How cryopreservation techniques might sustain a threatened species, and us in the process.
How cryopreservation techniques might sustain a threatened species, and us in the process.
November 2, 2016
On a hot evening in June, Washington State University (WSU) entomologist Brandon Hopkins sat in front of a microscope in Orland, California, handling one honeybee after another as each committed one of life’s most important acts. Hopkins squeezed one drone at a time, contracting the male’s abdominal muscles to mimic a natural mating event. As the pressure exposed the drone’s penis and a speck of semen, Hopkins vacuumed it off carefully. “You do that hundreds and hundreds of times as quickly as possible,” he said.
The process is much more technical than the actual reproductive rituals of bees (which usually happen in mid-air), but the outcome is the same: The drone gives his life, and the species lives on. Rather than immediately contributing to the growth of the colony, however, this bee’s semen will be stored in liquid nitrogen and shipped to another state.
Hopkins is collecting the first-ever honeybee samples to deposit into the National Animal Germplasm Program, a national livestock gene bank run by the Agricultural Research Service (ARS), the main research arm of the U.S. Department of Agriculture (USDA). The bank contains the genetic material of approximately 31,000 species that have been deemed agriculturally important in the United States.
Housed in Fort Collins, Colorado, the repository began in 1957 as a seed library, but in 1999, the ARS started collecting the genetic material of animals used for food or fiber as well, including various kinds of beef cattle, freshwater fish, yaks, and bison. Researchers selecting for certain traits, or breeders trying to introduce greater variability to their stock, can draw from the ever-growing gene bank. And, in the event of catastrophic disease or man-made extinction, the library’s stock could be used to rebuild a population.
Until recently, honeybees were left out of the national repository because researchers did not have a way to indefinitely preserve their genetic material. Previous attempts to freeze bee sperm had left it hardly useable just a year later, said Harvey Blackburn, ARS program leader. Such a short shelf life defeats the purpose of the repository, Blackburn said. “We’re interested in long-term storage—not something we’d have to do every year to keep samples around.”
About five years ago, however, Hopkins developed his technique to freeze—or cryopreserve—the sperm, making it possible not only to create an emergency supply for the repository, but also to breed better bees for the field.
Hopkins’ technological advance came at a critical time. As beekeepers continue to lose a third of their bees every year on average, many are counting on breeding to combat some of the threats to honeybees, namely pests and disease.
Cryopreservation Attempts Through the Years
In the 1970s and ’80s, when researchers first began working on methods for freezing bee sperm, there wasn’t enough industry demand for the product to justify the effort, and they discontinued the work. Hopkins initially took up the long-abandoned project in hopes of addressing a problem that was growing worse—the lack of diversity in the honeybee populations.
The diversity issue traces back to 1922, when the U.S. banned the importation of live bees in an attempt to prevent the spread of pests and diseases afflicting bees around the world. The pests made it to the U.S. anyway, but the ban remains in place, and some entomologists worried the geographical limitation would lead to inbreeding, which could weaken bees’ ability to adapt to threats.
Steve Sheppard, head of WSU’s entomology department, believed introducing genes from bees in the Old World, including Italy and the Republic of Georgia, would help breeders select for stronger, healthier specimens.
In 2008, Sheppard acquired a permit to import bee sperm, enabling him to artificially inseminate queens raised by several large West Coast producers. Still, he faced the logistical problem that fresh sperm only lasts a short time unless it’s frozen.
As a doctoral student at WSU five years ago, Hopkins wanted to solve that problem. Cryopreservation differs between animals, so Hopkins could not just apply the methods that created a cottage industry from bull semen. He found that bee sperm is susceptible to “cold shock”—a condition that occurs when it’s frozen too quickly and leaves it unviable. By slowly cooling the semen instead, Hopkins discovered it was still viable after thawing.
In 2015, when the ARS finally began exploring the idea of adding bees to the Fort Collins gene bank, Blackburn realized the WSU team had already done much of the heavy lifting; WSU samples dated back several years without any sign of deteriorated viability. From there, everything fell into place.
Blackburn convened a honeybee species committee made up of people in the field who will recommend what species and subspecies should be in the repository. Their aim is to create a broad collection that represents the variability found in the United States and to look for rare traits or subspecies that should be preserved before they disappear.
For now, Hopkins and Sheppard of WSU will be the ones tasked with traveling across the states collecting material for the gene bank. Later, the cryopreservation method may be employed by other researchers in those various locations, Blackburn said.
The bee sperm collected at each site is stored in a straw resembling a cocktail straw. Each one can hold about half a milliliter of liquid. The samples are cooled to minus-300 degrees Fahrenheit and sent to Colorado, where they’re stored in 6-foot-wide tanks of liquid nitrogen. The facility is filled with these tanks, holding material from all kinds of species.
The repository holds multiple types of material for many of its specimens. Working separately at a USDA lab in Fargo, North Dakota, researchers are working on a method to cryopreserve honeybee embryos. When they succeed, that material also will be added to the Fort Collins gene bank.
“As they get comfortable with that procedure, we’ll want to be doing that as well,” Blackburn said. “It gives us two avenues in how we might reconstitute populations.”
Applications for Hopkins’ Honeybee Breakthrough
Hopkins’ method offers a new opportunity for the people tasked with maintaining live colonies in the face of various threats.
Between pesticides, parasites, the loss of foraging, and greater demands in agriculture, particularly the growth of the almond industry, bees are under significant pressure, said Pat Heitkam, one of three California queen producers whose drones supplied the first contributions to the national gene bank.
Queen production—an important aspect of the beekeeping industry—involves breeding bees with desirable traits. Producers keep their own colonies for the purpose of making new queens that they can then sell to other beekeepers. Heitkam’s operation is responsible for producing 90,000 queens each year.
“The quality of queens has always been important but now it’s more so,” Heitkam said. Where queens used to last three to four years, beekeepers now tend to introduce a new queen every year. And they’re paying more attention to quality to ensure colonies remain in good shape.
An ARS breeding and genetics lab in Baton Rouge that focuses on selecting for mite-resistant traits could use cryopreservation for a different application. Founded in 1928, the Baton Rouge lab developed a lot of the technology used to artificially inseminate bees—an important step that allowed people to make controlled crosses of species. In the last couple of decades, however, the lab has focused completely on resistance to mites, which many researchers consider enemy number one in the effort to improve bee health.
Hopkins’ cryopreservation technique could allow researchers at the lab to save genetic material from good bees for later use, even if the line of bees it came from goes away. “You can’t put bees in a jar on a shelf,” said Bob Danka, an entomologist at the lab who is also on the honeybee species committee. “It’s a struggle to keep them alive, and we spend a lot of time just managing bees here while we do the research.”
Blackburn will have the final say on who can take and use samples from the national repository but, in the end, the gene bank signifies an important breakdown in the walls between research and application. As the repository grows and technology advances, selection for positive traits, like those in the lines Danka’s lab is developing, will be more easily crossed with valuable commercial traits—gentle, hardy bees that make honey and collect pollen.
Susan Cobey, a beekeeper and researcher in the WSU entomology department, works in both spheres and said collaboration between the two has become essential.
“There’s been a huge gap between those two worlds—the science community is slow to get their information out to the industry, and the industry is impatient,” she said. “The science is more applied now because the needs are so big.”
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