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Liquid nitrogen freezing is key to bee conservation

Posted by | July 22, 2016

Going through customs can be frustrating for many travelers. Imagine going through with a container filled with frozen bee sperm collected from Kazakhstan or Slovenia.

“It’s certainly a challenge,” said Brandon Hopkins, Washington State University entomology research associate and lab manager of the WSU Apiary Program. “Most customs agents aren’t used to seeing that, so it takes a lot of explaining.”

WSU researcher Brandon Hopkins lifts frozen honey bee germplasm out of the tank of liquid nitrogen, where the material is stored.
WSU researcher Brandon Hopkins lifts frozen honey bee germplasm out of the tank of liquid nitrogen, where the material is stored.

It’s complicated by the fact that WSU has the only permit issued by the USDA to import honey bee semen into the U.S.

Honey bees are not native to North or South America, and importing into the U.S. has been tightly restricted since the 1920s. As a result, current U.S. honey bees have limited genetic diversity.

“We’re importing germplasm from Old World populations around Europe to increase genetic diversity here,” Hopkins said. “The goal is to improve commercial breeding for bees, so they can better fight off diseases and parasites.”

Hopkins and other WSU entomologists have collected genetic material in Italy, Slovenia, the Republic of Georgia and Kazakhstan since they received a USDA permit to import honey bee semen in 2008.

Freezing bee semen

WSU scientists still transport fresh germplasm, but its shelf life is only about two weeks. Hopkins developed a method for freezing the material as part of his master’s degree at Eastern Washington University, and he refined it further when he came to WSU to earn his Ph.D.

“Cryogenic freezing has been used to preserve germplasm from animals like cattle for decades,” Hopkins said. “I adapted it for honey bees. Right now we are the only repository for bee germplasm in the world.”

The importation process starts with a trip to collect material overseas. Once they arrive in a country, they work with local beekeepers or government agencies equivalent to the USDA to visit a variety of hives.

They collect mature male bees, called drones, and then extract semen. Each male produces about one microliter. For comparison, a single drop of water is approximately 100 microliters.

Hopkins talks about the cryogenic freezing program in the current WSU honey bee lab.
Hopkins talks about the cryogenic freezing program in the current WSU honey bee lab.

“We try to collect hundreds of microliters of sperm every day we’re there, so those are long days,” Hopkins said.

The collected semen is frozen in the origin country because freezing fresh material yields the best results. A special substance is added to the collected semen to avoid damaging the cells during freezing. Once back at WSU, the samples are stored at -196°C (-320°F) in a tank of liquid nitrogen.

Theoretically, it can stay viable at that temperature for 10,000 years or more, Hopkins said. It can then be thawed out and used to breed honey bees here.

Ensuring genetic improvement

Unfortunately, the breeding process is not as simple as inseminating one queen and then providing the second generation of bees to breeders. Second generation queens contain only 50 percent of the imported European DNA.

If those queens are released, the imported genes would quickly become diluted as they breed with U.S. bees. To prevent genetic dilution, Hopkins and WSU researchers inseminate second generation queens with imported material as well, ensuring that third generation bees have 75 percent imported DNA. Then that generation of bees is inseminated as well. The iterative process results in dozens of queens with more than 85 percent imported genetic material.

These multiple rounds of insemination are where the frozen semen is most helpful.

“Without frozen semen, this process would require trips back to Europe every year, or multiple times per year,” Hopkins said. “With frozen semen, we simply thaw the semen to use for each generation.”

The collected semen is submerged in liquid nitrogen in a small tank first, before being placed in the larger tank for long-term storage.
The collected semen is submerged in liquid nitrogen in a small tank first, before being placed in the larger tank for long-term storage.

The genetically diverse bees can then be provided to U.S. bee breeders to breed future generations that are adapted for certain geographic regions.

“We want to improve the genetic background of honey bees so they can fight off diseases and be more likely to survive in their climates,” Hopkins said. “Everything we do in this effort is to ensure bees survive to pollinate our food sources forever.”

Honey bee conservation

Beyond increasing disease resistance, the cryogenic program also has a conservation component. A few countries and regions are interested in preserving the genetic material of their distinct bee varieties.

“You can’t put a fence around bees,” Hopkins said. “So in Italy and France and other locations, they’re trying to conserve their unique sub-species. Freezing semen conserves those specific genetic lines or entire sub-species inexpensively  for a long time.”

Hopkins said the WSU bee program has worked with researchers from around the world to create more honey bee genetic repositories, like the one at WSU. With a potential new Honey Bee & Pollinator Research Facility at WSU, he thinks they’ll be able to do even more research to diversify the gene pool for U.S. honey bees.

“We’re still really early in studying this,” Hopkins said. “But we’re learning more every day, and that center will be a huge boost to our program and the body of knowledge about saving the honey bees here.”

To learn more and to donate to the WSU bee program, visit http://bees.wsu.edu/.