Preventing Further Frog Extinctions And Initiating De-Extinctions

Since the 1960s, more than 200 amphibian species globally have become extinct, and the majority of these extinctions have occurred in the past 20 years. Rabb’s Fringe-limbed tree frog was recently added to this growing list when Toughie, the last of his kind, died in September 2016 at his adopted home in the Atlanta Botanical Gardens, Georgia.

There are many similar recent examples, and it is a tragedy that the loss of such unique biological character and diversity is occurring before our eyes. At least another 950 frog species are living on the brink of extinction, ready to go the same way as Toughie, and circumstances are so dire that interventional animal husbandry protection appears to be their only chance of survival. Unfortunately, sufficient resources presently exist to care for only about 50 of these species¹. So what will happen to the other 900 species?

The main driver of the amphibian extinction crisis is human activity through direct habitat destruction and the associated introduction of disease (e.g chytrid fungus), pesticide exposure, and competitive speciation. The implementation of habitat preservation initiatives has been successful in securing selected populations². As the most reliable and enduring protection tools, these programs must be maintained and expanded with the highest priority. However, it is clear that a wider suite of complementary techniques requires rapid development to address the current short-fallings.

Assisted reproduction techniques such as IVF (in vitro fertilization) are complementary to conservation programs and represent one area where more progress is essential. Reliable egg and/or embryo freezing techniques are required for long-term species survival so that IVF material can be safeguarded until habitats have been sufficiently restored. However, due to their complexity, progress on this front has been slow. So what can be done in the meantime?

Frog cell culture and cell freezing are obvious developmental paths to explore because, in this way, permanent genetic and cellular records of each representative frog species can be stored indefinitely, giving other techniques, as well as our awareness of the critical nature of this problem, sufficient time to catch up. Furthermore, such cells would represent a source for induced pluripotent stem cell (iPSC) production and hence artificial gamete construction as well as IVF and somatic cell nuclear transfer (SCNT) cloning techniques³.

Surprisingly, the successful culture of frog cells has historically been a rare accomplishment⁴. Recently, we have spent time developing techniques relevant to frog cell culture and cryopreservation. We described how tissue samples can be collected from frogs euthanized due to sickness or injury. Multiple vials from cultures of each species are frozen at -196 °C in liquid nitrogen. After several months, one representative tube is thawed, and cultured again to show that the cells are still alive and have a normal chromosomal complement. Because these cells are stored in liquid nitrogen, a living cellular record of that specific species as well as material for assisted reproduction, repopulation, and de-extinction initiatives can, in theory, exist for at least 100 000 years.

These techniques are OK for frog species that still exist. But what about for frogs like Toughie who was the last of his species, and for which cell storage techniques had not already been developed? Not knowing what else to do, researchers have sometimes placed tissues from “last of their kind” frog species in -80C freezers for storage, to wait for a time when techniques may be developed to resurrect them. In anticipation of such events, we have used these culture techniques in a preliminary de-extinction program, extracting living cells from tissues that were placed at -80 °C in the absence of cryoprotectant.

In an unpublished proof of principle exercise, we stored tissue from a dead Jervis Bay tree frog in a -80 °C freezer for five months. (Jervis Bay tree frogs are currently not threatened with extinction). We applied our culture techniques to this tissue and were able to isolate living cells. From a random cell population sample, all 33 cells analyzed had a normal chromosomal content, meaning that, in theory, these cells are suitable at least for cloning, and therefore the “de-extinction” of this one frog.

Historically, frogs were used in possibly the first successful IVF experiment conducted by Spallanzini in 1770. The culture of frog nerves by Harrisson in 1907 is recorded as the first example of successful animal cell culture. Briggs and King used frog cells in the first successful cloning experiment in 1952. It would be rewarding if all of these landmark scientific breakthroughs could be used to save frogs from extinctions and bring back those that have recently disappeared from our planet. It would be even more rewarding if sufficient awareness was created to protect our environment so that frogs like Toughie could save themselves.

These culture techniques are described in the articles entitled: (i) Culture, cryobanking and passaging of karyotypically validated native Australian amphibian cells, recently published in Cryobiology, and (ii) Karyomaps of cultured and cryobanked Litoria infrafrenata frog and tadpole cells, recently published in Data in Brief. The work was conducted by Richard Mollard, an Honorary Fellow with the Faculty of Veterinary and Agricultural Sciences at the University of Melbourne.

References:

1. J. Clulow, S. Clulow, Cryopreservation and other assisted reproductive technologies for the conservation of threatened amphibians and reptiles: bringing the ARTs up to speed, Reprod. Fertil. Dev., DOI 10.1071/rd15466 (2016).
2. L.E. Rose, G.W. Heard, Y.E. Chee, B.A. Wintle, Cost-effective conservation of an endangered frog under uncertainty, Conserv. Biol., 30 (2016) 350-361.
3. J. Saragusty, S. Diecke, M. Drukker, B. Durrant, I. Friedrich Ben-Nun, C. Galli, F. Goritz, K. Hayashi, R. Hermes, S. Holtze, S. Johnson, G. Lazzari, P. Loi, J.F. Loring, K. Okita, M.B. Renfree, S. Seet, T. Voracek, J. Stejskal, O.A. Ryder, T.B. Hildebrandt, Rewinding the process of mammalian extinction, Zoo Biol., 35 (2016) 280-292
4. L.R.E. Kouba A. J., Houck M. L., Silla A. J., Calatayud N., Trudeau V. L., Clulow J., Molinia F., Langhorne C., Vance C., Arregui L., Germano J., Lermen D., Della Togna G., Emerging trends for biobanking amphibian genetic resources: The hope, reality and challenges for the next decade, Biol. Conserv., 164 (2013) 12.