# When is the sex of a dart frog determined



## Ron Jung (Mar 29, 2005)

Hey everyone here is a question I haven't been able to find an answer to. When is the sex of a dart frog determined? Is it at fertilization, when the tad is growing, when it morphs? Does raising the tads in groups have any effect on sex? I raise all my tads communially and get huge tads.

The reason I ask is 2 yrs. back I bred Epip. Azureiventris and got 60 offspring from my last remaining female before she died  . I sold most of them and held back 14 for myself. I tracked down with some help all the people that got the frogs from me either directly or indirectly and all 60 juvs. that I had turned out male. Not even 1 female out of 60 offspring.

I know with reptiles temp. can determine the majority of the sexes in a clutch of eggs but with darts what determines it? I raised all my tads at the same temp and food and with all my other 20 diff. species I got a mix of m/f except with the Azureiventris

Any insite would be great.

PS is there anyone out there with a SPARE FEMALE AZUREIVENTRIS? :lol:

Thanks in advance for your responses.

Best.
Ron Jung at RAINFOREST DESIGNS UNLTD.


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## Ben_C (Jun 25, 2004)

It is determined at fertilization.

hope this helps,
B


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## kyle1745 (Feb 15, 2004)

Ben do you have any data on this?


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## Ben_C (Jun 25, 2004)

Not on me, you can find it (i think) in 'herpetology' by zug, but i can't remember if that was the book. That would be specific to Dendrobates, but I don't believe there is a single case of ESD in amphibians, so the only remainder is genetic sex determination. Wheter or not males are XY or ZZ, I have no idea, but i'm 99.999999999% certain it is genetic.


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## Ron Jung (Mar 29, 2005)

I still can't figure out why out of 60 offspring they were all male? Do you know the chances of that with a 50/50 chance of m/f for every egg? I was told by a lady in the US that is studying the Azureiventris that the Azureiventris actually has no toxins like other darts do, and I know the males like to sit up high in the open and call very loud and if it is true that they don't carry any "dart" toxins then it could be that the males get picked off at an alarming rate and maybe that is why the outragous ratio of m/f so that in the wild if they get eaten the ratio would be even by the time they are of breeding age? Just a thought. Has anyone else hear that Epip. Azureiventris doesn't have toxins in the wild?

Any insight would be great.

Thanks again for your thoughts.

Best.
Ron Jung at RAINFOREST DESIGNS UNLTD.


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## kyle1745 (Feb 15, 2004)

The reason I posted the question for resources is that it has been rumored that environment conditions during the tadpole stages and young froglets can determine sex.


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## aquascott (Oct 18, 2006)

i have been interested to see if in the last three years anyone has bred some females???


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## Ed (Sep 19, 2004)

To date there have been no cases of TSD found in amphibians.. frog sex determination is genetic.. (either XX/XY (male heterogamety) or ZZ (male)/ZW (female heterogamety). 
However this does not mean that temperature may not play a role in expression of sexual characteristics. It is known in several amphibian species (for example the genus Pleurodeles) that temperature can change the expression of the sex (in Pleurodeles this is species dependent). What this means is that you may have an animal that is genetically a female but due to changes in hormone expression brought about by temperature it may behave and function as a male (or vice versa). 

If this is the case (and I'm only theorizing here) then breeding two frogs (both genetically the same sex but one expressing the opposite sex) together would result in an extreme disproportion of the frogs being one sex. If this is further combined with with a thermal modification of expression of gender then you could easily see all of one sex or another. 

Ed


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## aquascott (Oct 18, 2006)

so would that be the same as fish who (change) sex in response to external stimulus.


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## Ed (Sep 19, 2004)

Probably not... 
Sex determination in fish may be due to a lot of different factors including multiple sex chromosomes or none at all.. in the cases where sex is not genetically determined then there may not be any sex chromosomes. 
There are also TSD cases in fish.. 

If the theory is true, then in the wild the frogs are not exposed to temperatures that cause a enzyme or receptor to malfunction resulting in the skewed sex ratio. If the theory is correct, we are keeping the tadpoles/metamorphs at the wrong temperatures and are forcing a malfunction in development resulting in the skewed sex seen in this species. 

Other viable hypothetical alternatives are endocrine disruption and epigenetic factors. 

The closest to the scenario practiced by protogynous fish is a Hyperolius (see JSTOR: An Error Occurred Setting Your User Cookie) but this is a seperate case from is being seen in the dendrobatid in question. 


Ed


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## Sherman (Oct 29, 2008)

I have heard there is a disproportionately large number of captive bred male pumilio as well.


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## Ed (Sep 19, 2004)

It depends on what you mean by disproportional... In the original post there was 100% male from all of the tapdoles produced. 

There can be differences in survivial rates between sexes that can skew a sex ratio in one direction or another.. in some animals, maternal diet can affect offspring sex ratios (for one citation see Rosenfeld CS, Grimm KM, Livingston KA, Brokman AM, Lamberson WE & Roberts RM 2003 Striking variation in the sex ratio of pups born to mice according to whether maternal diet is high in fat or carbohydrate. PNAS 100 4628–4632 )

This has been noted in a number of species.. and this doesn't include potential effects on survivial of one sex or another based on some of the husbandry methods used. For example, the anecodotal reports of high mortality of pumilio metamorphs less than six months of age can skew the sex ratio if the greater proportion of survivors are males.

Ed


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## Sherman (Oct 29, 2008)

Ron Jung said:


> I raised all my tads at the same temp and food and with all my other 20 diff. species I got a mix of m/f except with the Azureiventris


Ron, do you know at what temperature your azureiventris developed?


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## Sherman (Oct 29, 2008)

In Lotters Poison Frogs, under notes for _Hyloxalus azureventris_, *S*CHULTE (1999) had noted a skew in sex ratio of captive-hatched offspring. Frogs in the wild deposited their tadpoles into "subterranean rivulets and pools" that were around 17-18 deg C. He presumed the temperature had a determining effect on the formation of the sex. There is no mention of toxicity.

Ed, is this what you described as changing the expression of the sex? This would not change the genetic make-up, only it's behavior and function.

If you started genetically with a 50/50 mix(male/female), then subjected them to, in this case, temperatures that are too high during development, you could effectively end up with a 100males? And of those males, 50% would still be genetically female?


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## Ed (Sep 19, 2004)

Sherman said:


> Ed, is this what you described as changing the expression of the sex? This would not change the genetic make-up, only it's behavior and function.
> 
> If you started genetically with a 50/50 mix(male/female), then subjected them to, in this case, temperatures that are too high during development, you could effectively end up with a 100males? And of those males, 50% would still be genetically female?


I remembered the bit in Lotters as well which was parthly behind my explination. 

This is exactly what I meant in my post above. The problem is that while the frogs may be genetically female they are not going to produce ova, behave or be able to function as a female. This is assuming that there is not some other reason that there is a skewing of the sex ratio such as endocrine disruption or excessive mortality in female egg, tadpoles or froglets. 

Now if this species is of the ZZ (male)/ZW (female) system then a breeding between a functional female and a (I don't want to use the word phenotype as its not correct in this usage) temperature altered female would only produce 25% genetic males... 

Some comments

Ed


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## Sherman (Oct 29, 2008)

So, if you bred a normal female (ZW) to an environmentally altered female (ZW) and find a way to eliminate the environmental alteration, you would end up with 1 male (ZZ); 2 females (ZW&ZW); and 1 (WW). What would the (WW) be?

Are the temperature altered Pleurodeles fertile? Can a genetic female produce viable sperm, and a genetic male produce ova?

As for azureiventris, assuming you had real males and females (not altered ones), wouldn't it be simple to confirm or eliminate temperature as a source for the large percentage of males. The other possible causes seem more difficult to test (i.e. endocrine disruption, epigenetics, survivability and maternal diet.)


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## Ed (Sep 19, 2004)

Hi Sherman,



Sherman said:


> What would the (WW) be?


In genetic sexing systems like this, the embryo doesn't develop or dies early (at least in non-fish vertebrates). 




Sherman said:


> Are the temperature altered Pleurodeles fertile? Can a genetic female produce viable sperm, and a genetic male produce ova?


Yes the temperature altered Pleurodeles are supposed to be fertile. The reproductive system development is rerouted early so it still develops fully just the opposite sex. 




Sherman said:


> As for azureiventris, assuming you had real males and females (not altered ones), wouldn't it be simple to confirm or eliminate temperature as a source for the large percentage of males. The other possible causes seem more difficult to test (i.e. endocrine disruption, epigenetics, survivability and maternal diet.)


Yes, temperature would be an easy way to tell. As an alternative, one could also perform karyotyping on adults of known sexes to see if there are some different genetic sexes. 

Even if you are breeding of the same genetic sex but different expressed sex and have the correct temperatures for the tadpoles you could look for about 33% of the one sex to show up. (statistically about 75/100 tadpoles should survive and 25/100 of tadpoles would be one sex while 50/100 would be the other and 25 don't survive so 25/75=33%) 

Ed


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## heatfreakk3 (Oct 15, 2008)

i thought you found out the sex when they were adults


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## Sherman (Oct 29, 2008)

Thank you Ed. 

Karyotyping sounds like it requires a lot of lab work beyond the abilities of most hobbyists. Any data would be great though!

Has anyone tried developing_ azureiventris_ tadpoles at 17 deg C? If so, what became of them?



> i thought you found out the sex when they were adults


It is true, you normally need to wait until a frog is an adult to determine sex visually or behaviorally. A young frog does not have large toe pads, nor will it call. There is a set point in time, long before visual cues show up, when the sex becomes fixed (maybe). As per Ed's first response to this thread, genetically, sex is determined at fertilization. There are, however, certain environmental and chemical conditions that would cause disruption in the normal development of the reproductive organs. This disruption can cause a female to "turn" into a male, and vice versa.
One possible cause for this disruption is temperature. It has been speculated that temperatures that are too hot during development cause an abundance of male _azureiventris_.


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## Ed (Sep 19, 2004)

I agree that karotyping is beyond the average hobbyist but there are some people here who do work in labs with access to the materials. I have not heard of anyone in the US keeping the tadpoles down between 15 and 17 C to see what happens with them. 

Ed


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## aquascott (Oct 18, 2006)

i know one group that is starting an experiment with raising tads at the lower temp and a control group at a more "normal" temp. that's why i was asking about people that breed and got female offspring. They are splitting each clutch in half between the two conditions. The cold tank is on a chiller loop maintaining temps at around 17 C (temperature being recorded via data loggers).

Hey Ed, this is another thought/question on possible causes of these "perceived" male gender bias. Azuriventris males are very good parents, and if allowed to care for and guard their clutch, will readily allow the tads to piggyback for quite some time. Even when the male is given tad deposition sites in the enclosure the tads will still ride on the male for 10 days or longer. In this time the tadpoles noticeably increase in size, as if they are feeding off of the males back. Could it be possible that they are picking up regulating hormones excreted by the male? It was just another possibility other than temperature that may be different in captivity. Has anyone allowed complete parental transport with these frogs? If so are you still seeing all male offspring? Our next clutch will be completely tank raised as another group.

Ed thanks a ton for your valuable information that you continually provide!


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## Ed (Sep 19, 2004)

aquascott said:


> i know one group that is starting an experiment with raising tads at the lower temp and a control group at a more "normal" temp. that's why i was asking about people that breed and got female offspring. They are splitting each clutch in half between the two conditions. The cold tank is on a chiller loop maintaining temps at around 17 C (temperature being recorded via data loggers).



Sounds like a good article for Leaf Litter. Regardless keep good records because if you do, that would publishable in a number of research journals... 



aquascott said:


> Hey Ed, this is another thought/question on possible causes of these "perceived" male gender bias. Azuriventris males are very good parents, and if allowed to care for and guard their clutch, will readily allow the tads to piggyback for quite some time. Even when the male is given tad deposition sites in the enclosure the tads will still ride on the male for 10 days or longer. In this time the tadpoles noticeably increase in size, as if they are feeding off of the males back.).


Have you necropsied any to see if they have exhausted the yolk before climbing on the back of the male? I suspect this is the source of the nutrition that allows the tadpoles to grow. If you observe the tadpoles you should be able to see if they feed on the males skin (after a few days they should be passing fecal strings..) 
I would be very surprised if the expressed sex is affected during that period as gonadal differentiation occurs later in development 

see 

IngentaConnect Pattern of Gonadal Sex Differentiation, Development, and Onset of...


ScienceDirect - General and Comparative Endocrinology : Pattern of Gonadal Sex Differentiation, Development, and Onset of Steroidogenesis in the Frog, Rana curtipes


Ed


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## Sherman (Oct 29, 2008)

> I agree that karotyping is beyond the average hobbyist but there are some people here who do work in labs with access to the materials.


I better understand the value of karotyping now. It would not require a large sample size to be tested to be valuable. Finding female genes in a group of "all male" frogs would show that there is a disruption of sexual expression.
Finding the "all male" clutch to be genetically male would lead to survivability issues of the females.


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## JWerner (Feb 17, 2004)

I remember Rthis being discussed before. When the azureiventris were brought in by Sean, I got some and bred them. I held back a group of 8 and exactly half became female. This test group was reared in the coolest area in my FR - on the floor, where temps in the 50's is very common. All morphed out with yellow markings. Another group of tads from the same clutch were reared in cups and kept a fairly constant 70 degrees and higher. All morphed out bright red/orange. The tads on the floor took twice as long to morph.


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## Ed (Sep 19, 2004)

Sherman said:


> I better understand the value of karotyping now. It would not require a large sample size to be tested to be valuable. Finding female genes in a group of "all male" frogs would show that there is a disruption of sexual expression.
> Finding the "all male" clutch to be genetically male would lead to survivability issues of the females.


Exactly. 

Ed


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## Ed (Sep 19, 2004)

JWerner said:


> I remember Rthis being discussed before. When the azureiventris were brought in by Sean, I got some and bred them. I held back a group of 8 and exactly half became female. This test group was reared in the coolest area in my FR - on the floor, where temps in the 50's is very common. All morphed out with yellow markings. Another group of tads from the same clutch were reared in cups and kept a fairly constant 70 degrees and higher. All morphed out bright red/orange. The tads on the floor took twice as long to morph.


Good information. If this can be repeated using some form of documentation of temperatures (like data loggers) then this would be real information that has some good value. 

There is some information that I need to follow up on for possible mechansims but in short, some anurans appear to develop one form of gonads which in the opposite genetic sex, regress and then form the genetically expressed sex. 

This article discusses what we are theorizing in a salamander 
P450 aromatase expression in the temperature-sensitive sexual differentiation of salamander (Hynobius retardatus) gonads 

Ed


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## Sherman (Oct 29, 2008)

Great paper Ed. Thank you.

It has been shown that sexual expression can be manipulated by temperatures. It has been suggested that it has a role in _azureiventris_ development. Some have had "success" by producing a balanced male/female ratio at low temperatures.

Why doesn't everyone have a thermometer on their developing _azureiventris_?


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