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Publication Number
US-12484575-B2
Publication Date
2025-12-02
Expiration Date
Abstract
Herein is described a method devised to cryopreserve mosquito eggs, viable upon subsequent thawing. The method reproducibly yields >25% hatched first instar larvae of Anopheles-species mosquitoes from cryopreserved eggs. The majority of hatched larvae continue to develop normally (87% pupation), through to adults (98%, 50% of which are female) which subsequently blood feed and produce viable second generation embryos that also develop normally. Adult mosquitoes obtained from cryopreserved embryos are able to be infected with Plasmodium falciparum gametocytes and produce salivary gland sporozoites in similar numbers to control mosquitoes.
Core Innovation
The invention provides cryopreserved viable anopheline eggs combined with a cryoprotectant additive (CPA). The CPA is used at a concentration of 50% to 100% v/v and is an isotopic variant of methanol, an isotopic variant of ethylene glycol, or a combination thereof.
In one aspect, a composition includes a plurality of cryopreserved anopheline eggs and the CPA at 50% to 100% v/v, where the CPA is an isotopic variant of methanol or an isotopic variant of ethylene glycol. The cryopreserved eggs are stored at or below about −135° C in cryopreserved storage and are characterized by post-thaw hatching and release of viable first instar larvae after thawed, diluted and incubated conditions.
In another aspect, the invention provides a method for the cryopreservation of a viable anopheline egg using staged incubations with methanol followed by transfer to liquid nitrogen and storage at a temperature equal to or less than −135° C. After cryopreserved storage, warming and dilution are performed, followed by incubation and hatching to release viable first instar larvae, and development to adult mosquitoes that can be infected with Plasmodium falciparum gametocytes and produce salivary gland sporozoites.
Claims Coverage
The document includes three independent claims for a composition and two cryopreservation methods. The claims center on cryopreserved anopheline eggs with CPA at 50% to 100% v/v using isotopic variants, and on staged methanol incubation followed by transfer to liquid nitrogen and cryopreserved storage at or below −135° C.
Cryopreserved anopheline eggs with isotopic CPA concentration 50% to 100% v/v
A composition comprising a plurality of cryopreserved anopheline eggs and a cryoprotectant additive (CPA) at a concentration of 50% to 100% v/v, wherein the CPA is an isotopic variant of methanol, an isotopic variant of ethylene glycol, or a combination thereof.
Staged methanol incubations and transfer to liquid nitrogen with storage at or below −135° C
A method for the cryopreservation of a viable anopheline egg comprising staged methanol incubations, transfer to liquid nitrogen, and cryopreserved storage at a temperature equal to or less than −135° C.
High-concentration methanol incubations with specific temperature and time windows, then liquid nitrogen and storage at or below −135° C
A method for the cryopreservation of a viable anopheline egg comprising high-concentration methanol incubations with specific temperature and time windows, followed by transfer to liquid nitrogen and cryopreserved storage at a temperature equal to or less than −135° C.
Across the independent claims, the core coverage is cryopreserved anopheline egg compositions and cryopreservation methods that employ isotopic CPA or staged high-concentration methanol incubations, followed by transfer to liquid nitrogen and cryopreserved storage at or below −135° C.
Stated Advantages
Enables post-thaw hatching and release of viable first instar larvae from cryopreserved anopheline eggs.
Supports development to adult mosquitoes that can be infected with Plasmodium falciparum gametocytes and generate salivary gland sporozoites.
Documented Applications
Cryopreserved anopheline eggs to generate adult mosquitoes for infection with Plasmodium falciparum gametocytes and downstream production of sporozoites.
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