To prepare screwworm
eggs for penetration by
a cryoprotectant, entomologist
Dennis Berkebile and
technician Staci Bohling
remove the eggs' outer
Using cryopreservation techniques, ARS
entomologists can now freeze screwworm fly embryosto later thaw
and rear them to adulthood. The advance should cut the costs of maintaining
fly colonies for research or of programs using the sterile-insect technique.
Considered the backbone of screwworm eradication, sterile-insect releases
have helped eliminate this parasitic fly species from the United States
and Central America as far south as the Isthmus of Panama. In the approach,
both male and female screwworms are reared under factorylike conditions
and then sterilized by irradiation. The sterile insects are then released
to mate with wild flies, resulting in no progeny. Eventually, the fly
population collapses. (See "Squeezing
Out Screwworm," Agricultural Research, April 2001, pp.
"Freezing embryos eliminates the need to continuously rear flies
used in research or held in reserve as backup strains for the mass-rearing
and release program. This is costly, especially if there's no immediate
need for them," notes Roger Leopold, an entomologist with the Insect
Genetics and Biochemistry Research Unit at ARS's Red River Valley Agricultural
Research Center, Fargo, North Dakota. Leopold's efforts to cryopreserve
fly embryos first began there in the mid-1980s, "but it didn't
really get going until the early 1990s," he says. That's when he
consistently began reviving frozen embryos of house flies and other
While visiting the Midwest
Livestock Insects Research
Unit in Lincoln, Nebraska,
Arun Rajamohan (from Fargo)
prepares screwworm embryos
with a cryoprotectant before
Around 1996, he teamed up with Dennis Berkebile, an entomologist specializing
in screwworm genetics at ARS's Midwest Livestock Insects Research Unit,
Lincoln, Nebraska. Along with two other collaboratorsHarvey Blackburn,
an animal geneticist at ARS's National Center for Genetic Resources
Preservation, Fort Collins, Colorado; and Arun Rajamohan, a postdoctoral
researcher at North Dakota State UniversityLeopold and Berkebile
explored creating an embryo-based repository to back up colonies of
10 key screwworm strains. Up until August 2004, these strains were kept
at ARS's Lincoln site and the screwworm mass-rearing plant in Tuxtla
Gutierrez, Mexico. (In August, the Lincoln unit concluded its screwworm
program with the transfer of some of its last remaining colonies and
biocontainment equipment to a new facility being built in Panama.)
According to Berkebile, some of the Lincoln colonies date back 20 years
and include strains with useful mutations, including colored eyes and
curly wing deformities. Scientists prize such mutations because they
can serve as markers to important genetic traits, like fitness, and
can be used to create all-male strains of screwworm with potential to
cut fly-rearing costs. Such strains could prove useful if screwworm
eradication expands into South America and the Caribbean.
In Lincoln, Nebraska,
Roger Leopold (from Fargo)
places screwworm embryos
in a liquid-nitrogen
storage unit to be
transported to the
germplasm unit in Fort
Berkebile says creating a repository addressed fears of losing the
strains to calamities such as disease outbreaks, fire, or even earthquake
damage to facilities in which the flies are keptderailing years
of research in support of programs to eradicate the parasite and prevent
its reintroduction. "The idea behind using cryopreservation was
that we'd be able to preserve genetic material encoded within the embryos'
DNA," says Berkebile. "So, if something were to happen to
the strains they're mass-producing in Mexico, for example, we'd have
these strains in storage to bring back and start the colonies all over
again." The alternative would be to capture wild flies in field
traps, screen them, and breed them in hopes they would be genetically
similar to the lost strain.
Embryo cryopreservation also offers a way to safeguard against unwelcome
mutations and inadvertent selection. Both events can change a colony's
unique genetic makeup, rendering it less useful to research or sterile-insect
applications. "If there are any genetic changes (either positive
or negative) in the live populations, we would have the original genetic
material to refer to in evaluating that change," says Blackburn,
who leads the Fort Collins center's National Animal Germplasm Program.
"Having a genetic repository for insects would allow breeders to
determine what genes may have been lost in the selection process."
So why not freeze adult flies, rather than the embryos, and thaw them
out as needed? Because embryos lack a cuticlethe outer covering
of an insect that protects it from water loss and environmental elements.
This makes it possible for scientists to extract water from the embryo
and replace it with a cryoprotectant like ethylene glycol, which prevents
the embryo's 40,000 to 50,000 cells from being damaged by ice crystals
"We're working with an insect stage that's comparable to a 7-month-old
mammalian fetusif you consider the stage of differentiation of
the cells and organs," Leopold explains. Differentiation is the
process whereby cells begin to form specific tissues and organs. Even
with embryos, he adds, "Cryopreservation at this stage of differentiation
is usually very difficult."
The process begins with collecting eggs soon after they're deposited
by caged female flies. The eggs are incubated to the correct developmental
stage; loaded with the cryoprotectant, which eliminates most of the
cell water; and then "vitrified," meaning they are cooled
so rapidly that the cryoprotectant and any remaining water turn to a
glass rather than to ice crystals.
The scientists then plunge the embryos into liquid nitrogen (-196°C)
and store the vitrified eggs in metal cassettes within a tank of liquid
nitrogen. According to Leopold, the eggs can be stored there indefinitely
at a cost of pennies per day. Depending on the number of strains in
culture, maintaining live caged colonies, by comparison, can cost thousands
of dollars per month.
To revive the embryos, scientists remove the cassettes from liquid
nitrogen, hold them in nitrogen vapor for a short time, and then immerse
the embryos in fetal bovine serum, which removes the cryoprotectant.
Thirty to 45 minutes later, the embryos begin moving within the eggs.
About 8 to 12 hours later, they hatch as larvae. The hatchlings are
then reared on an artificial diet of dried blood, egg, milk, and other
ingredients that simulate their natural diet of animal flesh.
So far, the longest the scientists have stored the cryopreserved eggs
is just over 1 year. But Leopold reckons these eggs, like embryos of
other animals currently in storage, can last indefinitely in their frozen
state. When he and Berkebile first tried this technique on two screwworm
strains, about 30 to 40 percent of the eggs hatched after being frozen.
Now, oftentimes 70 to 80 percent hatch, with 30 to 60 percent of the
larvae developing into adult flies. The researchers expect to bolster
that rate with a new, improved diet that they devised for recovering
Meanwhile, they've also begun storing cryopreserved embryos inside
tanks at ARS's Fort Collins germplasm unit. Over the next couple years,
they'll monitor the embryos' viability and use revived specimens to
check for genetic changes in live colonies.
Historically, the focus of ARS's National Center for Genetic Resources
Preservation has been to preserve germplasm from plants in the form
of seeds or pollen. The focus has recently been expanded to include
livestock and aquatic germplasm as frozen sperm and embryos.
"We see potential for expanding the program to include cryopreservation
and conservation of insect genetic resources," says Blackburn.
"For example, we've already had discussions with several labs about
cryopreserving bee semen. Clearly, the spectrum is limitless for both
harmful and beneficial insects."By Jan
Suszkiw, Agricultural Research Service Information Staff.
This research is part of Veterinary, Medical, and Urban Entomology,
an ARS National Program (#104) described on the World Wide Web at www.nps.ars.usda.gov.
To reach scientists mentioned in this story, contact Jan
Suszkiw, USDA-ARS Information
Staff, 5601 Sunnyside Ave., Beltsville, MD 20705-5129; phone (301)
504-1630, fax (301) 504-1641.
"Frozen Flies Safeguard Research, Screwworm Eradication Efforts"
was published in the February
2005 issue of Agricultural Research magazine.