Cryopreservation of Anopheles stephensi embryos

The ability to cryopreserve mosquitoes would revolutionize work on these vectors of major human infectious diseases by conserving stocks, new isolates, lab-bred strains, and transgenic lines that currently require continuous life cycle maintenance. Efforts over several decades to develop a method fo...

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Bibliographic Details
Published in:	Scientific reports Vol. 12; no. 1; p. 43
Main Authors:	James, Eric R., Wen, Yingda, Overby, James, Pluchino, Kristen, McTighe, Shane, Matheny, Stephen, Eappen, Abraham, Hoffman, Stephen L., Billingsley, Peter F.
Format:	Journal Article
Language:	English
Published:	London Nature Publishing Group UK 07-01-2022 Nature Publishing Group Nature Portfolio
Subjects:	631/1647/2234 692/699/255/1629 Animals Anopheles - embryology Anopheles - parasitology Anopheles stephensi Aquatic insects Cryopreservation Cryopreservation - methods Cryoprotective Agents Cryoprotectors Culicidae Eggs Embryo, Nonmammalian - embryology Embryos Humanities and Social Sciences Infectious diseases Larva - growth & development Larvae Life cycles Mosquitoes multidisciplinary Oviposition Plasmodium falciparum - parasitology Science Science (multidisciplinary) Vectors
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Summary:	The ability to cryopreserve mosquitoes would revolutionize work on these vectors of major human infectious diseases by conserving stocks, new isolates, lab-bred strains, and transgenic lines that currently require continuous life cycle maintenance. Efforts over several decades to develop a method for cryopreservation have, until now, been fruitless: we describe here a method for the cryopreservation of Anopheles stephensi embryos yielding hatch rates of ~ 25%, stable for > 5 years. Hatched larvae developed into fertile, fecund adults and blood-fed females, produced fully viable second generation eggs, that could be infected with Plasmodium falciparum at high intensities. The key components of the cryopreservation method are: embryos at 15–30 min post oviposition, two incubation steps in 100% deuterated methanol at − 7 °C and − 14.5 °C, and rapid cooling. Eggs are recovered by rapid warming with concomitant dilution of cryoprotectant. Eggs of genetically modified A. stephensi and of A. gambiae were also successfully cryopreserved. This enabling methodology will allow long-term conservation of mosquitoes as well as acceleration of genetic studies and facilitation of mass storage of anopheline mosquitoes for release programs.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	2045-2322 2045-2322
DOI:	10.1038/s41598-021-04113-x