Hugo Claessen
Belgian Herpetological Society
Antwerp
August 1999
Last
update: 17 August, 2002
Spindle leg syndrome, a condition that was identified as a problem many years ago, affects the front legs of metamorphosing tadpoles. The result is froglets which have weak, deformed limbs and which are seldom able to feed effectively and thus die. Last year I attended the 'Anuren Taggung 1998' in Germany which I have investigated and now produce my conclusions. A German vet, Dr Thomas Wohrmann, from the University of Aachen, has examined, by histology, hundreds of affected Dendrobatids and has found the following: 1) The thyroid gland is always normal. |
Because I am an amateur, but wanting to fully understand what this meant, I spoke with veterinary professors in Belgium and the Netherlands and these are my conclusions:
1. Whilst the thyroid is histologically normal this does not confirm normal secretion of hormones which requires further investigation.
2. The muscular atrophy is the histological examination of the visible effect of spindle leg. The 'elbows' are fixed and the musculature has too few fibres, which are also small. (Gouda and Hak, University of Utrecht, 1995.)
3. The spinal column has sub-total dystrophy; it is not completely closed.
4. The medulla oblongata is not closed at all. This is the section of the brain that connects with the spinal chord.
The Biology
(Development stages are the standard stages of early development of
anurans according to Gosner, 1960). In the development of frogs' eggs the
closing of the Spinal column starts at stage 13, when the neural plate is
formed. This closing is complete at stage 15 when a neural tube is completely
formed. The Medulla oblongata stays open which is normal for amphibians. The
closing of the Spinal column and the formation of the forelegs are controlled by
a gene, the Homoobox-Gene XLHBox
If there is a disturbance of this gene, the frogs develop spindly legs. The German investigators were able to generate this in the laboratory and each time they introduced a disrupter the frogs developed Spindly Legs. The external influences on that gene are not known, but it is possible that the disrupter is introduced by the parents, by high temperature or radiation (perhaps UV?), after the eggs have been laid, but prior to the tadpoles becoming free swimming at Stage 25.
If this is true, it means that it is too late to change the process once tadpoles have formed as the genetic information to produce Spindly Legs is set in the egg. Only the influences of temperature or radiation can generate a further disruption of the gene making it impossible to repair a disruption that has existed from stages 13 to 15. Therefore, in my opinion, if we wish to find the reasons for spindly leg, and its cure, we must look at the parents, not the tadpoles.
The human condition, Spina bifida, in which babies are born with a spinal column not completely closed, is also caused by the XLHBox 1 gene. The incidence of the condition has been demonstrated to be reduced by giving the mother large quantities of Folic Acid, and often Vitamin E, as a prophylactic. Both Spindly Legs and Spina bifida are conditions generated by a disruption of the same gene making it feasible that the same prophylactic could work, a possibility which must be worth trying with frog parents.
The Question
It is questionable how the frogs obtain these substances in the wild
but it should be possible to give the frogs fruit flies dusted with Folic Acid
and vitamin E powders but how, and in what quantities, as there is no
information on dosage for amphibians? Pregnant women are frequently given a
medication called OMNIBIOTA Prenatal which gives a dose of Folic Acid of 5mg and
12mg of Vitamin E for a woman of 55Kg body weight. This equates to O.lmg and
0.22mg/Kg respectively.
Folic Acid
Because Folic acid is one of the vitamins from the B complex, there is
little risk of hypervitaminosis (overdosing). Information for fish and poultry
gives a dosage of about 10 to 20 mg Folic acid /kg food. Daily doses of 20 mg/kg
body weight would seem to be sufficient for fish and poultry but this is over
100 times the level indicated for humans.
Vitamin E
In a professional fish food for Tilapia and Trout, a dosage of
50-100mg/kg body weight of fish is given. This is again, at least, 200 times the
level indicated for humans. Vitamin E prevents the formation of free radicals
that can initiate the disruption of the relevant gene.
As our normally used vitamin and mineral supplements contain very low levels of both Folic Acid and Vitamin E, I suggest that some form of supplementation may be necessary, though a considerable amount of work will be needed to ascertain dosages. It may be that, in the first instance, a simple addition to the usual vitamin/mineral supplements might be tried.
The next text is from Brander G. C. & Pugh D. M 1977 Veterinary Applied Pharmacology and Therapeutics:
Folic acid, folinic acid, vitamin C and cyanocobalamin are connected with nucleic acid synthesis. The acid is also involved in ansmethylating actions along with cyanocobalamin, both in nucleoprotein formation and in fat metabolism through methylation in the synthesis of choline and methionine. Through this latter action folic acid and cyanocobalamin reduce nutritional requirements of choline and methionine. Clinically the symptoms shown in experimentally induced deficiencies are unlikely to occur. These symptoms include macrocytic anaemias with reduction in the numbers of all cellular elements of the blood, diarrhoea, skin, lesions, reduced growth rates in experimental animals and birds. It should be noted, however, that, as with para-aminobenzoic acid, which is a precursor of folic acid, small quantities of the gut 'active' sulphonamides would promote a deficiency, probably by reducing bacterial numbers and therefore the synthesis of this and other vitamins.
Therapy when an outbreak of (Vitamin E) deficiency disease is suspected usually consists of immediate administration of vitamin E and/or selenium in whatever form is appropriate and convenient. Nutritional requirements are not defined. This is possibly because the availability of the vitamin is conditioned by the composition of the diet. Where the level of unsaturated fatty acids in the diet is high, the vitamin requirements will also be high, but it should be remembered that other compounds and conditions besides these acids will depress absorption of vitamin E, including triorthocresylphosphate (in some species), possibly sulphaguanidine and gastro-enteritis. It must also be borne in mind that variations in mineral and carbohydrate concentrations in a diet will modify the symptoms resulting from a vitamin E deficiency or insufficiency. Deficiencies other vitamins such as choline may predispose muscles to the effects of vitamin E deficiencies.
Conclusions
Many people are trying to solve the problems of spindly leg and,
whilst not suggesting that my ideas are the answer, I feel that they are worthy
of further investigations. If you have any further information, ideas or
criticisms please let us discuss them together.
Editor's note
Whilst some of Hugo's article above is conjecture, it is well thought
out and would form an excellent basis for further research. If there are any
members currently producing froglets with spindle leg it would be worth adding
vitamin E and Folic Acid to the usual vitamin and mineral supplements to see if
this alleviates the problem.
Lars Osterdahl
Swedish Dendrobates Society
Last update: 17 August, 2002
In April 1997 I became a member of the Swedish Dendrobatid Society, and when I exposed my status as a rookie (I had only bred D. auratus) by asking what spindle legs looked like, my fellow board members decided I needed a lesson. I was given two clutches of D. leucomelas eggs that were from parents that had given a very high percentage of spindle legs in their offspring.
I decided to treat these eggs exactly like those of auratus. The two clutches consisted of 7 and 5 eggs respectively, the first group AĘ having tails (Gosner stage ~ 18), the second had no visual sign of development. I left them in their Petri dishes until they hatched, with the dishes kept in shallow plastic boxes with lids to maintain a humid atmosphere.
After 8 days the first group began to hatch, at which point the tadpoles were moved to individual glass jars with 2 cm tap water. On this same day we left for Gotland, an island in the Baltic. This was a journey that was to take 14 hours, during which time the temperature in my frog box fell to only +10°C. However, there was no visible effect on the tadpoles.
On day 12 all eggs in group A had hatched. The tadpoles were moved to glass jars and the water level was increased to 25mm with well-water. In contrast to the tap-water in Stockholm, which is fairly soft, the well on Gotland produced water with a high Calcium content.
Group B started to hatch on day 15. As the tadpoles grew bigger the water level was increased to 12 cm.
On the 65th day two A-groupers front legs appeared. The others followed successively until day 95 when the last two metamorphosed. None of the 12 small frogs had abnormal legs.
Notes on the way I treat tadpoles
Cannibalistic tadpoles are either kept solitarily or in tanks with a
large water volume. For solitary breeding I use 1-litre wide-mouth glass
jars. I do not change water as often as suggested by most literature. This
is left until such time as when too much debris has collected. I then
siphon this debris out and top-up with fresh water. If the water takes
on an opaque or milky colour (meaning too much infusoria, and consequent oxygen
depletion) I perform an almost total water change.
The food used in the case described here was small pieces of earthworm, given every second day. No vitamins, Spirulina, or fish food were given. I have Daphnia in the jars, partly as an indicator of water quality and partly as a source of food. Also, the algal growth on the sides and bottom of the jar is a food source used by the tadpoles.
Peter Marsters,
Division of Clinical Chemistry,
School of Clinical Laboratory Sciences,
University Hospital,
Queen's Medical Centre,
Nottingham, NG7 2UH.
21. 08. 99
Last update: 17 August, 2002
I have been handed a paper entitled Developments in the study of Spindle leg syndrome, and have been requested to forward helpful suggestions. Although I am very happy to do this I must state from the start that this is not my area of expertise and my comments must therefore be generalised.
I have not come across the XLHBox 1 gene but assume that it expresses a protein essential to limb development. As you may know all homeobox genes express developmental proteins, which may have no function in the adult organism. You say that when this gene is disrupted under laboratory conditions Spindle leg can be induced. I assume that the gene is actually āknocked out in the spawn so that the developing embryo does not receive any expressed protein from the XLHBox 1 gene.
It is well known that some genes are much more susceptible to environmental effects than others. The reasons for this have not yet been fully elucidated. However the expressing genes must be accessible to the nuclear transcription machinery and as such are certainly on exposed locations in the chromosomes. Therefore mutations of this gene could occur during the spawn stage. Spawn is often very exposed to environmental and external influences, such as toxins in the water or high exposure to harmful ultraviolet radiation. Also in support of this is the fact that during development, genome replication occurs with high frequency and mutations commonly occur due to errors in the replicate DNA. If vitamin E does have a protective role during DNA replication, then it follows that its presence would be most beneficial during the developmental stages when crucial differentiation of pluripotent cells is occurring and replication is frequent.
Folic acid may have a role in the switching on of developmental genes. Thus, in the case of oviparous creatures, it seems possible that the mother donates it and vitamin E to the oocytes prior to spawning. On that basis your suggestion to treat the mother frogs, in the period directly before spawning, with Folic acid and vitamin E would seem sound.
It would be most interesting to test your theory, but impractical for you to do so alone since statistical evidence would require a large cohort of results. Studies of this type need to be strictly governed. Ideally they should be āblind in so much that the individuals responsible for feeding the frogs should not be aware of which frogs receive the supplement and which the placebo until after the results have been recorded. Statistical significance would need to be established and it would be necessary to repeat the experiment several times using different amounts of Folic acid and vitamin E in order to optimise dosage. However if a study of this nature is not possible I see no reason why your frogs should not be treated with Folic acid and Vitamin E, prior to spawning, as a prophylactic.
It seems from your most interesting article that the similarities between Spina bifida and spindle leg do suggest that incidences of spindle leg may indeed be reduced by a similar treatment of the supplement used during human gestation. You appear to have collected your evidence scientifically and made reasonable deductions. It may be of use to you to further evaluate the roles of vitamin E and Folic acid in expectant humans, as it seems likely that these may have an application in the gravid frog and her spawn.
I do hope that my comments may be of some use to you and I wish you every success in your endeavours.
Peter Marsters
Carlos Cabrera
Sweden
I am a student of molecular biology and found Hugo Claessen's text interesting so checked Folic Acid in one of my chemistry books. The description given was the following: "Folic acid, pteroyl-L-glutamic acid, vitamin B. Folic acid and its derivatives (mostly the tri- and heptaglutamyl peptides) are widespread in nature. It is a specific growth factor for certain micro organisms, but in animals the intestinal bacteria provide the small quantity needed for growth. The coenzyme forms are actually the reduced products of folic acid. The main function of 5,6,7,8, -tetrahydrofolate (THFA) is a carrier of a C1 (methanoate) unit in the biosynthesis of purines, serine and glycine. The nitrogen atoms at positions 5 and 10 are the reactive sites of the molecule. Green leaves are specially rich in this molecule".
In my opinion this description suggests two possibilities:
a) The lack of a complete intestinal bacterial flora in captive bred or wild caught livestock in hobbyists' terraria could be one of the causes of deficiency in folic acid intake by the organism. Those bacteria could exist in the natural dendrobatid biotopes but may not survive in terraria. It would be quite interesting to compare the rates of spindle-leg-syndrome in the offspring of newly captured frogs, those of captive bred animals and those that have been kept in captivity for longer periods.
b) The lack of folic acid should imply the lack of 5,6,7,8, -tetrahydrofolate that could affect the synthesis of the two amino acids, serine and glycine. This in turn could affect the synthesis of lipids and proteins and thus be one of the factors causing the described muscular atrophy. Vitamin E is essential for fertility and reproduction. Deficiency in rats leads to abortion in the female and loss of fertility in the male. The richest sources are seeds and their oils, and green leaves. The vitamin works as an antioxidant". You may remember from Mr Claessen's article that vitamin E prevents the formation of free radicals (anti oxidating effect), which are one of the most active mutagens (substances able to change the genetic information in the DNA).
Reference
Dictionary of Chemistry D.W.A. Shap Penguin Books