Zoology has a discipline: evolution; zoology is vertically integrated, concerned with biological organisation at the level of organisms in their environment, organs, tissues, cells and molecules. This blog meanders through the animal kingdom, from aardvarks and anoles, through mouse and man, to zorillas and zebras.
Sometimes more and more information comes to light on incidents and people I have written about or drawn attention to previously. One I found recently throws light on an event in 1957 that shocked academics in Britain and Australia (22 June 2019 Frog Hearts and University Politics in 1950s London).
Academic zoological circles in London were much quieter in the 1960s than in the 1950s. Alan John "Jock" Marshall had taken umbrage at his treatment by the University of London and had left to return to Australia and become the first Professor of Zoology at Monash University. A self-confessed larrikin in his younger days, the word most usually used to describe him to me was ‘wild’. I only knew of his reputation because he died in 1967, aged 56.
Jock Marshall was graded Reader in the university; he was head of the very small zoology department at St Bartholomew’s Hospital Medical School. In 1957 Marshall was put forward to the University by his medical school for promotion. But Marshall’s promotion to Professor was blocked by one external member and one internal member of the university committee, as explained in detail by his widow (who edited his autobiographical notes) here. Marshall was incandescent with rage at his treatment and the account of the meeting given to him afterwards by the dean of the medical school who was present. The Cambridge mafia he blamed; the formerly supportive internal member was not only a bosom friend but also reliant on the external member for supporting his candidature for the Royal Society. Marshall before his departure managed a parting shot at the internal member of the committee (elected FRS in 1958) who approached him at a meeting of the Zoological Club in a friendly manner: 'I told him to "Piss off you little bastard". He pissed off.’
Those who blocked Marshall’s promotion were the external Carl Frederick Abel Pantin FRS (1899–1967) of Cambridge and the internal James Eric Smith (1909-1990) of Queen Mary College, the one elected to the Royal Society in 1958. Marshall’s strong supporter on the committee was Alastair Graham (FRS 1979) of Reading who also pointed out that Marshall’s research record was much stronger than that of another head of a zoology department in a London medical school who had already been promoted. It is interesting to note that Pantin, although elected to the Royal Society in 1937 was, like Marshall, a reader until his own elevation to the professoriate in 1959.
Reading about Pantin and Smith it is difficult to reconcile this description of Smith in his biographical memoir for the Royal Society, ‘Like Carl Pantin he was liked by all who came in contact with him for the same reason that both exuded a warm humanity’, with the goings-on at the promotion committee. The former though, it was alleged, did have something of a reputation for damning non-Cambridge candidates for jobs. Marshall’s supporters in London—and he had many of great distinction—considered the whole affair, in the words of J.Z. Young, ‘utterly disgraceful’.
Jock Marshall had already made his name in studying birds in the field and in the lab to examine fundamental biological questions, like, for example, what controls the onset of a breeding season in the tropics. His research and books were very well known and many like his 1954 classic on Bower-birds* which were well received at the time have come to be regarded as classics, linking the breeding behaviour of these remarkable birds to changes in the endocrine system. That some of his books, as on his travels in New Guinea and birds-of-paradise, were popular may though have counted against him in the weird world of academic snobbery prevalent in that era.
Marshall’s life had been spent studying birds and I can only imagine his reaction to what I found in Smith’s biographical memoir:
Eric [Smith] later used to remark that he and Pantin were perhaps the only professors of zoology who would have had difficulty in identifying a blackbird when they went on walks, as both were invertebrate zoologists and had not been bird watchers when young.
We can now only imagine Jock Marshall’s reaction. Incandescent would not get anywhere near it.
Sadly, I think Smith and Pantin were wrong about Jock Marshall and about professors of zoology of the time. I suspect that, with a few notable exceptions, relatively few would have had any knowledge of birds or their natural history—rather like, I am told, modern students of biology in British universities.
*Bower-birds, their displays and breeding cycles. A preliminary statement. Oxford: Clarendon Press, 1954.
A pair of Woodpigeons (Columba palumbus) provide endless interest and entertainment in our garden. The male, instantly recognisable by a deformation which results in some feathers on his upper back pointing outwards, is fiercely territorial seeing off all comers with, if necessary, wing-to-wing combat. He, surprisingly, was the offspring of the resident pair a couple of years ago. The young sometimes fall prey to the local Sparrowhawks (Accipiter nisus) (two dead in the garden so far this year).
We have four species of columbiform within walking distance. Apart from the ubiquitous Woodpigeon, there are Stock Doves (Columba oenas) in a nearby park, Collared Doves (Streptopelia decaocto) in gardens (although now less common than, say, 20 years ago) and small flocks of Rock Doves (Columba livia) on the coastal cliffs which do appear to be the wild form rather than the feral pigeons which occur in towns and country.
It was while watching the Woodpigeons doing their regular, almost constant, patrol under the bird feeders in the hope of dropped sunflower hearts, that I began to get itchy feet and think of when, with covid still in full flow in most parts of the world, we might hope to see a pigeon or dove other than in deepest Ayrshire. Columbiforms have been highly successful in reaching virtually all parts of the world and we have watched them in very different circumstances and locations—from single birds on a number of South Pacific islands to vast flocks of African Green Pigeons (Treron calvus) in the Republic of Congo. Most of my photography is video but here are a couple of photographs of pigeons we have seen in vastly different habitats and with vastly different ranges: from Bhutan in 2016, Snow Pigeons (Columba leuconota), which have a wide distribution around the Himalayas; from 2010, a Henderson Island Fruit Dove (Ptilinopus insularis) endemic to the scrub forest of that 47 square-kilometre patch of raised coral reef in the Pacific.
Pigeons usually featured in my lectures to undergraduates on lactation since they feed their young ‘milk’ produced by the lining of crop. The mechanism of production, sloughing off whole cells, differs from mammalian milk secretion but the major hormonal control is the same. Oscar Riddle (1877-1968) not only discovered the pituitary hormone which he named prolactin but also the fact that it stimulated milk secretion by the mammary gland and the pigeon crop. As a result of this discovery in 1932, the original way to assay mammalian prolactin was by using the response of the pigeon crop sac to pituitary extracts and blood samples. In its various formats, the pigeon crop sac assay was used in physiology and medicine for 40 years until the early 1970s when it was superseded by radioimmunoassay. Kindly remember that when you next see a pigeon or dove, be it a feral pigeon scouring an inner city or a fruit dove on an exotic atoll. There is more to a pigeon than meets the eye.
Snow Pigeons, Bhutan 2016
Henderson Island Fruit Dove, 2010 Photograph by Michael Moore of the Noble Caledonia Expedition Team
The covid war has had a galvanising effect on communications. Not only can lectures be seen remotely with a degree of interaction with the speakers but the whole recording can be watched later on YouTube. The Zoological Society of London has been broadcasting its scientific meetings and I particularly enjoyed the recent ‘Why do eggs fail?’. The details are here and the YouTube video is below:
The speakers illustrate the way in which research findings on the fertilization of eggs and on evolutionary trade-offs are being combined with information from the wild to address a problem in practical conservation.
When writing recently about ‘Amo’ (Emmanuel Ciprian Amoroso FRS, 1901-1982) and his interest in amphibians I remembered that that he had been involved in making a film about the Marsupial Frog, Gastrotheca sp, which he had kept and studied at the Royal Veterinary College in the 1950s. Amo’s life’s work was studying viviparity, particularly the mammalian placenta. The question was to what extent the mother provides oxygen and nutrients to the young of other vertebrates that are born alive or may, as in amphibians be stored in the body until they are released as tadpoles or young frogs. He was interested in this species because the eggs after fertilization are manoeuvred by the male into the mouth, just above the cloaca, of a backward-facing pouch on the back of the female. The eggs hatch there and the tadpoles grow are released into water at a stage shortly before development of the hind legs.
Amo was very proud of the film. Although the phenomenon of storing eggs in the pouch in these South American frogs was well documented, it is believed this was the first time the whole process had been observed and filmed. I then wondered if the film still existed and where it had been shown.
The first clue was the list of publications in Roger Short’s biographical memoir on Amo. There it is described as ‘1957 Reproductive phenomena in Gastrotheca marsupiata. R. Soc. Conversazione (23 May)’. Notes and Records of the Royal Society (12, 154-159, 1957) provides more information:
The second film was shown by Professor E.C. Amoroso, F.R.S., and Miss J.H Austin, of the Royal Veterinary College, and Dr. J.F.D. Frazer of Charing Cross Hospital, and was concerned with the reproduction methods of the frog, Gastrotheca marsupiatum, which is partially independent of water for its reproductive processes.
I then found that the film had been shown earlier, at a meeting of the Physiological Society at the Royal Veterinary College on 14-15 December 1956 (Journal of Physiology 135, 38P, 1957). There was no other information other than the title and ‘authors’ since the presenters could choose to have their contribution recorded ‘by title only’. The authors were shown as EC Amoroso, J Austin, A Goffin and E Langford. From the acknowledgements I have found that A.R. Goffin was a technician in Amo’s department of Physiology (he is thanked for taking photographs). Miss J.H. Austin in 1960-61 was then on the staff of the department of anatomy, possibly as a lecturer, at the RVC where she is listed in Scientific Research in British Universities and working on ‘development of gastrotheca’. She was not in the 1962-63 volume and I can find no further information on her or on E Langford.
Eventually I found that the film still exists. It is in the collection of the British Film Institute under the title 'Natural history of the large South American pouched tree frog Gastrotheca [originally listed as Gastortheca] marsupiatum'. BFI Identifier 21716. The entry in the catalogue indicates the original was a 16 mm silent film of a surprisingly long 1500 feet, i.e. 42 minute running time. It is dated as 1955 with a ‘release date’ of 1954 which is unlikely. There is no indication in the BFI catalogue of who made the film or where it was obtained from.
Given the costs of licensing from the BFI I cannot see it ever being digitised or being made available for viewing.
There are lots of videos of Gastrotheca frogs on YouTube but I have not found one that shows the life cycle. In that important respect it seems that Amo’s film is so far unique. Beware though that in searching for ‘marsupial frog’ the best video is one from a BBC series which shows an Australian species, Assa darlingtoni, that has ‘hip pickets’ for its eggs, not a dorsal sac, and is unrelated to Gastrotheca species from South America.
A Publication
In 1957 Amo’s friend and erstwhile collaborator on reproduction and lactation in the Grey Seal (of which stories of their adventures were often related), Leo Harrison Matthews FRS (1901-1986) then Scientific Director at the Zoological Society of London, published a paper in Bulletin de la Société de France entitled, ‘Viviparity in Gastrotheca (Amphibia, Anura) and some considerations on the evolution of viviparity’. I have not read the paper (it falls into that category of too young to be in online archives and too old to be digital) but it clearly covered the same ground and probably the same frogs as the film. It is the only written record covering the breeding of these frogs in London around 1955-56 and has been referred to in later papers in relation to the identity of the frogs (see below).
Amo referred to his work on Gastrotheca in several of his reviews and conference papers but not in a full paper. In 1959 a photograph appeared in the published version of a talk he had given at a Josiah Macy Jr symposium on gestation in Princeton in 1958—again a paper I have been unable to find online. That was reproduced—and is shown below—in another paper, ‘The evolution of viviparity’, published in Proceedings of the Royal Society of Medicine in 1968.
Amo argued that because the eggs of Gastrotheca contain plenty of yolk, the developing young receive oxygen but little if any additional food from the parent. That may be the case in the species he studied but in another, Gastrotheca excubitor, which retains the young throughout the tadpole stage and in which froglets emerge from the pouch, recent evidence suggests the possible transfer of nutrients from the mother to the young through the wall of the pouch. Definitive evidence, though, is still lacking.
Origin of the marsupial frogs in London
Where did Amo get his frogs? Was it via Deryk Frazer who appeared as presenting the film at the Royal Society along with Amo and Austin? A clue exists in an article written by Bob Bustard for The Aquarist in 1958; he wrote:
…An example of this is Gastrotheca marsupiatum, appropriately called the pouched tree frog. I imported this frog into Britain for the first time in 1955, and it has become remarkably popular among vivarium keepers already. This is possibly because it does so well at about 65°F and will breed readily in a small indoor vivarium.
My bet is that the frogs at the Royal Veterinary College came directly or indirectly from Bob Bustard.
A paper also appeared in British Journal of Herpetology describing the breeding in captivity of ‘Gastrotheca marsupiatum’ in 1957 by a J. Walker. I have not seen this paper but my guess it describes animals obtained from Bob Bustard also.
Marsupial frogs were kept in UK throughout the 1980s and early 1990s at least. I do not know if they were newly imported or had been bred in captivity, or indeed were descendants of those imported by Bob Bustard in 1955. Gillett records captive-bred individuals for sale around 1957 and I knew of somebody with a pair in the early 1990s
Which species of frog?
The frogs kept and bred in Britain and in continental Europe in the 1950s and later were all called Gastrotheca marsupiatum or Gastrotheca marsupiata. Before moving on it is worth noting that the currently accepted name is G. marsupiata was that coined by Duméril and Bibron in 1841. The same species was called Nototrema marsupiatum by Günther in 1859. G. marsupiatum appears to have been mistakenly used as Gastrotheca came to replace Nototrema; there may also have been a little discussion over the gender of the specific name matching that of the genus.
But that is by the by. During the 1970s doubt was thrown on the identity of the frogs studied in the 1950s and beyond as G. marsupiata, notably by Eugenia M del Pino in 1975 and by William Duellman and Scott Maness in 1980. The latter listed a number of papers on the reproductive habits of, supposedly, G. marsupiata. That included the paper by Harrison Matthews and, therefore, by implication those in Amo’s film, although by then awareness of the existence of the film seems to have been lost. ‘All those works in which the origin of the specimens was given cite Quito, Ecuador’, Duellman and Scott wrote, continuing, ‘G. marsupiata does not occur north of Central Peru; the Ecuadorian frogs formerly associated with that species are G. riobambae’. As a result of this statement, the animals in captivity became to be referred to as G. riobambae. That view still obtains: captive-bred animals in the USA, Britain and continental Europe are listed as Gastrotheca riobambae—the Andean Marsupial Frog.
What I do not know is where the frogs Amo used for his film were originally imported from and if Duellman was correct in assuming that the frogs in captivity around and after that time were G. riobambae rather than G. marsupiata.
This is the first photograph of a marsupial frog I remember seeing. It is from Doris Cochran's Living Amphibians of the World, published in 1961 by Hamish Hamilton. The photographer was the American, John H. Tashjian. Also in that volume were black-and-white photographs by Wilhelm Hoppe. All are captioned G. marsupiata. Are the photographs of G. marsupiata or G. riobambae?
This discussion serves to illustrate the value of ‘voucher specimens’ in research and captive-breeding projects. The same consideration applies to photographs since it is very difficult to know whether the animals has been correctly identified, especially those in captivity. With preserved specimens there would be no doubt about which species was being studied or bred. Does anybody have any preserved specimens from these early days of breeding marsupial frogs?
I will return to marsupial frogs in a future article, because their reproductive biology is so fascinating and because their storage of eggs and tadpoles presents physiological challenges that require further study, not the least of which is how the female’s own young avoid the fate of being rejected as non-self while being held in intimate contact with the mother’s body, a biological problem that haunted Amo and one which drove his interest not only in mammals but in reptiles, amphibians and fish.
I found this photograph of the meeting at which Amo (2nd right, front row) talked about marsupial frogs. It is from the website on placentation by the late Kurt Benirschke (1924-2018)
For those seeking further information:
Amoroso EC. 1968. The evolution of viviparity. Proceedings of the Royal Society of Medicine 61, 1188-1200.
Bustard HR. 1958. Tree frogs. Aquarist and Pondkeeper 23 (4, July 1958), 81-82.
Duellman WE, Maness SJ. 1980. The reproductive behavior of some hylid marsupial frogs. Journal of Herpetology 14, 213-222.
Gillett L. 1995. The good old/bad old days. A survey of reptile and amphibian species traded during the period 1948-1957. British Herpetological Society Bulletin 1995 (52), 26-29.
Kirk BR. 1985. Observations on the breeding of the marsupial frog, Gastrotheca marsupiata. British Herpetological Society Bulletin 1985 (14), 22-24.
Matthews LH. 1957. Viviparity in Gastrotheca (Amphibia, Anura) and some considerations on the evolution of viviparity. Bulletin de la Société de France 82, 317-320.
Pino EM del, Galarza ML, Albuja CM de, Humphries AA. 1975. The maternal pouch and development in the marsupial frog Gastrotheca riobambae (Fowler). Biological Bulletin 149, 480-491.
Warne RW, Catenazzi A. 2016 Pouch brooding marsupial frogs transfer nutrients to developing embryos. Biology Letters 12: 20160673. http://dx.doi.org/10.1098/rsbl.2016.0673
I am ending this series of articles on Maxwell Savage by discussing one small part of his research on the life of the Common Frog, Rana temporaria, in Britain: frog spawn.
Common Frogs lay spawn in clumps, not as single strands of eggs like the Common Toad, Bufo bufo. Savage realised that because the swollen jelly surrounding the egg is 99% water, the mass of spawn acts as a heat store for the developing embryos within. Unlike water in the pond, the water in the jelly is held and thus constitutes ‘a relatively enormous mass of solidified water’. Therefore, in sunlight the mass of spawn warms up. At night, because the warmed ‘solidified water’ cannot go anywhere, the only way for heat to be lost is by conduction and since water is a very poor conductor the heat is retained. Savage did experiments at home and in the field. He showed that in 63 cases out of 73, the spawn was warmer than the surrounding water. The average difference 0.63°C, not a big difference Savage noted, but one which could have given an important advantage to repopulation after the last Ice Age.
He also investigated the structure of the mass of spawn itself. It is not an amorphous mass but structured in a way ‘which may be likened to a bunch of grapes glued together only where they touch’. Therefore, water circulates freely within the mass of spawn and oxygen for respiration only has to pass across the jelly the distance of one half of the diameter of the individual egg. He demonstrated this structure by dropping Indian ink on a freely floating mass; the ink passed through in a very short time.
The corollary of this demonstration of the structure of the spawn mass is that if a mass of frog spawn is laid or later held in water of insufficient depth the inner channels are blocked by collapse of the mass.
Savage also became involved in studying the events that lead to the ovum being surrounded by coats of jelly on its passage through the oviduct, which swell on contact with pond water after fertilization. I will not dwell on the aspect he examined—the inner gel that surrounds the ovum—but it is of historical interest and possible physiological importance. Savage wrote:
By a coincidence, Dr. Burgess Barnett and I shared a common interest in two unrelated scientific fields—the study of amphibia and of blood-clotting. In May 1939, Dr. Barnett, who had just left the Zoological Society of London, where he had been the Curator of Reptiles, for a similar post at Rangoon, wrote to say that he had ‘‘stumbled upon* a curious property of the frog’s egg. The jelly contained a clotting factor which he likened to the prothrombin of blood. He invited me to collaborate m its investigation, and when I accepted he sent me the only notes that he had. They consisted of some tests on normal and on haemophilic blood. In 1939, the subject of blood-coagulation, although it already had a large literature, had not grown to the enormous extent that it has now assumed, and in the light of more recent knowledge, it is clear that the factor he had discovered was not prothrombin, but a variety of thromboplastin [thrombokinase]. Thromboplastins are rather widely distributed in nature, sometimes occurring in unexpected places, and if this had been the only point, there would have been only a mild interest in the fact. From Dr. Barnett’s letters it seems clear that he had not any idea of the possible function of the factor, and very tentatively he suggested that it might be protective…Before we reached the point of actually collaborating, Dr. Barnett died. There the matter remained, until in planning this chapter I realized the probable function of the factor and the part it may play in the ecology of the frog and very likely in the lives of other species as well.
Savage’s experimental evidence suggested that some form of clotting process was involved in the solidification of the inner layer of jelly. I have found no particularly relevant further research but for several descriptions of various numbers of layers of jelly in different species and some of their molecular components. I will leave the last word on this topic to Savage:
This rather complicated story of the formation of the jelly envelope may, then, be summarized as follows. The vitelli [ova] pass into the oviduct. There they form the nuclei round which collects the insoluble product of a clotting system, in which a factor resembling the thromboplastin of blood contributes to a transformation that has something in common with the similar transformation that occurs when blood clots. The protein is not the same, and the rest of the process probably differs considerably. This protein layer is fairly concentrated. When it has formed, the eggs pass into the ovisac, where they become distributed in a fairly concentrated solution of a different protein. When they are ejected by the frog, the mass passes into water, salt is removed and the protein precipitates, forming the second and outer layer of jelly. Both layers now imbibe water, and in twenty-four hours or so achieve maximum size. In this process, the outer layer is stretched by the swelling of the inner layer, and gives way except at the points of contact of the spheres, where there is no stress. The final result is that a mass of adherent spheres is formed, with channels in between. The final percentage of structural protein in a blood-clot and in frog-jelly is about the same, but the clot and the jelly approach this concentration from opposite directions: the jelly swells, but the blood-clot is formed full size at once and, indeed, tends to shrink.
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This is the last in my series on Maxwell Savage, the forgotten man of herpetology in Britain. After searching for information on Savage and producing a short biography Trevor Beebee wrote:
“Ecology and Life History of the Common Frog” is a seminal achievement, bringing all his earlier work together in one book as a very readable monograph. The research is all the more remarkable because it was carried out in his own time, with no external funding – just his own energy and enthusiasm. Among other things, the book also hints at how dramatically frogs declined in the countryside between the 1930s/1940s, when he found ponds with thousands of spawn clumps, and the late 1950s, when only a few of his old study ponds had any spawn at all.
I place Savage at the pinnacle of the small band of people in Britain who have investigated the lives of amphibians and reptiles. He asked simple, important questions about the life of the Common Frog at all stages of his life history which he attempted to answer by any means necessary. Ronald Henry Maxwell Savage cast light. Every time we see a frog we should remember him, his research, and encourage others to take up the challenge of testing his hypotheses and thereby illuminate still further the life of amphibians and how they work.
Maxwell Savage argued that not only does the smell of algae attract Common Frogs to ponds but that a chemical from algae actually initiates spawning. However, the Common Frog is not a convenient species in the wild or in captivity to test that hypothesis and so in the 1960s he shifted his entire effort to Xenopus laevis which he kept at home in banks of linked aquarium tanks.
Xenopus laevis By Brian Gatwicke on Flickr
There is, of course, no reason to suppose that the demonstration of an effect of algae on the highly aquatic Xenopus would indicate that a similar mechanism was at play in Rana temporaria only that if it happened in one species the possibility that it could apply to another would have to be entertained. Breeding in Xenopus is very different from the annual spawning of Rana temporaria. In warmer parts of Africa, spawn may be produced all-year round, or during the whole breeding season in South Africa.
Savage published two papers on Xenopus, his last papers on any subject. The first, a letter to Nature, was in 1965; the second, in Proceedings of the Zoological Society of London, in 1971. Sadly, it appears that both were poorly refereed and edited since they both suffer from the defects noted by reviewers of his 1961 book: immense detail on some matters; scant detail on others. This is a great pity because it makes drawing conclusions difficult for those reading his papers 50 years later. I suspect the problem is that, as good a scientist as Savage was, he never went through that scientific writing apprenticeship experienced by those employed in research institutes and universities. That may be one of the reasons his work has been set aside or ignored by later workers.
His experimental set up at home, first in Hadley Wood and then, after he retired, at Welwyn, was designed to hold pairs of frogs in different compartments through which water was recirculated. Some compartments were lit; others were kept dark. He noted that hornwort, Ceratophyllum, and filamentous algae grew in the lighted compartments but there was never a bloom of free-living algae. There were a number of complications in the set-ups and management the two houses which make interpretation of the results more difficult. For example, water was partially changed for fresh well water at the same time as pairs were moved to different compartments; the temperature regime at the two houses was different; pairs spawned sometimes without apparent stimulus. Some compartments suffered low dissolved oxygen concentrations and low numbers of spawning occasions. This reliance on dissolved oxygen in the water was explained by the fact that the male in amplexus is kept with its head under the water and needs to rely on oxygen uptake through the skin. With insufficient oxygen in the water the male frog either drowns or breaks his grip and breathe air. Not surprisingly most males, but not all, chose to live and fertilise spawn another day.
Savage went to great lengths to ensure the randomisation of pairs of animals in the various compartments of his tanks, with each pair being reallocated a different compartment each week or, later, 10 days, according to random number tables. He also initially relied on the presence of spawn to test whether and external substance had worked or not. Then he realised that changes in behaviour may occur if not spawning itself, particularly in males; wach evening he scored each male ranging from 0 (no activity) up to full amplexus (4).
In his Nature paper there appears to have been no hornwort or other ‘higher’ plant present initially. He then compared the effect of adding ‘weed from natural ponds’ to compartments upstream of the ones in containing the animals. The nature of the ‘weed’ was undefined. One half of each bank of compartments was brightly illuminated; the other was covered with a dark cloth. Savage thus had four treatments: no weed, dark; no weed, light; +weed, dark; +weed, light. Spawning’ was attributed to weed when it occurred within four days from the addition’. These were his results:
Savage interpreted these findings as showing that a substance produced by algae, part of the ‘weed’ from natural ponds stimulated spawning. Assuming that the ‘weed’ was some ‘higher’ water plant like hornwort and not just a mass of filamentous algae, he does not appear to have considered the ‘higher’ plant could have been the source of a stimulatory material. His next step was to add algal monocultures to see if they induced spawning. He used a variety of cultures and treatments of the culture including, for example, two species of Chlamydomonas, an unidentified species found in ponds in which frogs spawn, culture medium sterilized three weeks earlier to kill the algae.
Savage bulked all these treatments which in the table of results he called ‘weed’ and compared them with untreated ‘no weed’ frogs. Observations were made each day for a total of 157 days on six pairs of frogs (randomised as described above). He counted each day as a trial, giving a grand total of 943 (157 x 6) trials. For ‘weed’ (i.e algal culture added) days he multiplied the number of additions of ‘weed’ by 6 and by 4, the number of days after treatment a positive score could be recorded. That product came to 312, and the number of trials on ‘no-weed’ days was, by difference, 630. Days when spawn was observed were 18 for ‘weed’ and 10 for ‘no-weed’ indicating an approximately 4-fold effect of adding algal cultures on the incidence of spawning. From the constructed 2 x 2 table, he obtained a P value of <0.005,
In Savage’s 1971 paper a similar regime was followed but with 8 pairs of animals and 10-day periods between water changes and pairs being allocated to a different compartment. Again he bulked results from all the preparations of algae, filtrates, concentrates and pure chemical (see below) and compared them with frogs not exposed to the additions on day 5 of the 10-day period. The incidence of spawning in the treated frogs was approximately 2.5-fold higher (eggs produced on 76 of 2120 days with the addition of algal preparations vs eggs on 47 of 3192 days)
The calculated effect was likely to have been be an underestimate, as Savage explained:
…(1) The same female does not spawn every night of the stimulus period. Often, other females spawn when she does not. Although, clearly, there was activity all this time, the total number of nights when some of the females do not spawn contribute to the cell “stimulus-no eggs”. (2) There was no way of knowing whether the substance in any particular experiment was active. Some preparations were probably not active, and contributed to the same cell as in (1).(3) The dissolved oxygen effect [see above]…had not been discovered in this period, although it was known that sections 1 and 2 were relatively ineffective. This again distorts the Table.
He went on:
The results are unquestionably significant, but not sharp, in the sense that the additions of substances did not always produce spawning in every female every night.
After getting these results he went on to make an attempt at finding out what the chemical was that was stimulating spawning. For all of his work on Xenopus he had turned himself into an algologist, culturing various species for long periods. He also used his chemical knowledge to deduce from the various extraction, dfistillation and analytical procedures the possible nature and identity of the molecule(s) involved.
He knew, for example, that some freshwater algae produce steroids as well as metabolites which they release into the water. One of the latter is glycollic acid (or glycolic acid) which is secreted during photosynthesis and then taken up again at night by the same or other algae. He filtered an algal culture, acidified it to pH 5 and distilled the solution under reduced pressure. Both filtrate and the residue were active in stimulating spawning. After testing with various reagents he realised he had isolated glycollic acid. Therefore, he tested pure glycollic acid (it is used in cosmetic preparations as a skin exfoliant) to see if, like the algae and extracts, it would stimulate spawning. He used spawning and behaviour of the males as indices of activity. His results showed clearly that glycollic acid in the water stimulated reproductive behaviour and, when the dose was high enough, spawning. The incidence of eggs being produced was 5-fold higher compared with untreated control animals.
A eureka moment one might have thought. However, Savage realised argued that glycollic acid could not be his putative substance from algae that attracted frogs to the breeding pond. It is non-volatile and odourless. He thought it far more likely that glycollic acid is an intermediate in the synthesis of the actual stimulating chemical by the plants. That, he wrote, would explain why glycollic acid was far more effective when given in the spring, when plant growth is high, rather than in the autumn.
Scendesmus, one of the algae cultured by Savage
In the summary of his 1971 paper Savage described which algal preparations were active when introduced into the aquarium water, although he presented no statistical data on this point:
(a) unialgal cultures of Chlamydomonas pulsatilla and of a species of Scenedesmus; (b) filtrates from cultures of Scenedesmus; (c) isopropanol extracts of dried cells of Scenedesmus; (d) glycollic acid; (e) a fatty or waxy material isolated from Scenedesmus filtrates, or from aquarium water to which a culture has been added, by means of reversed phase column chromatography.
The best material is an extract from the media, and this has been effective at one part in two million of aquarium water. It is still impure, and the true activity may be greater
by a factor of 100.
But Savage had not quite finished. I suspect he was still doing more work while the paper was in preparation for publication since he added a ‘Biochemical Appendix’. In it he provided crude chemical evidence that the algal extracts contain one or more steroid hormones. He also knew that addition of progesterone to the water, like a shot of gonadotrophins used to breed Xenopus for pregnancy tests, was followed by a single spawning and then a cessation of activity. That cessation was unlike natural spawning or that seen after the addition of algal extracts. He proposed a hypothesis to explain the puzzling effect of water changes on spawning activity:
…Let it be supposed that two substances are involved, one of which is not a hormone but a precursor, and is of fairly high stability in the aquaria, and the other, unstable, is a hormone derived from it by microbiological processes in the aquarium water. Microbiological processes are of great importance in steroid chemistry, sometimes providing the only route to a desired structure. The concentration of hormone in the water will then depend on the resultant of the two rates of formation and destruction of the hormone, and could be greatly influenced by the profound modification of the micro biological environment produced by WC/R [water change/re-randomisation]. A very small concentration of hormone could be effective in frogs living continuously in the water.
The first part of the hypothesis has been confirmed by adding an extract to the aquaria, waiting three days, running off 10 1. of the water and recovering the usual ketone.
The second part has been confirmed, without any intention, by an experiment in the isolation of such a substance from a natural pond at the time when R. temporaria was spawning there, and by this means to establish a connection between the two species in their sexual activity. At the same time, the opportunity of fractionating the extract was taken, almost all the extracts having been used on the frogs in a crude state.
10 l of the water from the pond was processed in the usual way. The crude extract (165 mg) was dissolved in cyclohexane (10 ml.) and extracted with five lots, each of 10 ml. of N/1.NaOH. The alkaline extract was acidified, and extracted with five lots, each of 10 ml. of 1.1.1 trichlorethane, and the product (10 mg) subjected to Girard fractionation. The ketone fraction (0.6 mg) (clearly impure) and the non-ketones (3-6 mg) were added to line A and B respectively, on 24 March…The final activity was very similar in both—a male score totalling 23 in line A and 26 in line B, with four lots of eggs in line A and three in line B. The timing was quite different. All the activity in line B was before WC/R, but all the eggs, and more than half the male activity in line A was after WC/R. The correspondence with the hypothesis seems good.
Fast forward to 2021 and we know that algae are indeed a rich source of biologically active phyto-oestrogens.
It would seem that Savage was searching for a single chemical constituent secreted by algae to explain the attraction of Common Frogs to ponds and the timing of actual spawning in both the Common Frog and in Xenopus. That seems an unnecessary assumption and it may be better to think in terms of several putative substances released by algae that could act in different ways and at different times on the animals.
I have described Maxwell Savage’s work at some length because it shows how he built up the case of a causal link, not just an association, between the presence of green algae and breeding in both the Common Frog and Xenopus. These experiments on Xenopus (with that final link between the two species in which he tested extracts taken from pond water in which Common Frogs were spawning) were Savage’s last published word from his 40-year study. However, before moving to the final section of this article, I think it is worth pointing out that Savage had found an earlier report of algae having a possible stimulating effect on Xenopus. Edward Bles was the first person to breed Xenopus under controlled conditions and Savage wrote:
Some observations by Bles seem to have been overlooked. His paper seems to show that he sometimes added a pure culture of Chlamydomonas to his aquaria to induce spawning, and that he suspected that some event in the microflora influenced his animals.
Although the 1971 is the last I have found, Trevor Beebee noted that Savage was ‘still experimenting with Xenopus in 1974. probably in the same garage laboratory where one of his grandsons recalled sleeping on a camp bed “under the whir of the aquariums where my dreams were soaked in croaks and plops”’.
Getting to the end of revisiting Maxwell Savage’s research on frogs and algae—but not quite at the end of the series—I remain amazed that with so many ways in which his observations, inferences and experiments could be followed up by direct, simple experiments nobody has actually done so. There are experiments crying out to be done. For example, would Common Frogs be attracted to a pondless area of ground in by the smell of chemicals now known to be responsible for the odour of various algae? Does glycolic acid stimulate Xenopus to spawn directly, rather than as argued by Savage, being involved in the synthesis of substances that do?
Neither the seemingly endless observational studies nor or the current fixation with genomics will provide the answers. But simple experimental biology can. We owe it to Maxwell Savage to just get on with it, and settle once and for all whether there is a causal link between algae and reproduction in any species of amphibian.
Beebee TJC. 2010. Ronald Maxwell Savage, 1900-1985: a tribute. Herpetological Journal 20, 115-116.
Savage RM. 1965. External stimulus of the natural spawning of Xenopus laevis. Nature 205, 618-619.
Savage RM. 1971. The natural stimulus for spawning in Xenopus laevis (Amphibia). Proceedings of the Zoological Society of London 165, 245-260.
Sychrová E, Štěpánková T, Nováková K, Bláha L, Giesy JP, Hilscherová K. 2012. Estrogenic activity in extracts and exudates of cyanobacteria and green algae. Environment International 39, 134-140.
And Chlamydomonas, another algal species cultured by Maxwell Savage:
