{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T14:38:07Z","timestamp":1787236687719,"version":"build-2736575974"},"reference-count":57,"publisher":"Oxford University Press (OUP)","issue":"4","license":[{"start":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T00:00:00Z","timestamp":1787184000000},"content-version":"vor","delay-in-days":50,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"name":"U.S. Department of Agriculture, Agriculture Research Service","award":["3060\u201330500-001-001-T"],"award-info":[{"award-number":["3060\u201330500-001-001-T"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,7,1]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Anopheles gambiae (Giles) is a medically important mosquito species and a vector of the malaria Plasmodium parasite. There have been many advancements in vector control research that involves genetic manipulation of the mosquito. Malaria vector research requires rearing continuous mosquito colonies that are costly and carry risks to maintain. Therefore, we studied the feasibility of germplasm cryopreservation as a way to maintain genetic background as well as restore mosquito colonies. One part of germplasm cryopreservation is the cryopreservation of the whole germinal organ such as the testis as this would preserve half the genome. The aim of this study was to develop a simple method to cryopreserve whole testicular-seminal vesicle complex from which one could derive both the viable spermatozoa as well as primordial and stem cells from male mosquitos. This is the first study which demonstrates the successful cryopreservation of a whole organ in an insect system. In this study, various cryopreservation protocols were tested to determine which resulted in the highest testicular cell survival. The methods tested included different freezing rates, different incubation protocols, and use of different non-permeating cryoprotectants. The cooling rate that was found to have the highest survival was cooling to \u221240\u2009\u00b0C at a rate of 1\u2009\u00b0C min\u22121 followed by a plunge into liquid nitrogen, although there was little difference in survival based on cooling rate.<\/jats:p>","DOI":"10.1093\/jme\/tjag135","type":"journal-article","created":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T13:53:44Z","timestamp":1787234024000},"source":"Crossref","is-referenced-by-count":0,"title":["Whole testicular organ and seminal cryopreservation in the malarial mosquito\n                    <i>Anopheles gambiae<\/i>\n                    (Diptera: Culicidae)"],"prefix":"10.1093","volume":"63","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4711-1942","authenticated-orcid":false,"given":"Courtney C","family":"Grula","sequence":"first","affiliation":[{"name":"Department of Biological Sciences, North Dakota State University , Fargo, ND,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1587-2891","authenticated-orcid":false,"given":"Arun","family":"Rajamohan","sequence":"additional","affiliation":[{"name":"Weed and Insect Biology Unit, Edward T. Schafer Research Center, U.S. Department of Agriculture\/Agricultural Research Center , Fargo, ND,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Melanie","family":"Tischleder","sequence":"additional","affiliation":[{"name":"Weed and Insect Biology Unit, Edward T. Schafer Research Center, U.S. Department of Agriculture\/Agricultural Research Center , Fargo, ND,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joseph P","family":"Rinehart","sequence":"additional","affiliation":[{"name":"Weed and Insect Biology Unit, Edward T. Schafer Research Center, U.S. Department of Agriculture\/Agricultural Research Center , Fargo, ND,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2026,8,20]]},"reference":[{"key":"2026082009533585800_tjag135-B1","doi-asserted-by":"publisher","first-page":"1079","DOI":"10.1097\/01.ju.0000084820.98430.b8","article-title":"Cryopreservation of sperm: indications, methods and results","volume":"170","author":"Anger","year":"2003","journal-title":"J. Urol."},{"key":"2026082009533585800_tjag135-B2","doi-asserted-by":"publisher","first-page":"3","DOI":"10.1007\/978-94-009-1535-0_1","article-title":"Care and maintenance of anopheline mosquito colonies","author":"Benedict","year":"1997","journal-title":"Mol Biol Insect Dis Vectors"},{"key":"2026082009533585800_tjag135-B3","doi-asserted-by":"publisher","first-page":"274","DOI":"10.3390\/biom10020274","article-title":"Ice binding proteins: diverse biological roles and applications in different types of industry","volume":"10","author":"Bia\u0142kowska","year":"2020","journal-title":"Biomolecules"},{"key":"2026082009533585800_tjag135-B4","doi-asserted-by":"publisher","first-page":"1164","DOI":"10.1095\/biolreprod63.4.1164","article-title":"Species variation in osmotic, cryoprotectants, and cooling rate tolerance in poultry, eagle, and peregrine falcon, spermatozoa","volume":"63","author":"Blanco","year":"2000","journal-title":"Biol. Reprod."},{"key":"2026082009533585800_tjag135-B5","doi-asserted-by":"publisher","first-page":"110","DOI":"10.1002\/mrd.1080400114","article-title":"Effects of cooling and equilibration in DMSO, and cryopreservation of mouse oocytes on the rates of in vitro fertilization, development, and chromosomal abnormalities","volume":"40","author":"Boquet","year":"1995","journal-title":"Mol Reprod Dev"},{"key":"2026082009533585800_tjag135-B6","doi-asserted-by":"publisher","first-page":"114","DOI":"10.1016\/j.cryobiol.2020.12.001","article-title":"Physiological responses to cryoprotectant treatment in an early larval stage of the malaria mosquito, Anopheles gambiae","volume":"99","author":"Campbell","year":"2021","journal-title":"Cryobiology"},{"key":"2026082009533585800_tjag135-B7","doi-asserted-by":"publisher","first-page":"136","DOI":"10.1016\/j.cryobiol.2021.06.002","article-title":"Cryopreservation of seminal vesicle derived spermatozoa from Bombus impatiens and Apis mellifera\u2013Implications for artificial insemination of bumble bees","volume":"102","author":"Campion","year":"2021","journal-title":"Cryobiology"},{"key":"2026082009533585800_tjag135-B8","author":"Centers for Disease Control and Prevention","year":"2023"},{"key":"2026082009533585800_tjag135-B9","doi-asserted-by":"publisher","first-page":"719","DOI":"10.1071\/RD07035","article-title":"Novel gamete storage","volume":"19","author":"Dinneys","year":"2007","journal-title":"Reprod. Fertil. Dev."},{"key":"2026082009533585800_tjag135-B10","doi-asserted-by":"publisher","first-page":"743","DOI":"10.1016\/s0015-0282(16)59879-3","article-title":"Cryopreservation of embryos and ova","volume":"49","author":"Friedler","year":"1988","journal-title":"Fertil. Steril"},{"key":"2026082009533585800_tjag135-B10b","doi-asserted-by":"publisher","DOI":"10.11416\/jibs.91.1_013","article-title":"Differences between strains in the tolerance to testis cryopreservation in the silkworm, Bombyx mori","volume":"91","author":"Fukumori","year":"2022","journal-title":"J Insect Biotechnol Sericol"},{"key":"2026082009533585800_tjag135-B11","first-page":"375","article-title":"Cryoprotectants: the essential antifreezes to protect life in the frozen state","volume":"25","author":"Fuller","year":"2004","journal-title":"Cryo Lett"},{"key":"2026082009533585800_tjag135-B12","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2211744119","article-title":"Stabilization of insect cell membranes and soluble enzymes by accumulated cryoprotectants during freezing stress","volume":"119","author":"Grgac","year":"2022","journal-title":"Proc Natl Acad Sci USA"},{"key":"2026082009533585800_tjag135-B13","doi-asserted-by":"publisher","first-page":"14920","DOI":"10.1038\/s41598-024-65624-x","article-title":"Monarch butterfly conservation through male germplasm cryopreservation","volume":"14","author":"Grula","year":"2024","journal-title":"Sci. Rep"},{"key":"2026082009533585800_tjag135-B14","doi-asserted-by":"publisher","first-page":"47","DOI":"10.1016\/s0093-691x(99)00239-3","article-title":"Fundamental aspects of sperm cryobiology: the importance of species and individual differences","volume":"53","author":"Holt","year":"2000","journal-title":"Theriogenology"},{"key":"2026082009533585800_tjag135-B15","doi-asserted-by":"publisher","first-page":"102","DOI":"10.1111\/acv.12229","article-title":"Recovery of gene diversity using long-term cryopreserved spermatozoa and artificial insemination in the endangered black-footed ferret","volume":"19","author":"Howard","year":"2016","journal-title":"Anim. Conserv"},{"key":"2026082009533585800_tjag135-B16","doi-asserted-by":"publisher","first-page":"432","DOI":"10.1089\/bio.2017.0029","article-title":"Improvement of a simple and cost-effective passive cooling rate-controlled device for cell\/tissue cryopreservation","volume":"15","author":"Huang","year":"2017","journal-title":"Biopreserv. Biobank"},{"key":"2026082009533585800_tjag135-B17","doi-asserted-by":"publisher","first-page":"41","DOI":"10.1007\/978-1-4939-6921-0_5","article-title":"Cryopreservation: vitrification and controlled rate cooling","volume":"1590","author":"Hunt","year":"2017","journal-title":"Methods Mol. Biol"},{"key":"2026082009533585800_tjag135-B18","doi-asserted-by":"publisher","first-page":"53","DOI":"10.4103\/2229-5070.129167","article-title":"The basic rules and methods of mosquito rearing (Aedes aegypti)","volume":"4","author":"Imam","year":"2014","journal-title":"Trop. Parasitol"},{"key":"2026082009533585800_tjag135-B19","doi-asserted-by":"publisher","first-page":"43","DOI":"10.1038\/s41598-021-04113-x","article-title":"Cryopreservation of Anopheles stephensi embryos","volume":"12","author":"James","year":"2022","journal-title":"Sci. Rep"},{"key":"2026082009533585800_tjag135-B20","doi-asserted-by":"publisher","first-page":"e00633","DOI":"10.1002\/adbi.202500633","article-title":"Cryopreservation of Anopheles gambiae Larvae","volume":"10","author":"Joshi","year":"2026","journal-title":"Adv. Biol. (Weinh)"},{"key":"2026082009533585800_tjag135-B21","doi-asserted-by":"publisher","first-page":"e2542","DOI":"10.21769\/BioProtoc.2542","article-title":"Rearing of Culex spp. and Aedes spp. mosquitoes","volume":"7","author":"Kauffman","year":"2017","journal-title":"Bio Protoc"},{"key":"2026082009533585800_tjag135-B23","doi-asserted-by":"publisher","first-page":"109","DOI":"10.1095\/biolreprod.113.111195","article-title":"Cryopreservation of mouse spermatogonial stem cells in dimethylsulfoxide and polyethylene glycol","volume":"89","author":"Lee","year":"2013","journal-title":"Biol. Reprod"},{"key":"2026082009533585800_tjag135-B24","first-page":"149","article-title":"Colony maintenance and mass-rearing: using cold storage technology for extending the shelf-life of insects. Area-Wide Control Insect Pests","author":"Leopold","year":"2007"},{"key":"2026082009533585800_tjag135-B25","doi-asserted-by":"publisher","first-page":"695","DOI":"10.1603\/0013-8746(2001)094[0695:COEOTN]2.0.CO;2","article-title":"Cryopreservation of embryos of the New World Screwworm, Cochliomyia hominivorax (Diptera: Calliphoridae)","volume":"94","author":"Leopold","year":"2001","journal-title":"Ann Entomol Soc Am"},{"key":"2026082009533585800_tjag135-B26","doi-asserted-by":"publisher","first-page":"264","DOI":"10.1603\/AN09094","article-title":"Quality testing of three species of tcphritid fruit flies after embryo clyopreservation","volume":"103","author":"Leopold","year":"2010","journal-title":"Ann. Entomol. Soc. Am"},{"key":"2026082009533585800_tjag135-B27","first-page":"325","article-title":"Low Temperature Biology of Insects. Cambridge University Press","author":"Leopold","year":"2010"},{"key":"2026082009533585800_tjag135-B28","doi-asserted-by":"publisher","first-page":"I333","DOI":"10.1016\/j.cryobiol.2021.01.013","article-title":"Passive slow freezing is an easy, cost-effective and efficacious alternative freezing method for slow controlled cryopreservation of ovarian tissue","volume":"35","author":"Lierman","year":"2020","journal-title":"Hum Reprod"},{"key":"2026082009533585800_tjag135-B29","doi-asserted-by":"publisher","first-page":"1394","DOI":"10.1038\/1831394a0","article-title":"Prevention of freezing damage to living cells by dimethyl sulphoxide","volume":"183","author":"Lovelock","year":"1959","journal-title":"Nature"},{"key":"2026082009533585800_tjag135-B30","doi-asserted-by":"publisher","first-page":"347","DOI":"10.1085\/jgp.47.2.347","article-title":"Kinetics of water loss from cells at subzero temperatures and the likelihood of intracellular freezing","volume":"47","author":"Mazur","year":"1963","journal-title":"J. Gen. Physiol"},{"key":"2026082009533585800_tjag135-B30b","doi-asserted-by":"crossref","first-page":"939","DOI":"10.1126\/science.168.3934.939","article-title":"Cryobiology: The Freezing of Biological Systems","volume":"168","author":"Mazur","year":"1970","journal-title":"Science"},{"key":"2026082009533585800_tjag135-B31","doi-asserted-by":"publisher","first-page":"345","DOI":"10.1016\/0014-4827(72)90303-5","article-title":"A two-factor hypothesis of freezing injury\u2013evidence from Chinese hamster tissue culture cells","volume":"71","author":"Mazur","year":"1972","journal-title":"Exp. Cell Res"},{"key":"2026082009533585800_tjag135-B32","doi-asserted-by":"publisher","first-page":"C125","DOI":"10.1152\/ajpcell.1984.247.3.C125","article-title":"Freezing of living cells: mechanisms and implications","volume":"247","author":"Mazur","year":"1984","journal-title":"Am. J. Physiol"},{"key":"2026082009533585800_tjag135-B33","first-page":"3","article-title":"Life Frozen State","author":"Mazur","year":"2004"},{"key":"2026082009533585800_tjag135-B34","doi-asserted-by":"publisher","first-page":"327","DOI":"10.1016\/s0093-691x(01)00674-4","article-title":"Current status of sperm cryopreservation: why isn\u2019t it better?","volume":"57","author":"Medeiros","year":"2002","journal-title":"Theriogenology"},{"key":"2026082009533585800_tjag135-B35","doi-asserted-by":"publisher","first-page":"515","DOI":"10.1126\/science.124.3221.515","article-title":"Mechanics of freezing in living cells and tissues","volume":"124","author":"Meryman","year":"1956","journal-title":"Science (1979)"},{"key":"2026082009533585800_tjag135-B36","doi-asserted-by":"publisher","first-page":"71","DOI":"10.1016\/j.cryobiol.2011.12.002","article-title":"Freezing injury: the special case of the sperm cell","volume":"64","author":"Morris","year":"2012","journal-title":"Cryobiology"},{"key":"2026082009533585800_tjag135-B37","doi-asserted-by":"publisher","first-page":"106","DOI":"10.1016\/j.cryobiol.2020.12.007","article-title":"Cryoprotectant toxicity and hypothermic sensitivity among Anopheles larvae","volume":"99","author":"Nesbitt","year":"2021","journal-title":"Cryobiology"},{"key":"2026082009533585800_tjag135-B38","doi-asserted-by":"publisher","first-page":"613","DOI":"10.3390\/ani15050613","article-title":"Endangered Przewalski\u2019s horse, Equus przewalskii, cloned from historically cryopreserved cells","volume":"15","author":"Novak","year":"2025","journal-title":"Animals (Basel)"},{"key":"2026082009533585800_tjag135-B39","doi-asserted-by":"publisher","first-page":"1231","DOI":"10.3727\/096368910X552835","article-title":"Effect of dimethyl sulfoxide (DMSO) on cryopreservation of porcine mesenchymal stem cells (pMSCs)","volume":"20","author":"Ock","year":"2011","journal-title":"Cell Transplant"},{"key":"2026082009533585800_tjag135-B40","author":"Pichette","year":"2010"},{"key":"2026082009533585800_tjag135-B41","doi-asserted-by":"publisher","first-page":"44","DOI":"10.1016\/j.cryobiol.2006.10.192","article-title":"Cryopreservation of Mexican fruit flies by vitrification: stage selection and avoidance of thermal stress","volume":"54","author":"Rajamohan","year":"2007","journal-title":"Cryobiology"},{"key":"2026082009533585800_tjag135-B42","first-page":"125","article-title":"Cryopreservation of Mediterranean fruit fly embryos","volume":"24","author":"Rajamohan","year":"2003","journal-title":"Cryo Lett"},{"key":"2026082009533585800_tjag135-B43","doi-asserted-by":"publisher","first-page":"261","DOI":"10.1016\/j.cryobiol.2020.10.053","article-title":"Designing a template cryopreservation procedure for insect germplasm: studies with the honey bee embryos","volume":"97","author":"Rajamohan","year":"2020","journal-title":"Cryobiology"},{"key":"2026082009533585800_tjag135-B44","doi-asserted-by":"publisher","first-page":"573","DOI":"10.1038\/313573a0","article-title":"Ice-free cryopreservation of mouse embryos at \u2212196 C by vitrification","volume":"313","author":"Rall","year":"1985","journal-title":"Nature"},{"key":"2026082009533585800_tjag135-B45","doi-asserted-by":"publisher","first-page":"1183","DOI":"10.1038\/s41467-021-21349-3","article-title":"Ice-nucleating proteins are activated by low temperatures to control the structure of interfacial water","volume":"12","author":"Roeters","year":"2021","journal-title":"Nat Commun"},{"key":"2026082009533585800_tjag135-B46","doi-asserted-by":"publisher","first-page":"529","DOI":"10.1186\/s12859-017-1934-z","article-title":"ImageJ2: ImageJ for the next generation of scientific image data","volume":"18","author":"Rueden","year":"2017","journal-title":"BMC Bioinformatics"},{"key":"2026082009533585800_tjag135-B47b","doi-asserted-by":"publisher","first-page":"104936","DOI":"10.1016\/j.cryobiol.2024.104936","article-title":"Vitrification of medaka whole testis with a trehalose-containing solution and production of medaka individuals derived from the vitrified cells","volume":"116","author":"Seki","year":"2024","journal-title":"Cryobiology"},{"key":"2026082009533585800_tjag135-B47","doi-asserted-by":"publisher","first-page":"312","DOI":"10.1093\/oxfordjournals.humrep.a136895","article-title":"Cryopreservation of human and rabbit oocytes and one-cell embryos: a comparison of DMSO and propanediol","volume":"4","author":"Siebzehnruebl","year":"1989","journal-title":"Hum. Reprod"},{"key":"2026082009533585800_tjag135-B48","doi-asserted-by":"publisher","first-page":"64","DOI":"10.1007\/978-1-4757-0190-6_4","article-title":"Biochemistry of cryoprotectants","author":"Storey","year":"1991","journal-title":"Insects Low Temp"},{"key":"2026082009533585800_tjag135-B49","doi-asserted-by":"publisher","first-page":"20190050","DOI":"10.1098\/rspb.2019.0050","article-title":"Evidence for non-colligative function of small cryoprotectants in a freeze-tolerant insect","volume":"286","author":"Toxopeus","year":"2019","journal-title":"Proc. Biol. Sci"},{"key":"2026082009533585800_tjag135-B50","doi-asserted-by":"publisher","first-page":"1192","DOI":"10.1016\/j.theriogenology.2007.08.024","article-title":"Development of cryopreservation protocol for long-term storage of black tiger shrimp (Penaeus monodori) spermatophores","volume":"68","author":"Vuthiphandchai","year":"2007","journal-title":"Theriogenology"},{"key":"2026082009533585800_tjag135-B51","doi-asserted-by":"publisher","first-page":"481","DOI":"10.1016\/s0378-4320(00)00099-3","article-title":"The causes of reduced fertility with cryopreserved semen","volume":"60\u201361","author":"Watson","year":"2000","journal-title":"Anim. Reprod. Sci."},{"key":"2026082009533585800_tjag135-B52","doi-asserted-by":"publisher","first-page":"963689721999617","DOI":"10.1177\/0963689721999617","article-title":"Cryopreservation: an overview of principles and cell-specific considerations","volume":"30","author":"Whaley","year":"2021","journal-title":"Cell Transplant"},{"key":"2026082009533585800_tjag135-B52b","doi-asserted-by":"publisher","author":"Wolkers","year":"2021","DOI":"10.1007\/978-1-0716-0783-1_1"},{"key":"2026082009533585800_tjag135-B53","author":"World Health Organization","year":"2019"},{"key":"2026082009533585800_tjag135-B54","author":"World Health Organization","year":"2025"}],"container-title":["Journal of Medical Entomology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/academic.oup.com\/jme\/article-pdf\/63\/4\/tjag135\/70723211\/tjag135.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/jme\/article-pdf\/63\/4\/tjag135\/70723211\/tjag135.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T13:53:46Z","timestamp":1787234026000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/jme\/article\/doi\/10.1093\/jme\/tjag135\/8767058"}},"subtitle":[],"editor":[{"given":"Julian","family":"Hillyer","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2026,7,1]]},"references-count":57,"journal-issue":{"issue":"4","published-print":{"date-parts":[[2026,7,1]]}},"URL":"https:\/\/doi.org\/10.1093\/jme\/tjag135","relation":{},"ISSN":["0022-2585","1938-2928"],"issn-type":[{"value":"0022-2585","type":"print"},{"value":"1938-2928","type":"electronic"}],"subject":[],"published-other":{"date-parts":[[2026,8]]},"published":{"date-parts":[[2026,7,1]]},"article-number":"tjag135"}}