Location: Insect Genetics and Biochemistry Research
2015 Annual Report
Objectives
Develop means for long-term storage of bee germplasm, including cryopreservation of embryos. [NP 305, Component 2, Problem Statements 2A, 2B, 2C]
Approach
Pollinating insects are important to the U.S. agricultural economy, contributing an estimated $16 billion to annual crop production. This critical component is currently at risk due to a dramatic decrease in managed and native pollinators, and will face additional challenges due to climate change in the future. Despite this importance, there is no organized germplasm biorepository (genebank) for bees. This project will develop protocols that support the establishment of a National Bee Germplasm Repository. Specifically, the project will focus on: 1) the development of an improved protocol for the cryopreservation of bee spermatozoa, 2) the development of a novel protocol for the cryopreservation of bee embryos, and 3) the development of a protocol for the in vitro rearing of embryos after storage into reproductively viable adults. This research will ultimately result in the preservation of elite and genetically diverse pollinator strains, the development of a cryogenically-based system for the safe importation of bee germplasm, and the systematic delivery of high-quality germplasm and insects to end users.
Progress Report
The establishment of an improved spermatozoa protocol is critical to the objective of this project of developing means for long-term storage of bee germplasm. Towards this end, we have developed and tested a unique USDA Honeybee Extender Medium. Unlike traditional formulations, this potentially proprietary formula does not activate the spermatozoa, thereby substantially improving spermatozoa longevity after recovery from cryo-storage in both room temperature and low temperature conditions. It can be inferred that similar results are likely when recovered sperm is stored in the spermatheca of a queen bee, thereby improving the fecundity of a queen artificially inseminated with spermatozoa that had been previous cryopreserved.
In concert with our spermatozoa studies, the development of a novel protocol for the cryopreservation of bee embryos is also critical to the establishment of the comprehensive long-term storage of bee germplasm. The embryonic insect cryopreservation procedure consists of three main processes: permeabilization of the outer layers, cryoprotectant loading to prevent ice nucleation, and vitrification in liquid nitrogen. Our recent research has focused on the permeability assessment of honey bee embryos. Our results indicate that the chorion typically present on insect eggs is either absent or is highly permeable in the honey bee, and that the underlying wax layer is highly fluid and easily dissolved. This means that the traditional three step method of the permeabilization process, which has been used in all insect embryonic cryopreservation protocols to date, is acutely toxic to honey bee embryos. To accommodate the unique honey bee embryonic physiology, we have developed a two-step permeabilization process, with a precisely timed wax removal step, that results in little to no acute toxicity.
Finally, the development of a honey bee embryonic storage protocol is of little practical use without the concurrent development of a protocol for the in vitro rearing of embryos after storage into reproductively viable adult queens. We have developed a protocol that reproducibly results in at least 90% of bees surviving to the pupal stage and at least 80% successfully emerging as adults. A significant component of this protocol is artificial diet optimization: the ratio of carbohydrates to protein in the artificial diet significantly affects survival while dietary manipulation, especially in the later stages of development, can reliably lead to the development of adult queens.
Accomplishments
1. Improved cryopreservation of honeybee spermatozoa. The establishment of a standardized spermatozoa protocol is critical to the establishment of a honeybee germplasm repository. Current efforts use Harbo Extender Medium, and although it does produce viable spermatozoa after cryopreservation, efficacy is limited by the fact that it is based on mammalian spermatozoa physiology. Researchers at USDA laboratories in Fargo, ND have recently developed an improved extender medium that is based on insect spermatozoa physiology. Although the improvement to survival immediately after storage is modest (70-80% survival for Harbo Extender Medium vs. ~90% for USDA Honeybee Extender Medium), the effects on sperm longevity after storage are more profound. While spermatozoa preserved with Harbo Extender Medium rapidly succumb at room temperature, more than 75% of those preserved in USDA Honeybee Extender Medium remain viable even after 6 months at room temperature. This is critical to the practical success of a honeybee germplasm repository: honeybee queens store spermatozoa for extended periods of time after insemination in their spermatheca, and a queen storing unviable sperm will result in the rapid demise of a colony.
