Waste Not; Want Not. Do Frogs Recycle Their Antifreeze?

Many advances in the biological sciences have come from what can be classified as—and funded as part of—agricultural research. Reproductive biology is one prime example but there are many others.

One advance the origins of which lie firmly in agricultural production is the recycling of urea. We were all taught at school that excess protein in the diet ends up as urea in urine with the nitrogen contained therein lost forever. But things are not that simple. Now it is known that in many animals, the urea is not simple dumped but can be broken down by bacteria in the gut and the nitrogen it contains recycled into protein.

It was the demonstration that urea, a form of non-protein nitrogen, added to the feed of farm ruminants, can be used within the rumen to make protein for growth or milk production that set off the interest in the recycling of urea under different nutritional and environmental regimes. It is not the ruminant’s tissues that are involved in breaking down the urea but the bacteria of the rumen. The bacteria then produce protein using urea as a source of nitrogen and this protein is digested and absorbed further down the alimentary canal. The history of the use of urea rather than expensive protein nitrogen sources was explained by my predecessor but one as Director of the Hannah Research Institute, James Andrew Buchan Smith (1906-2006), known throughout the world as ‘JABS’. Since those early days the recycling of urea nitrogen has been recognised in more and more organisms that produce urea as a nitrogenous waste product.

Amphibian species use high concentrations of urea in the blood for two main purposes: protection against dehydration and as an antifreeze. Some, notably the famous Crab-eating Frog (Fejervarya cancrivora) which can survive in sea water, build up high concentrations of urea in their body fluids in order to prevent the osmotic loss of water into the surrounding salty water. Other frogs that hibernate on land in sub-zero temperatures build up high concentrations of urea to act as a cryoprotectant. The high concentration lowers the freezing point of body fluids, preventing the formation of ice crystals.

In a paper published last year, a team in the USA produced preliminary evidence that the urea accumulated during hibernation in the Wood Frog, Rana sylvatica or Lithobates sylvaticus from northern North America, can be recycled into protein. Bacteria in the hind gut were found which could produce urease, an enzyme not produced by vertebrate tissues but essential for the recycling of urea nitrogen; more were present in winter than summer. Thus it is possible that as temperatures rise in spring, the bacteria become active, the urea is broken down and the products of that breakdown are converted into amino acids and hence protein.

Wood Frog
Brian Gratwicke [CC BY 2.0 (https://creativecommons.org/licenses/by/2.0)]

The findings on this frog indicate what could happen. However, there are some qualitative and quantitative pieces of evidence that would be more fully convincing of the first demonstration of urea recycling in amphibians. In mammals, specific urea transporters carry urea into the interior of the gut. Are they present in amphibians? Similarly, we do not know the magnitude and, therefore, the quantitative importance of any breakdown of urea to the overall nitrogen metabolism of these frogs at the end of hibernation. However, this could be another case of waste not, want not.  

Wiebler JM, Kohl KD, Lee RE, Costanzo JP. 2018 Urea hydrolysis by gut bacteria in a hibernating frog: evidence for urea-nitrogen recycling in Amphibia. Proceedings of the Royal Society B 285: 20180241. http://dx.doi.org/10.1098/rspb.2018.0241 

Frog Hearts and University Politics in 1950s London

When writing about an article in the famous series, New Biology, published by Penguin between 1945 and 1960, I was reminded of another influential article of the time and how its author came to feature—completely unintentionally—in a London academic promotion battle that turned into a loss to Britain and a win for Australia.

The article in New Biology in 1952 was on the working of the frog’s heart which has three chambers, two atria and just one ventricle into which both atria empty. George Eric Howard Foxon (1908-1982*), then Reader in Biology at Guy’s Hospital Medical School, outlined what he called The Old Story, dating from the mid-1800s, of how oxygenated blood from the lungs is preferentially diverted to the rest of the body after it (along with blood from the body) enters the single ventricle. According to this scheme, oxygen-rich and oxygen-poor blood do not mix to an great extent in the ventricle, and blood is pumped sequentially into the three major arteries: blood low in oxygen from the body is the first to leave for oxygenation in the lungs and skin; as the ventricle continues to contract, the next lot goes to the body generally while the blood remaining until the final squeeze of the ventricle, that rich in oxygen having arrived from the lungs, goes to the brain. Mixing of oxygen-rich and oxygen-poor from from the left and right atria was, it was argued, prevented by the trabeculae of the ventricle forming rough compartments.

Over the decades there had been doubt cast on this ‘old story’ notable amongst them the observation that there was no difference in time of movement of the blood into the three main arteries. In other words, the story of a sequential separation of flow into those arteries appeared to be wrong.

Foxon sat himself the task of trying determining if the blood from the right atrium was kept largely separate in the ventricle from that entering from the left atrium. He was following up work published in Paris in 1933 in which it had been found that particles from Indian-ink injected into the pulmonary vein were found in all three arteries; on other words there was mixing. Similar results, using starch grains, from the other side of the heart had also been reported.

This is Foxon's diagram of the frog's heart

Foxon used an X-ray opaque suspension of thorium dioxide to follow its passage through the heart by taking a series of X-ray photographs in rapid succession—pioneering technology in those days. While blood from the lungs via left atrium was found to fill the left side of the ventricle that from the body filled the right side. However, during ventricular contraction the movement was found to be so violent that all the blood was mixed together in the conus arteriosus, from which the major arteries (three on each side of the body) arise, such that there was no separation as the blood was forced into the three arteries.

But that story did not last. Later research using more modern technology showed that deoxygenated blood from the right atrium is directed preferentially to the lungs and skin through the pulmo-cutaneous artery. More and more evidence was produced to indicate Foxon was wrong; the ‘old story’ was right in essence if not in detail. It is that version of events—of partial separation of blood streams from the two atria during ventricular relaxation and contraction and flow through the conus arteriosus—which has entered the textbooks.

However, circulation within the heart and major arteries of the amphibian heart has continued to excite the interest of those exploring what happens in different environmental and physiological conditions, in diving and conditions where concentrations of oxygen in the immediate environment are low for example, as well as in several different species. Technological advances in measuring blood flow and pressures in small blood vessels have meant that physiological measurements can be done in conscious undisturbed animals, in the relatively large Cane Toad^† (Rhinella marina) for example. In Foxon’s day, ‘pithed’ frogs—those in which the brain has been destroyed—were used perforce. Thus physiological control mechanisms affecting pressures in the various blood vessels may have been disrupted compared with an intact animal in the wild.

The story does not end there. More recent research indicates that there are conditions in which there is a high degree of mixing of the two blood streams within the heart, as claimed by Foxon, and others in which there is a high degree of separation. One example will suffice: in the resting Cane Toad at 10°C mixing was 85% complete but at 30°C mixing was only 17%.

So having been dismissed as anomalous results, Foxon’s conclusions have been supported—in some conditions. The great mistake by a number of authors was to assume that what happens under particular experimental conditions happens under all, as an invariant mechanism for operation of the frog’s heart. Variation in the amount of blood directed to various parts of the body through the three major arteries and variations in mixing of the two streams of venous blood, provide a whole host of different tactics that can be be employed by an individual frog, or by different species, to enable them to adapt to the vicissitudes of life amphibian.

It is, it should be noted, misleading to state that blood arriving into the right side of the heart is, as in mammals, deoxygenated after passing through the tissues of the body. This is because some of it has passed through the skin and skin is an important site of oxygen uptake in amphibians. I have read studies in which the skin in resting animals accounts for a third of all oxygen uptake. The lungs provide a greater proportion during activity or if the oxygen content of the water or the water film over the skin falls. The implication is that blood from the right side of the heart recirculated through the body will still be supplying some oxygen to the tissues in frogs but not, of course, in mammals.

As an aside, a dangerous side effect of the study of ‘types’ as the main part of courses in biology was the impression created in the student that as one moved from the type fish to the type amphibian and so on to the type mammal, there was a progress from a primitive to an advanced organism. It was, therefore, easy to get the impression or to be told that the poor old frog having just three chambers in its heart was in some way inferior to the mammal or bird which had evolved four and had achieved the perfect double circulation, i.e. complete separation of blood streams through the body and through the lungs. Foxon was quick to dispel this line of thinking:

..the heart of the frog does not represent an unsuccessful attempt at the division of the heart into arterial and venous sides. It seems we must regard the frog not as any form of intermediate stage between fish and higher vertebrates but as an animal suited par excellence for a true amphibious made of life…

Foxon appeared in the biography A.J. ‘Jock’ Marshall (1911-1967), his opposite number at St Bartholomew’s Hospital School. Both schools fell under the aegis of the University of London. Foxon and Marshall were Readers and heads of their respective departments. The biology departments within the old medical school were, even by the standards of the day, very small with formally a lowly rôle; their teaching consisted of instilling a knowledge of elementary biology in very junior medical students who had taken on the study of medicine never having studied a biological subject at school. They had no honours students but could ,of course, do research and supervise postgraduate students. Through the latter route they could be promoted to Professor. Indeed Mr Foxon (he did not have a Ph.D. just like many British academics of the time) became Professor Foxon in 1955.

In 1957 Jock Marshall was put forward to the University by his medical school for promotion. Foxon encouraged Marshall, thinking the whole matter a formality for one so clearly qualified. But Marshall’s case was blocked by one external member and one internal member of the committee, as explained in detail by his widow here. Marshall was incandescent with anger at his treatment. The Cambridge mafia he blamed; the formerly supportive internal member was reliant on the external member for supporting his candidature for the Royal Society. The treatment Marshall received in London appears to have been the main reason for his looking for a job in his native Australia. He left to become the first Professor of Zoology at Monash University in Melbourne in 1960. However, he managed a parting shot at the internal member of the committee (who was elected FRS in 1958) who later approached him in a friendly manner: 'I told him to "Piss off you little bastard". He pissed off.’

Foxon, a Cambridge graduate who had previously worked in Glasgow University in the unenviable job of Assistant in Zoology (essentially the Professor’s dogsbody in a Scottish university) and at University College, Cardiff before, being appointed to Guy’s, continued his research on the heart and circulation in vertebrates.

Günther's Golden-backed Frog (Indosylvirana temporalis)
Sri Lanka, 2013
How was its heart working?

*Date of death is incorrect in the archives here

†The world seems to have adopted the Australian name for this South American species, introduced into the cane fields with a devastating impact on the wildlife. Until recently it was also and inaccurately called the Marine Toad (Bufo marinus).

Foxon GEH. 1952. The mode of action of the heart in the frog. New Biology 12, 113-126

The following can be referred to for some idea of the amount of work that has gone into determining how the frog heart and circulation work:

Gamperl AK, Milsom WK, Farrell AP, Wang T. 1999. Cardiorespiratory responses of the toad (Bufo marinus) to hypoxia at two different temperatures.

Graaf AR de. 1957. Investigations into the distribution of blood in the hear and aortic arches of Xenopus laevis (Daud.). Journal of Experimental Biology 34 143-172

Hedrick MS, Palioca WB, Hillman SS. 1999. Effects of temperature and physical activity of blood flow shunts and intracardiac mixing in the toad Bufo marinus. Physiological and Biochemical Zoology 72, 509-519

Hillman SS, Hedrick MS, Kohl ZF. 2014. Net cardiac shunts in anuran amphibians: physiology or physics? Journal of Experimental Biology 217, 2844-2847

Langille BL, Jones DR. 1977. Dynamics of blood flow through the hearts and arterial systems of anuran amphibians. Journal of Experimental Biology 68, 1-17

Pinder AW, Burggren WW. 1986. Ventilation and partitioning of oxygen uptake in the frog Rana pipiens: effects of hypoxia and activity. Journal of Experimental Biology 126, 453-468

Captive breeding of water frogs

There have been enormous advances since I started to keep amphibians sixty years ago. Recently, I was pleased to see further evidence of progress in a paper describing scientifically-based methods for the captive breeding of water frogs.

Even developing the methodology for common species to breed under controlled conditions gives a pretty good idea of where to start with the rescue of an endangered related species should that be necessary. In addition, of course, it tells you a lot about the physiological requirements and the conditions that trigger reproduction or, if conditions are not right, that inhibit it. 

This paper in the BHS’s Herpetological Bulletin by Christopher Michaels (now at the Zoological Society of London) and Kristofer Försäter working in England and Sweden describes the breeding of four species of water frog, Pelophylax. This is how they began the Discussion section of the paper:

Pelophylax sp. rely on well warmed, sunny areas of relatively still water with rafts of  floating vegetation and rarely stray far from water. They are heliophiles and actively bask, exposing themselves to the heat and UVB irradiation of direct sunlight (Michaels & Preziosi, 2013). Historically, indoors enclosures for amphibians were typically lacking in UVB provision and thermal gradients. With increasing understanding of amphibian lighting requirements and the availability of technology to meet them, indoors husbandry for water frogs is now much more easily achievable. Our captive enclosures were designed to recreate the UVB rich, brightly lit and warm environments inhabited by water frogs in nature and these conditions proved successful in maintaining and breeding this genus indoors.

Michaels CJ, Försäter K. 2017. Captive breeding of Pelophylax water frogs under controlled conditions indoors. Herpetological Bulletin  142, 29-34.

Dorothy Sladden (1907-1937): Ernest W. MacBride, Evolution and Eugenics. Part 2. Frogs’ eggs, sports and monstrosities

As well as for the inheritance of acquired characters, MacBride was also looking for environmental factors that caused mutations, in order, it seems, to provide an explanation for the findings of the Mendelian geneticists. The basis is explained by the opening paragraph of his article, The work of Tornier as affording a possible explanation of the causes of mutations, written in 1924 for Eugenics Review (15, 545-555):

Gustav
Tornier

In a previous communication to the Society I gave an outline of recent discoveries which tended to show that the effects of habits acquired during the lifetime of the individual were transmitted to posterity. Since that time more and more evidence pointing in the same direction has come in, and finally the recent experiments of Pavlov may be regarded as decisive on the question. The inheritance of environmental effect is therefore a vera causa, and has probably been the chief, if not the only cause, of the evolution of the plant and animal kingdoms, but it is certainly not the only cause of variation. The conspicuous deviations from the normal, known as sports or monstrosities, which appear suddenly, which are inherited strongly, and which "mendelize" when crossed with the type, still demand an explanation of the cause of their occurrence. It is on characters such as these that the breeder seizes when he wishes to produce a new strain; and it is the offspring of similar "sports" in the human race that fill the slums of our great cities.

     To say that these sports owe their origin to "mutations" in the chromosome-complex of the type is merely playing with the question; it is, in the words of Darwin, to substitute a form of words for an explanation. Granted that if the hereditary potentiality of a stock is changed, the nuclei of the germ cells have undergone a change; to assert this is merely to push the difficulty one step further back. What has changed the nuclei? Till we can answer that question all talk of an explanation of mutations is futile.

     …In view of these facts it occurred to Gustav Tornier [1858-1938; he worked in Berlin] that there must be some general underlying cause for the production of these abnormalities. It seemed to him futile to attribute each one to the separate appearance by chance of a '*factor." He was thus led to formulate what Milewski calls an “epoch-making theory” to the confirmation of which Milewski has himself largely contributed. Like the Mendelian theory itself the Tornierian hypothesis did not at first attract the notice to which it was entitled. This was due to two causes—first because the theory conflicts with all preconceived ideas as to the origin of mutations, and secondly because Tornier has published his results for the most part in journals which have not a sufficiently wide circulation to reach zoologists in general. Tornier's theory is as follows:—Every embryo is endowed at the beginning of its existence with a certain quantity of protoplasmic energy. This energy manifests itself in two ways: (1) by the early beginning and vigorous character of the movements of the embryo; (2) by the ability of the embryo to resist the tendency of all its tissues, especially those of a less active growth, to absorb an excess of water—in a word to regulate the intake of water. In practically all eggs the portion of less active growth is that in which the yolk globules are stored, and hence Tornier speaks rather too loosely of an absorption of water by "the yolk" and its consequent swelling. The swelling is not confined to yolk-containing cells, nor strictly speaking is it the yolk itself which swells, since this consists chiefly of globules of a lecithin-like substance insoluble in water. The very same swelling takes place in mammalian embryos in which there is no yolk. Davenport and Parker showed long ago that a large part of the increase in bulk which embryos undergo during the earlier period of their development is due to the imbibition of water.

     Now according to Tornier, when an embryo is exposed in the earliest period of existence to scarcity of oxygen, the protoplasm becomes weakened and is not able to prevent the overswelling of the less active portions of the body by water. This abnormal swelling— since the embryo is confined within a relatively inextensible membrane—leads to pressure on the actively growing parts which impedes their growth, distorts their shape, and in extreme cases, prevents their growth altogether. If we examine in detail its effects in goldfish embryos we find that all the natural cavities of the body, such as the buccal cavity, the gill cavity (beneath the operculum), and the body cavity become enlarged beyond their natural sizes, whilst strong pressure is exerted by the swollen yolk-sac against the growing embryo in an antero-posterior direction, so as to impede the natural tendency of the embryo to grow in length.

     …The interest in all studies of heredity to the Eugenics Education Society is, of course, their   applicability to human conditions.

     Tornier's work suggests strongly that bad conditions during the period of conception and the early phases of development may be the original cause of the degenerative mutants in man, including under that head not only mental defect, but haemophilia, night-blindness, colour-blindness, &c. But the important fact is that, however caused, the plasma-weakness is handed on to posterity. It is the object of negative eugenics to prevent these plasma-weak stocks from adding their quota to the race, by discouraging their reproduction. In the future we may hope that eugenics, in conjunction with medical research, may perhaps detect and isolate the specific causes of plasma-weakness, and thus diminish, if not entirely prevent, the appearance of plasma-weakness in the race.

It is difficult, when looked at from 2015, to believe that this was written by anything other than a crank. It is also difficult to believe, again when looked at from 2015, that the work in the first two papers, on the development of frog embryos after an environmental insult, were other than fanciful and difficult to not believe that they were thought as fanciful at the time by those not involved directly with MacBride. The introduction to Dorothy Sladden’s first paper summarises the thoughts of Tornier as expressed in MacBride’s article and explains the line of reasoning for the experiments she did:

In 1908, Tornier published a paper on the probable causes of the formation of the abnormal "fancy" races of goldfish. All these races originated in China where the wild ancestor (Carausius [sic] auratus*) still abounds in the streams, It had been supposed that these races were produced by a secret process known only to the Chinese breeders. The fish during winter were kept crowded in earthenware pots, on shelves in dark and insanitary huts; in summer they were transferred to small and filthy tanks overgrown with weeds. In these tanks they spawned and much of the spawn perished; amongst the fraction which survived, however, all sorts of abnormalities were found. By selecting the most striking of these, the breeders secured the parents of their “fancy" breeds, which showed in every succeeding generation a strong tendency to revert to the normal; only by the most rigid selection was anything like a "pure" race obtained.

    Tomier drew the conclusion that, the abnormalities were due to the effects of lack of oxygen in very early stages of development. This lack induced what he called "plasma-weakness" in different parts of the formative area of the very young embryo. In consequence of these localised areas of weakness the protoplasmic part of the egg was liable to mechanical distortion, induced by abnormal pressure from the yolk ; the latter absorbed water and swelled, thus crushing and destroying the protoplasmic structures from which the future organs of the adult arc formed.

The practicalities of the initial experiments involved keeping frog eggs in a 10% sucrose solution for four hours and seeing how the eggs and tadpoles developed. the sucrose solution was used in the belief—as in Tornier’s experiments outlined in MacBride’s article—that it would reduce the concentration of oxygen in the water sufficiently to affect the developing embryo. This is the summary of that paper:

Eggs of Rana temporaria were exposed, at the end of segmentation, 24 hours after fertilisation, to a 10 per cent, solution of sugar in tap-water for 4 hours; they were then transferred to normal aerated water. The resulting larvae exhibited marked structural abnormalities, although these might not be obvious for a prolonged period, e.g., 3 to 4 months after fertilisation. These abnormalities have, been described as (a) distention of body-cavity; (b) rupture of gut and extrusion of yolk; (c) flexure of tail; (d) distortion of sacral region; (e) non-appearance of limb.

However, in the second paper it was realised that the effect of immersion in sucrose was probably caused by an osmotic effect rather than lack of oxygen (I calculate the oxygen concentration in the water would have been reduced by only 9%):

Summarising these somewhat inconclusive results, it would seem that the effect of sugar is not the removal of oxygen as previously assumed, but very possibly osmotic action and is responsible for the early abnormalities in particular, while pH decrease may be responsible for the later ones…It now seems possible, however, that overcrowding may have seriously affected all the previous results.

The experiments show nothing other than when frogs’ eggs are damaged, the tadpoles do not develop normally, although I am sure that MacBride really fancied the notion that dirty conditions lead to ‘mutations’ (as proposed by Tornier for the genesis of the domestic strains of fancy goldfish) which are then embedded forever in the working class unless expunged by negative eugenics, and that is the reason why the newly-graduated Miss Sladden was given the project.

Dorothy Sladden’s skills in rearing frogs through metamorphosis are evident and were obviously put to good use by others in in the department. For example, Peter Gray in a paper on development of the development of the amphibian kidney also published, like her first paper, in 1930, thanks her profusely: ‘indeed, it is to her skill in the rearing of animals under artificial conditions that I am indebted for the greater part of my material.”

The studies described here have nothing of importance that I can see. By contrast the next set of experiments she did produced biologically significant results and should be seen in a different light to those on frogs' eggs. I will deal with them in my next post, Part 3.

View image | gettyimages.com

The two papers are:

Sladden, D.E. 1930. Experimental distortion of development in amphibian tadpoles. Proceedings of the Royal Society B 106, 318-325

Sladden, D.E. 1932. Experimental distortion of development in amphibian tadpoles. Part II. Proceedings of the Royal Society B 112, 1-12

*She was confusing the name of the goldfish (Carassius auratus) with the organism she wroekd on next, the stick insect (Carausius morosus)

Frog for lunch? Which species?

A correspondent in China sent me a photograph taken outside a restaurant with skinned frogs displayed. I would guess these are the Chinese Edible Frog or Bullfrog (Hoplobatrachus rugulosus previously Rana rugulosa). This species is eaten widely and is farmed for the table as well as taken from the wild. Baskets full of live frogs used to abound in Central Market in Hong Kong.

Brits note the uncanny resemblance of skinned frogs to the human body and seem more than willing to leave the frog on the rack rather than have it on the plate. However, physiologists will also note the basis for good sciatic-gastrocnemius preparations and those of a certain age will remember the smoked drum, the kymograph, the varnish and the blackened fingers.