang","family":"Dong","sequence":"additional","affiliation":[{"name":"W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, USA"},{"name":"Johns Hopkins Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Natalie","family":"Rutkowski","sequence":"additional","affiliation":[{"name":"W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, USA"},{"name":"Johns Hopkins Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6755-8111","authenticated-orcid":false,"given":"George","family":"Dimopoulos","sequence":"additional","affiliation":[{"name":"W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, USA"},{"name":"Johns Hopkins Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,7,13]]},"reference":[{"key":"ref_1","unstructured":"(2022, July 04). WHO Dengue Fact Sheet. Available online: https:\/\/www.who.int\/news-room\/fact-sheets\/detail\/dengue-and-severe-dengue."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"743","DOI":"10.4269\/ajtmh.2012.12-0157","article-title":"The Economic Burden of Dengue","volume":"86","author":"Gubler","year":"2012","journal-title":"Am. J. Trop. Med. Hyg."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"200","DOI":"10.4269\/ajtmh.2011.10-0503","article-title":"Economic Impact of Dengue Illness in the Americas","volume":"84","author":"Shepard","year":"2011","journal-title":"Am. J. Trop. Med. Hyg."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"156","DOI":"10.1016\/j.antiviral.2018.12.016","article-title":"Dengue Drug Discovery: Progress, Challenges and Outlook","volume":"163","author":"Lim","year":"2019","journal-title":"Antiviral Res."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1016\/j.coviro.2020.09.004","article-title":"Dengue Vaccine Development by the Year 2020: Challenges and Prospects","volume":"43","year":"2020","journal-title":"Curr. Opin. Virol."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Badolo, A., Sombi\u00e9, A., Pignatelli, P.M., Sanon, A., Yam\u00e9ogo, F., Wangrawa, D.W., Sanon, A., Kanuka, H., McCall, P.J., and Weetman, D. (2019). Insecticide Resistance Levels and Mechanisms in Aedes Aegypti Populations in and around Ouagadougou, Burkina Faso. PLoS Negl. Trop. Dis., 13.","DOI":"10.1371\/journal.pntd.0007439"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Marcombe, S., Fustec, B., Cattel, J., Chonephetsarath, S., Thammavong, P., Phommavanh, N., David, J.P., Corbel, V., Sutherland, I.W., and Hertz, J.C. (2019). Distribution of Insecticide Resistance and Mechanisms Involved in the Arbovirus Vector Aedes Aegypti in Laos and Implication for Vector Control. PLoS Negl. Trop. Dis., 13.","DOI":"10.1371\/journal.pntd.0007852"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Sene, N.M., Mavridis, K., Ndiaye, E.H., Diagne, C.T., Gaye, A., Ngom, E.H.M., Ba, Y., Diallo, D., Vontas, J., and Dia, I. (2021). Insecticide Resistance Status and Mechanisms in Aedes Aegypti Populations from Senegal. PLoS Negl. Trop. Dis., 15.","DOI":"10.1371\/journal.pntd.0009393"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"126","DOI":"10.1016\/j.pestbp.2012.05.008","article-title":"Insecticide Resistance in the Major Dengue Vectors Aedes Albopictus and Aedes Aegypti","volume":"104","author":"Vontas","year":"2012","journal-title":"Pestic. Biochem. Physiol."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Mukherjee, D., Das, S., Begum, F., Mal, S., and Ray, U. (2019). The Mosquito Immune System and the Life of Dengue Virus: What We Know and Do Not Know. Pathogens, 8.","DOI":"10.3390\/pathogens8020077"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"603","DOI":"10.1016\/j.pt.2018.04.011","article-title":"Diverse Host and Restriction Factors Regulate Mosquito-Pathogen Interactions","volume":"34","author":"Caragata","year":"2018","journal-title":"Trends Parasitol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"103964","DOI":"10.1016\/j.dci.2020.103964","article-title":"Mosquito Antiviral Immune Pathways","volume":"116","author":"Tikhe","year":"2021","journal-title":"Dev. Comp. Immunol."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Londono-Renteria, B., Troupin, A., Conway, M.J., Vesely, D., Ledizet, M., Roundy, C.M., Cloherty, E., Jameson, S., Vanlandingham, D., and Higgs, S. (2015). Dengue Virus Infection of Aedes Aegypti Requires a Putative Cysteine Rich Venom Protein. PLoS Pathog., 11.","DOI":"10.1371\/journal.ppat.1005202"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"227","DOI":"10.1016\/j.toxicon.2004.05.023","article-title":"Structure and Function of Snake Venom Cysteine-Rich Secretory Proteins","volume":"44","author":"Yamazaki","year":"2004","journal-title":"Toxicon"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"865","DOI":"10.1210\/er.2008-0032","article-title":"The CAP Superfamily: Cysteine-Rich Secretory Proteins, Antigen 5, and Pathogenesis-Related 1 Proteins\u2014Roles in Reproduction, Cancer, and Immune Defense","volume":"29","author":"Gibbs","year":"2008","journal-title":"Endocr. Rev."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"111","DOI":"10.1038\/aja.2010.77","article-title":"The Role of Cysteine-Rich Secretory Proteins in Male Fertility","volume":"13","author":"Koppers","year":"2011","journal-title":"Asian J. Androl."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"57","DOI":"10.1016\/j.pbi.2015.05.031","article-title":"Plant Cysteine-Rich Peptides That Inhibit Pathogen Growth and Control Rhizobial Differentiation in Legume Nodules","volume":"26","author":"Downie","year":"2015","journal-title":"Curr. Opin. Plant Biol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1514","DOI":"10.2174\/156802612802652411","article-title":"Cysteine-Rich Mini-Proteins in Human Biology","volume":"12","author":"Lavergne","year":"2012","journal-title":"Curr. Top. Med. Chem."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"256","DOI":"10.1002\/ptr.6823","article-title":"Cysteine-Rich Antimicrobial Peptides from Plants: The Future of Antimicrobial Therapy","volume":"35","author":"Srivastava","year":"2021","journal-title":"Phyther. Res."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"106","DOI":"10.1016\/j.peptides.2013.08.012","article-title":"Genome-Wide Search and Comparative Genomic Analysis of the Trypsin Inhibitor-like Cysteine-Rich Domain-Containing Peptides","volume":"53","author":"Zeng","year":"2014","journal-title":"Peptides"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"e0253121","DOI":"10.1128\/mBio.02531-21","article-title":"Pleiotropic Odorant-Binding Proteins Promote Aedes Aegypti Reproduction and Flavivirus Transmission","volume":"12","author":"Dong","year":"2021","journal-title":"MBio"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Dong, Y., Sim\u00f5es, M.L., Marois, E., and Dimopoulos, G. (2018). CRISPR\/Cas9 -Mediated Gene Knockout of Anopheles Gambiae FREP1 Suppresses Malaria Parasite Infection. PLoS Pathog., 14.","DOI":"10.1371\/journal.ppat.1006898"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Sim\u00f5es, M.L., Dong, Y., Mlambo, G., and Dimopoulos, G. (2022). C-Type Lectin 4 Regulates Broad-Spectrum Melanization-Based Refractoriness to Malaria Parasites. PLoS Biol., 20.","DOI":"10.1371\/journal.pbio.3001515"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"E10540","DOI":"10.1073\/pnas.1711538114","article-title":"Germline Cas9 Expression Yields Highly Efficient Genome Engineering in a Major Worldwide Disease Vector, Aedes Aegypti","volume":"114","author":"Li","year":"2017","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Isoe, J., Koch, L.E., Isoe, Y.E., Rasc\u00f3n, A.A., Brown, H.E., Massani, B.B., and Miesfeld, R.L. (2019). Identification and Characterization of a Mosquito-Specific Eggshell Organizing Factor in Aedes Aegypti Mosquitoes. PLoS Biol., 17.","DOI":"10.1371\/journal.pbio.3000068"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1016\/j.jinsphys.2019.04.009","article-title":"A Trypsin Inhibitor-like Protein Secreted by Cotesia Vestalis Teratocytes Inhibits Hemolymph Prophenoloxidase Activation of Plutella Xylostella","volume":"116","author":"Gu","year":"2019","journal-title":"J. Insect Physiol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"109042","DOI":"10.1016\/j.cbpc.2021.109042","article-title":"Anti-Fibrinolytic Activity of a Metalloprotease Inhibitor from Bumblebee (Bombus Ignitus) Venom","volume":"245","author":"Kim","year":"2021","journal-title":"Comp. Biochem. Physiol. Part C Toxicol. Pharmacol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"766","DOI":"10.1016\/j.ibmb.2012.07.004","article-title":"A Novel Protease Inhibitor in Bombyx Mori Is Involved in Defense against Beauveria Bassiana","volume":"42","author":"Li","year":"2012","journal-title":"Insect Biochem. Mol. Biol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1016\/j.peptides.2016.09.013","article-title":"Loss of Second and Sixth Conserved Cysteine Residues from Trypsin Inhibitor-like Cysteine-Rich Domain-Type Protease Inhibitors in Bombyx Mori May Induce Activity against Microbial Proteases","volume":"86","author":"Li","year":"2016","journal-title":"Peptides"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Mojzisch, A., and Brehm, M.A. (2021). The Manifold Cellular Functions of von Willebrand Factor. Cells, 10.","DOI":"10.3390\/cells10092351"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"449","DOI":"10.1182\/blood-2012-01-405134","article-title":"Sequence and Structure Relationships within von Willebrand Factor","volume":"120","author":"Zhou","year":"2012","journal-title":"Blood"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"209","DOI":"10.1007\/s13659-015-0069-z","article-title":"A Novel Trypsin Inhibitor-Like Cysteine-Rich Peptide from the Frog Lepidobatrachus Laevis Containing Proteinase-Inhibiting Activity","volume":"5","author":"Wang","year":"2015","journal-title":"Nat. Products Bioprospect."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/s41938-020-0208-7","article-title":"Molecular Identification, Characterization, and Expression Analysis of a Novel Trypsin Inhibitor-like Cysteine-Rich Peptide from the Cotton Bollworm, Helicoverpa Armigera (H\u00fcbner) (Lepidoptera: Noctuidae)","volume":"30","author":"Shakeel","year":"2020","journal-title":"Egypt. J. Biol. Pest Control"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"223","DOI":"10.1002\/arch.20412","article-title":"Expression of Trypsin-like Serine Peptidases in Pre-Imaginal Stages of Aedes Aegypti (Diptera: Culicidae)","volume":"76","author":"Cuervo","year":"2011","journal-title":"Arch. Insect Biochem. Physiol."},{"key":"ref_35","first-page":"1","article-title":"Trypsin-like Serine Peptidase Profiles in the Egg, Larval, and Pupal Stages of Aedes Albopictus","volume":"6","author":"Cuervo","year":"2013","journal-title":"Parasites Vectors"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"117","DOI":"10.1016\/0022-1910(71)90048-5","article-title":"Trypsin and Chymotrypsin during Metamorphosis in Aedes Aegypti and Properties of the Chymotrypsin","volume":"17","author":"Yang","year":"1971","journal-title":"J. Insect Physiol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1493","DOI":"10.1534\/g3.113.006742","article-title":"The Developmental Transcriptome of the Mosquito Aedes Aegypti, an Invasive Species and Major Arbovirus Vector","volume":"3","author":"Akbari","year":"2013","journal-title":"G3 (Bethesda)"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"e76132","DOI":"10.7554\/eLife.76132","article-title":"A Transcriptomic Atlas of Aedes Aegypti Reveals Detailed Functional Organization of Major Body Parts and Gut Regional Specializations in Sugar-Fed and Blood-Fed Adult Females","volume":"11","author":"Hixson","year":"2022","journal-title":"Elife"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"e2023470118","DOI":"10.1073\/pnas.2023470118","article-title":"Cross-Talk of Insulin-like Peptides, Juvenile Hormone, and 20-Hydroxyecdysone in Regulation of Metabolism in the Mosquito Aedes Aegypti","volume":"118","author":"Ling","year":"2021","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1007","DOI":"10.1016\/j.jinsphys.2012.05.005","article-title":"The Fate of Follicles after a Blood Meal Is Dependent on Previtellogenic Nutrition and Juvenile Hormone in Aedes Aegypti","volume":"58","author":"Clifton","year":"2012","journal-title":"J. Insect Physiol."},{"key":"ref_41","first-page":"1","article-title":"Juvenile Hormone Controls Ovarian Development in Female Anopheles Albimanus Mosquitoes","volume":"9","author":"Nouzova","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"190","DOI":"10.1016\/j.ibmb.2007.10.008","article-title":"Characterization of a Juvenile Hormone-Regulated Chymotrypsin-like Serine Protease Gene in Aedes Aegypti Mosquito","volume":"38","author":"Bian","year":"2008","journal-title":"Insect Biochem. Mol. Biol."},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Ji, J.-L., Han, S.-J., Zhang, R.-J., Yu, J.-B., Li, Y.-B., Yu, X.-P., Liu, G.-F., and Xu, Y.-P. (2022). Inter-Alpha-Trypsin Inhibitor Heavy Chain 4 Plays an Important Role in the Development and Reproduction of Nilaparvata Lugens. Insects, 13.","DOI":"10.3390\/insects13030303"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"W171","DOI":"10.1093\/nar\/gkz365","article-title":"CHOPCHOP v3: Expanding the CRISPR Web Toolbox beyond Genome Editing","volume":"47","author":"Labun","year":"2019","journal-title":"Nucleic Acids Res."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"103753","DOI":"10.1016\/j.dci.2020.103753","article-title":"DNA Synthesis Increases during the First Hours Post-Emergence in Anopheles Albimanus Mosquito Midgut","volume":"112","year":"2020","journal-title":"Dev. Comp. Immunol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"676","DOI":"10.1038\/nmeth.2019","article-title":"Fiji: An Open-Source Platform for Biological-Image Analysis","volume":"9","author":"Schindelin","year":"2012","journal-title":"Nat. Methods"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/14\/7736\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T23:49:34Z","timestamp":1760140174000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/14\/7736"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,7,13]]},"references-count":46,"journal-issue":{"issue":"14","published-online":{"date-parts":[[2022,7]]}},"alternative-id":["ijms23147736"],"URL":"https:\/\/doi.org\/10.3390\/ijms23147736","relation":{},"ISSN":["1422-0067"],"issn-type":[{"type":"electronic","value":"1422-0067"}],"subject":[],"published":{"date-parts":[[2022,7,13]]}}}