# Humane techniques to sterilize frogs??



## Woodsman (Jan 3, 2008)

Though I have only been keeping and breeding PDFs for about 6 years, I have had a life-long interest in evolutionary biology and find the origins of the many morphs of D. tinctorius an amazing story that could use a bit more work to be told. I know that there are VERY good reasons for anyone interested in the conservation of these frogs to not want to see inter-morph hybrids created in the hobby as this would greatly complicate any effort at some future conservation effort (though I'm not sure that the poor record keeping for localities and breeding would make most of the hobby frogs good candidates for such work). I do think, though, with D. tinctorius in particular, it is clear that a number of morphs that are currently in the hobby are intermediate between others and that some carefully-planned crosses could help elucidate the baseline ecology within the species.

So the question is, what do we recommend to hobbyists (or scientists) that happen to produce inter-morph animals? Of course we don't want fertile animals such as these to become established in the hobby, as they would muddy the waters terribly. Isn't there some way we could offer to barter these animals between established (and ethical) parties that are interested in this subject without the kind of "demonization" that I've seen leveled at some ("you have to euthanize those frogs immediately" was one comment I saw posted after a member up-loaded photos of his unfortunate cross)? 

My proposal would be to research techniques that would allow for the humane sterilization of inter-morph frogs that could then be traded to other study participants. Sterile frogs couldn't then be used by those without scruples for breeding for profit. I know that the researchers in Japan who created genetically-modified "glow-in-the-dark" fish did sterilize the fish before licensing them to the wider pet trade (for fear of a catastrophic release of such monsters into the wild). I'm sure similar techniques must be available for amphibians.

I am only making this suggestion as someone who has a serious scientific interest in learning more about the evolutionary biology of D. tinctorius, so I hope this will be reviewed by members of the board in this light. If there are others interested in such a project, of course I would be very interested to hear from you.

Richard T. Lynch, Botanist, Sweetbay Magnolia Conservancy, Staten Island, NYC
718.273.3740 ([email protected])


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## Julio (Oct 8, 2007)

well keeping them in a terrarium that is quite warm will cause them to go sterile over a period of time.


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## Dane (Aug 19, 2004)

I don't quite understand what you are hoping to achieve with these sterile hybrid tincs. Is it for some genetic comparison between a "pure" line and a hybrid? Mixed morphs don't just happen, and it's far easier to prevent them than to come up with some means of allowing them to be disseminated without harming established lines.


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## ColombianFrogger (Jul 9, 2004)

hibridizating morphs could be useful in biogeography studies, and elucidating the real identity of an species group. for example, if you take 2 morphs from point A and C and make them hibridate, they should give offsprings similar to the frogs in point B, working as a proof of your distribution hypotheses. It also works in pumilio or another highly polimorphic species, not only with colors, but also calls. If you, for example have a northern histrionicus and try to hibridize it with an Ecuador sylvaticus, will their call be alike to the northern, southern or middle populations? that could help delucidating the status of an species complex.

however, sorry, its hard for me to explain it in english


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## Dane (Aug 19, 2004)

ColombianFrogger said:


> hibridizating morphs could be useful in biogeography studies, and elucidating the real identity of an species group. for example, if you take 2 morphs from point A and C and make them hibridate, they should give offsprings similar to the frogs in point B, working as a proof of your distribution hypotheses. It also works in pumilio or another highly polimorphic species, not only with colors, but also calls. If you, for example have a northern histrionicus and try to hibridize it with an Ecuador sylvaticus, will their call be alike to the northern, southern or middle populations? that could help delucidating the status of an species complex.
> 
> however, sorry, its hard for me to explain it in english


Makes sense to me, but I just don't understand why hobbyists would be involved.


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## markbudde (Jan 4, 2008)

I am not an evolutionary biologist. 

However, it seems to me (again, only my opinion) that this would be better address through DNA sequencing. Different species have different mutations in their genes. A frequently used gene is cytochrome C oxidase, but there are many potential genes, some which are more rapidly evolving. If an intermediate morph (we'll call it B) is part of the evolutionary progress from one morph (A) to another (C), then we would expect B to have an intermediate sequence between A and C. If, on the other hand, B is the result of an interbreeding event between A and C, we would expect that at a specific site B would have either the sequence from A or C, but not a mixture of both. This is because DNA tends to mix in chunks much larger than individual genes. In this case, sequence analysis would be more informative and less of an ethical issue. But again, I'm not expert so people with more experience in evolutionary biology should weigh in.


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## ChrisK (Oct 28, 2008)

Dane said:


> Makes sense to me, but I just don't understand why hobbyists would be involved.


Yeah the english on that was pretty good haha -

I think he's not interested in it hobby-wise, but maybe more documented study-wise


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## Fishman (Jan 16, 2007)

Woodsman, while I share your interest in the study of this groups evolutionary biology I personally feel that this type of work should not be taken on by hobbyists. Outside of a controlled research institution there is far to much possibility of these animals making it out to the general public. Short of actually surgically castrating the frogs there is no way to insure that they are sterile. The Glowfish that you used as evidence are not sterile, the company that produces them breeds them in traditional manners (it would cost far to much to genetically modify every new batch of fish), I have personally kept and bred these fish. Instead the company use incredibly strict copy right infringement laws to protect their trademarked fish. In addition you can buy "sterile" triploid grass carp for in pond weed control, yet the occasional fish is not sterile to the point that these fish have now become established nuisance pests throughout US waterways. In short, there is far to much possibility for cross bred frogs to end up in reproducing in an unsuspecting hobbyists collection and so it should not be encouraged to produce them.


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## Nick_ (Mar 24, 2008)

markbudde said:


> However, it seems to me (again, only my opinion) that this would be better address through DNA sequencing.


I think this would be the most intelligent decision. I personally would not pay much if any attention to ecology reports and/or research pertaining to this area that was not backed by some sort of mapping and analysis.

Woodsman, I donot think that there are going to be any recomendations for situations in which the interbreeding is handled. Im not debating its nature, but rather the fact that even suggesting methods is unlikey due to the general stance of this board. The time and money invested in such a project could be more appropriatley funneled into cb to fund other true studies. Again, this is simply mo'.


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## sldunn (Jun 2, 2008)

Just to chime in, the question asked was are there any know techniques to humanely sterilize the offspring of two morphs. While I am sure this is not commonly practiced, it certainly can be done and it would be interesting to know how.


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## Woodsman (Jan 3, 2008)

I think the work of the TWI/ASN in involving hobbyists in the conservation of dendrobatids speaks to some of your concerns. It is clear that the vast number of frogs in the hobby today have little or no locality data associated with them (I understand that some "true sipaliwinis" from NAIB were collected with such data, but most were not), so the idea that frogs in the hobby today could ever be returned to the wild is highly unlikely (also, the potential for disseminating potentially devastating diseases is a real concern). I greatly appreciate the dedication to keeping the frogs as one might be able to see them in nature (this is akin to the "species orchids" versus "hybrid orchid" dichotomy that permeates another community of hobbyists), but the idea that our personal frogs would ever be appropriate for conservation purposes is quite small (here I am only referring to D. tinctorius, the only species I have worked with).

Other researchers who have visited D. tinctorius in the wild have suggested that many of the established bloodlines in the hobby today do not represent all of the frogs from a given locality, but only "types" that were selected by various exporters (i.e., that the morphs "Regina" and "Giant orange" likely do not represent different populations, but are only kept desperate in the hobby; similar situations probably exist for "Alanis" and "Inferanlanis", blue-leg "Powderblues" and Gray-leg "Powderblues, etc.) If we really don't have the locality data in a complete form, then isn't part of what we are doing in the hobby looking a little like the American Kennel Club, making selections based on what we like and perpetuating those selections through our own "bloodlines" (though I do love my Siberian Huskies dearly!)?

So many good people are putting in a great deal of time rearing and breeding these frogs (including myself!!), and I just believe that there are a great number of basic biological research questions that could be answered by the kinds of progeny studies that I am suggesting (I only wish I had this kind of interest in the federally-endangered native plant species I work with professionally, as I would be years ahead of where I should be with species that are actually becoming extinct in the wild!!)

Thanks for your comments, Richard.



Dane said:


> I don't quite understand what you are hoping to achieve with these sterile hybrid tincs. Is it for some genetic comparison between a "pure" line and a hybrid? Mixed morphs don't just happen, and it's far easier to prevent them than to come up with some means of allowing them to be disseminated without harming established lines.


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## Ed (Sep 19, 2004)

markbudde said:


> I am not an evolutionary biologist.
> 
> However, it seems to me (again, only my opinion) that this would be better address through DNA sequencing. Different species have different mutations in their genes..



Hi Mark,

There are some animals out there that throw curve balls when it comes to this sort of analysis. For example, one of the spring salamanders (Gyrinophilius (I forget which one off hand)) in the southeastern USA has a population where the adults only get between one half to two thirds the size of the rest of the population. On genetic analysis they are the same species but the populations are effectively reproductively isolated as the females cannot pick up the spermatophore deposited by the males of the other population due to the size difference. When the male attempts to lead the female so she can pick up the spermatophore the distance is either too far or too little and the spermatophore cannot be picked up in the females cloaca. When I last paid attention to it, it was making a big fuss out of if they were species or not... 
So until we fully understand a species we can't rely totally on DNA analysis as there are some oddities out there. 

In addition, herps have some other oddities out there once you break down the reproductive barriers, for example look at the fertile intergenera hybrids between Lampropeltis, Pituophis, Pantherophis or the suspected fertile hybrids between Python regius and Morelia or Aspidites. The hybridization method was used in the old days by killifish keepers to work out relationships between different populations but I suspect that given the natural occuring hybrids that can be found in the amphibian and reptile world that this is full of pitfalls when you break down the species boundries. Its possible it could provide some evidence that is useful but I'm not sure it could be gotten in a practical way by a hobbyist as there would need to be some fairly consistant controls on the breeding, and rearing of the tadpoles. 

Ed


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## Nick_ (Mar 24, 2008)

Ed said:


> Hi Mark,
> 
> There are some animals out there that throw curve balls when it comes to this sort of analysis. For example, one of the spring salamanders (Gyrinophilius (I forget which one off hand)) in the southeastern USA has a population where the adults only get between one half to two thirds the size of the rest of the population. On genetic analysis they are the same species but the populations are effectively reproductively isolated as the females cannot pick up the spermatophore deposited by the males of the other population due to the size difference. When the male attempts to lead the female so she can pick up the spermatophore the distance is either too far or too little and the spermatophore cannot be picked up in the females cloaca. When I last paid attention to it, it was making a big fuss out of if they were species or not...
> So until we fully understand a species we can't rely totally on DNA analysis as there are some oddities out there.
> ...


 They did not isolate the gene responsible or it simply did not exist? I am confused. I did not thnk that expressed traits could occur without the 'english' of a gene? That is pretty interesting regaurdless of implication.


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## Woodsman (Jan 3, 2008)

Ed said:


> Hi Mark,
> 
> There are some animals out there that throw curve balls when it comes to this sort of analysis. For example, one of the spring salamanders (Gyrinophilius (I forget which one off hand)) in the southeastern USA has a population where the adults only get between one half to two thirds the size of the rest of the population. On genetic analysis they are the same species but the populations are effectively reproductively isolated as the females cannot pick up the spermatophore deposited by the males of the other population due to the size difference. When the male attempts to lead the female so she can pick up the spermatophore the distance is either too far or too little and the spermatophore cannot be picked up in the females cloaca. When I last paid attention to it, it was making a big fuss out of if they were species or not...
> So until we fully understand a species we can't rely totally on DNA analysis as there are some oddities out there.
> ...


Thanks Ed for this note,

Though we are currently living in the "age" of molecular genetics, members should always keep in mind that Darwin uncovered the basic processes by which selection in nature occurs without any knowledge of the work of Gregor Mendel (of the famous garden Pea experiments), and (of course), neither of these icons had any knowledge of the future work of Watson and Crick (the double-helix guys). To my way of thinking, population ecology will always be the best basis for conservation of rare taxons. Molecular work can be used to clarify many fundamental questions (as can the kind of basic biological work I am proposing here), but it will ultimately be good field work conducted on dendrobatids that will provide the best plan of action for future efforts at conservation.

We should also perhaps at some point provide a primer on the differences between phenotypes and genotypes, for those who might be interested.

I look forward to fleshing out some of these ideas at length with all interested in the New Year. Take care, Richard.


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## Nick_ (Mar 24, 2008)

Woodsman said:


> Thanks Ed for this note,
> 
> Though we are currently living in the "age" of molecular genetics, members should always keep in mind that Darwin uncovered the basic processes by which selection in nature occurs without any knowledge of the work of Gregor Mendel (of the famous garden Pea experiments), and (of course), neither of these icons had any knowledge of the future work of Watson and Crick (the double-helix guys). To my way of thinking, population ecology will always be the best basis for conservation of rare taxons. Molecular work can be used to clarify many fundamental questions (as can the kind of basic biological work I am proposing here), but it will ultimately be good field work conducted on dendrobatids that will provide the best plan of action for future efforts at conservation.
> 
> ...



I definatley agree, especially with differentiating and actually identifeing the genos and phenos. With the price of current portable GPS units, a trip for someone wo has the time and funds would certainly be worth while. Going back to te op, I see y the issue would be one worth adressing for certain reasons, but simply think that there must be more efficient ways to reach the goal without sterilization. The fielding work of those "genetic pioneers" was certainly without aid of modern machinery, but also was on the level of middle school science. Advanced understanding of taxon seems to be more politically charged these days than actuall hard facts and "pudding" if you will. I could not think of a better way to spend 4-5 grand and a couple monthhs than to visit Central and South America (other than building another room for critters). Is there a current compilation scientific journals/findings on any one site, or peraps a menagerie of links for Dendrobates and others. Recent threads like tis one have sparked a fairly intense interest in this area for me.


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## Ed (Sep 19, 2004)

Nicholas OConnor said:


> They did not isolate the gene responsible or it simply did not exist? I am confused. I did not thnk that expressed traits could occur without the 'english' of a gene? That is pretty interesting regaurdless of implication.


When they looked there wasn't any significant gene variations... Its simply how that population developed. 

Ed


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## Woodsman (Jan 3, 2008)

Julio said:


> well keeping them in a terrarium that is quite warm will cause them to go sterile over a period of time.


Hi Julio,

Do you have an idea as to the temps involved? It has been established in a number of herps that the temps that eggs are incubated at can affect sex, so I did have the thought that higher egg incubation temps could cause the frogs to become sterile adults. 

There is also research ongoing now to try to sterilize eggs with UV irradiation (as a method of controlling invasive Coqui frogs in Hawaii), as well as a few others I'm looking into. I'll update as I find out more.

Thanks for the advice, Richard.


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## Ed (Sep 19, 2004)

Woodsman said:


> Hi Julio,
> 
> Do you have an idea as to the temps involved? It has been established in a number of herps that the temps that eggs are incubated at can affect sex, so I did have the thought that higher egg incubation temps could cause the frogs to become sterile adults.
> 
> There is also research ongoing now to try to sterilize eggs with UV irradiation (as a method of controlling invasive Coqui frogs in Hawaii), as well as a few others I'm looking into. I'll update as I find out more.


True TSD is currently unknown in amphibians. There are some cases where you can vary the phenotypic expression (but not the genotypic expression) of sex through exposure to extreme temperatures during critical developmental periods. For a classic example see 
Temperature sex-reversal in amphibians and reptile...[Int J Dev Biol. 1990] - PubMed Result 

There is often discussion around sex ratios in some dendrobatids being due to TSD but this would be somewhat surprising as this would be a change from not only other closely related dendrobatids but amphibians in general. That said, however I am unaware of any attempts to quantify if in those species there is a change from the normal genetic sex determination, or a temperature induced phenotypic change or some form of endocrine disruption causing the heavy ratios. 

Ed


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## rozdaboff (Feb 27, 2005)

I think that Julio may have been more referring to the susceptibility of spermatogonia/oogonia to increases in temperature, as opposed to skewing the sex ratio in one direction. 

One paper I found mentions that high temperatures can inhibit amphibian oogenesis - but there isn't a citation for this data, and it doesn't mention if this inhibition is irreversible (i.e. a temporary effect on the hormonal axis as opposed to problems at the level of the oocyte/oogonia). But a quick search didn't find anything about exposure amphibians to temperature extremes for sterilization. As they are ectotherms and all reproductive organs are internal - I would wonder if the temperature extremes needed for sterilization would not lead to other systemic issues.

Julio - where did you hear about this?


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## Julio (Oct 8, 2007)

Hey Oz,
this has been my personal experience as well as others in the hobby, that dart that are kept too warm will turn out sterile.


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## Dane (Aug 19, 2004)

Julio,
what species did you observe this in, and what were the temperatures/durations they were exposed to?



Julio said:


> Hey Oz,
> this has been my personal experience as well as others in the hobby, that dart that are kept too warm will turn out sterile.


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## Julio (Oct 8, 2007)

i have personally have this happen with some pamanian auratus and some cobalt tincts about 8-9 years ago when i had a huge heat wave during a really hot summer.


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## buddha (Oct 17, 2006)

*just found something that was neat..*

http://www.dendroboard.com/forum/science-conservation/35321-humane-techniques-sterilize-frogs-3.html

So many Big words i passed out twice.. Just thought it was neat. -buddha


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## kyle1745 (Feb 15, 2004)

Moved this post to the original thread rather than add clutter.


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