Jean Rostand. Frogs, glycerol, cryoprotection and how he might have scooped Parkes, Smith and Polge

Jean Rostand

In a previous article I described some of the work on frogs and toads done by the French biologist, Jean Rostand (1894-1977), who in later years has been described as ‘brilliant’ and ‘eccentric’. Rostand had another claim to fame that caused a flurry of excitement and embarrassment at the National Institute of Medical Research (NIMR) in London. In 1952 Rostand wrote, asking for offprints of papers written by members of the Institute concerning the use of glycerol as the first agent that protected spermatozoa and other cells from the effects of freezing to and thawing from very low temperatures. What happened is described by Sir Alan Parkes FRS (1900-1990) in his autobiography Off-beat Biologist:

     Early in May 1952 a most interesting and friendly new connection opened up - Audrey [Smith] had a request for offprints from Jean Rostand, the distinguished French biologist, who at the same time sent us a copy of a paper published by him as early as 1946 in the Comptes-Rendus de l'Academie des Sciences entitled 'Glycérine et résistance du sperme aux basses températures'. His paper was entirely unknown to us and the mere title put us in a twitter. Had we perpetrated some fearful scientific blunder? However, on reading the paper it seemed that, although we ought to have known of it, the work was in no way similar to ours. Rostand had used glycerol to keep suspensions of frog sperm from solidifying at temperatures slightly below freezing. Under such conditions the spermatozoa regained mobility, but had lost fertilising power. In acknowledging Audrey's reprints Rostand said that he would try removing the glycerol slowly by dialysis, but we never heard whether fertilising power was thus restored.

     Early in July 1955 Ruth Deanesly [Lady Parkes] and I were at a conference in Paris and took the opportunity of calling on Rostand. He received us most kindly at his combined home and laboratory and proved to be a delightful person. On leaving Paris on the Sunday morning we bought a newspaper which, to our surprise and pleasure, carried a detailed article about him, illustrated with sketches, from which we learnt that Jean was the son of Edmond Rostand, the playwright who had immortalised Cyrano de Bergerac, that on Wednesday of the previous week (the very day we had called on him) he had been awarded a prize of three million francs by the Singer-Polignac Foundation and that at one stage of his researches he had fallen foul of the gendarmes when he set free 70,000 toads in the woods of Saint-Cloud.

Thus Rostand is recorded as a pioneer in cryobiology. The work at NIMR became famous because, when applied to agriculture, it revolutionised artificial insemination and enabled the rapid genetic improvement of livestock. It was no accident that two of the triumvirate involved started off with degrees in agriculture, Parkes himself and Chris Polge, later FRS, (1926-2006). The other member was Audrey Ursula Smith (1915-1981) who was medically qualified with a degree in physiology.

Audrey Smith, Alan Parkes and Chris Polge
see below for sources

Accounts of how glycerol was found to be affective differ slightly. The Parkes team was following up work which suggested that some sugars might be protective against freezing and thawing. Fructose or laevulose, to use the terminology in use at the time, had been used to some effect in the early 1940s and this work was being checked. However, nothing of any great significance was found and the sugar solutions were put in the cold store for some months.

Parkes in 1956 wrote of what happened next:

Some months later work was resumed with the same material and negative results were again obtained with all of the solutions except one which almost completely preserved motility in fowl spermatozoa frozen to -79°C [the temperature of ‘dry ice’ solid carbon dioxide]. Further experiments confirmed this result and at this point, with some trepidation, the small amount (10 or 15 ml) of the miraculous solution remaining was handed over to our colleague Dr D. Elliott for chemical analysis. He reported that the solution contained glycerol, water, and a fair amount of protein! It was then realized that Meyer's albumen[*] - the glycerol and albumen of the histologist - had been used in the course of morphological work on the spermatozoa at the same time as the laevulose solutions were being tested, and with them had been put away in the cold-store. Tests with new material very soon showed that the albumen played no part in the protective effect.

Although we never discovered exactly what had happened it is very likely that during the long spell in the cold-store the labels had fallen off some of the bottles, a habit they have in cold-store, and had been replaced by some zealot on the wrong bottles, though there was no evidence that any of our own technicians had been involved. Be that as it may, there can have been few experiments in which blind chance stepped in more effectively.

Other accounts indicate that it was Audrey Smith who deduced that glycerol was a constituent of the ‘wrong bottle’ as the result of her accidentally dropping the bottle in the lab sink. A droplet of the contents flew out of the sink and landed on a hotplate which was indeed hot. She immediately recognised the smell from the puff of smoke as acrolein, the thermal degradation product of glycerol.

Labels falling off bottles was commonplace in laboratories of the time. Labelling glass was difficult. The usual method was a water-soluble gummed paper label for reagents. Sellotape, the UK equivalent of Scotch Tape may also have been used but at the time but was expensive and not widely available. It too suffered on bottles and the labels also often fell off especially in the cold. A mystery remains in who put the labels back the wrong—but fortuitously right—way round.

It would appear that in using glycerol to suspend frog spermatozoa, as Parkes indicated, Rostand also accidentally discovered its cryoprotective effects but only took his experiments to slightly below freezing point.

*The recipe I have is 50 ml white of egg, 50 ml glycerol, 1 g of sodium salicylate

Hunter RHF. 2008. Ernest John Christopher Polge. 16 August 1926 – 17 August 2006. Biographical Memoirs of Fellows of the Royal Society. 54, 275–296. doi:10.1098/rsbm.2008.0006

Parkes AS. 1985. Off-beat Biologist. Cambridge: Galton Foundation.

Polge C, Smith AU, Parkes AS. 1949. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature 164, 666.

Rostand J. 1946. Glycérine et résistance du sperme aux basses températures. Comptes-Rendus de l'Academie des Sciences 222, 1524-1525.

AS Parkes photograph: Wellcome Library, London. Wellcome Images images@wellcome.ac.uk http://wellcomeimages.org Portrait of Alan Sterling Parkes outside the Family Planning Offices, June 1957.  POlge photograph: Royal Society - from Hunter 2008.

Elliot’s Pheasant: a colour plate from 1962

In the days when colour printing was extremely expensive, the Avicultural Society had special appeals for funds to support the appearance in Avicultural Magazine of the occasional colour plate. A well-known bird artist was then commissioned. Although the whole run of the Society’s magazines can be found online, the plates rarely see the light of day. Therefore I decided to show one, now and again, on this site. This is the 21st in the series. 

 – – – – – – – – – –

The artist was John Cyril Harrison (1898-1985). For most of his life he lived in Norfolk. He trained at the Slade after the First World War and became well known for his wildlife paintings, especially birds. He was a regular visitor to Scotland, parts of Africa and Iceland. He was prolific and his work often appears in auctions.

The article accompanying this plate was written by Philip Wayre (1921-2014) who in 1959 had founded the Ornamental Pheasant Trust. He also had a small zoo at Great Witchingham, the Norfolk Wildlife Park.

Elliot's pheasant (Syrmaticus ellioti) was decreasing in numbers at the time Philip Wayre wrote his account. It is now classified as Near Threatened by IUCN because of forest habitat loss across its range in south-east China. It is relatively easy to breed in captivity.

Avicultural Magazine 68, 1962

 

THE LAST TIGERS OF HONG KONG by John Saeki

I am late to the party with this book which was published in Hong Kong in 2022. I was thanked by the author for providing our recollections of what we had been told about the 1965 sightings and evidence for what was possibly or even probably the last wild tiger to be seen in Hong Kong a few months before our arrival in 1965.

John Saeki did a great job in pulling together accounts of sightings and hunting of tigers in Hong Kong during the 20th century. He did so against a background of an incredible degree of incredulity on the part of journalists for the local newspapers who never seemed to be able to get it into their heads that Hong Kong was well within the natural range of the Tiger or that villagers or expats were not all stupid in mistaking tigers for domestic tabbies. Saeki also drew on evidence from regions and provinces in south China which indicated just how common and dangerous to the human population tigers had been and how steps had been made throughout China to wipe them out, an occupation that reached its peak in the 1950s with skins sold for foreign currency and body parts for the Chinese medicine trade.

Tigers in Hong Kong killed people: two villagers in 1937 and the policemen Ernest Goucher and Rutton Singh, who had a tiger cornered, in 1915 together with two more villagers.

Reports of Hong Kong Tigers were characterised by each incident, proven or otherwise, being of a relatively short duration.from days to a couple of months. Hong Kong did not have many wild ungulates and the reports often came to light as a results of attacks on domestic cattle, water buffalo and pigs. Tigers wandering across the border from mainland China would have to work very hard to find food.

It is possible that not all reports of tigers were tigers. Large animals in which the stripes were not seen could have been leopards, particularly melanistic ones, which did visit or live in Hong Kong in the past (see HERE). 

Not only have tigers now gone from South China, were they there their chances of even reaching the Hong Kong border have now also gone. In the mid-1960s tourists and newcomers were taken to the village of Lok Ma Chau to look across the closed border area and the Shum Chun, Sham Chung or Shenzhen River into China. There were fields, more fields, a small town and hills in the distance. Across that whole area is now the city of Shenzhen with more than 17.3 million people.

Coincidentally, there was a report from Lok Ma Chau of a tiger being seen at Lok Ma Chau in September 1976. The report said it was about three feet high, four feet long and dark; it had been seen ‘twice in ten days, roaming about after dark’. The old questions loom: was that a Tiger, a Leopard, a much smaller Leopard Cat or large domestic cat looking bigger in the dark? 

1966. The view from Lok Ma Chau into China. The river is the border 

2023. The view from the mouth of the river across the border with the city of Shenzhen behind

Saeki J. 2022. The Last Tigers of Hong Kong.Hong Kong: Blacksmith Books. Available on Amazon

Greater Painted-Snipe. Hong Kong January 2025

AJP spotted these Greater Painted-Snipe (Rostratula benghalensis) in Hong Kong last week. They were lurking at the edge of a reed bed. That’s a male in front—see the golden eyestripe—with a much more brightly coloured female almost hidden behind—see the white eyestripe. The species, found across South-East Asia, the Indian sub-continent and Africa, is polyandrous with the male incubating the eggs and rearing the young.

Honduras: Proboscis Bats hiding in plain sight

 

This sign board amongst the mangroves lining the Rio Cuero of the Cuero y Salada Wildlife Refuge in Honduras appears to have nothing goimg for it in the wildlife line. However, roosting in plain sight is a colony of Proboscis Bats (Rhynchonycteris naso). This bat goes by a variety of common names in the countries of Central and South America where it lives, often in wetlands: Brazilian Long-nosed Bat, Sharp-nosed Bat, River Bat and Long-nosed Proboscis Bat, the latter surely tautological. From the angle we viewed them it is not possible to see, because of foreshortening, the long nose.

This species is known to roost with the whole colony of 5-10 individuals lined up along branches, for example. It is small bat, around 6 cm long, nocturnal and insectivorous.

The Multi-Toed Frogs of Jean Rostand. An old problem solved by a fluke, literally by a fluke

I was looking at old copies of Animal Life magazine, published in Britain in the early 1960s when I came across an article I remembered reading 62 years ago. It was by the French author and self-funded biologist Jean Rostand  (1894-1977).

Animal Life No 2. October 1962 pp 34-38

 

Jean Rostand was wealthy enough to give up an ‘official’ career in science in order to continue research in his laboratory at home. Well known as a writer about science, the history of science and the human condition in the light of scientific discovery, much of his own research was on the development of amphibians from the egg. Some of his findings were overlooked, ignored or forgotten, only to be rediscovered decades later. He did achieve recognition in France, however, both scientific and literary.

In this article I will only consider one aspect of Rostand’s research: polydactyly in frogs and toads. He had found polydactyly of the hind limb (six toes in this case) in the Common Toad (Bufo bufo) and established it to be genetically determined. But then, in his own words:

...in a pond near Concarneau in Brittany I was surprised to find frogs with six, seven, eight and even nine toes. There was in fact a massive variation, affecting from ten to fifteen per cent of the population of the pond. Unlike the polydactyly of the toad, the polydactyly of the frog is not transmissible to its descendants: it is not, at any rate, transmitted in accordance with the accepted rules. It is simply the benign symptom of a much more serious anomaly which strikes the tadpole larvae and causes therein a considerable modification of the bone structure, the growth of supernumerary feet and the formation of various types of excrescence. All the tadpoles that are severely attacked perish before changing into frogs: the polydactylic adults are therefore survivors. What is the cause of this singular anomaly, which, in some respects, recalls certain malignant proliferations? Must one blame a physico-chemical factor, such as chemical substances or radiations, which are present in the surroundings? Or is it due to an infectious agent, a virus? The first hypothesis seems rather unlikely, because we know of no inorganic factor capable of producing such effects. If the second hypothesis can be proved—which is what I am trying to do at the moment—it is possible that the study of these abnormal larvae will throw some light on the formation mechanism of certain tumours. In any case the exhaustive study of anomalies, whether hereditary or acquired, among toads and frogs could, in one way or another, assist the study of human anomalies.

Rostand had in fact redescribed the syndrome. It had been first observed and reported in France in 1937.

Some observations were compatible with the variable presence of some agent. In some years ponds which had shown the anomaly in tadpoles were completely free of the condition but the cause remained unknown.

In 2017 Alain Dubois reviewed what was known and what was not known about Anomaly P. It has been found in a number of countries in Palaearctic water or green frogs, now separated off in their own genus, Pelophylax but not in frogs of the genus Rana. It may affect all species of Pelophylax including the kleptons between some of those species. The Edible Frog, Rana esculenta, now Pelophylax kl. esculentus, was the form studied by Rostand. Dubois wrote:

Much still remains to be known about the anomaly P: its cause, its geographic distribution, exactly which taxa are affected and why, what is the impact of this syndrome on frog populations, etc. Although this problem attracted the attention, especially of an amateur naturalist, Jean Rostand, mostly in the years 1950-1970, no studies are apparently under way nowadays, in any laboratory or European country, to elucidate these questions. This is surprising and even shocking, especially in view of the strong interest raised in recent years by amphibian anomalies in conservation biology.… Given the fact that this syndrome involves facts of cellular abnormal multiplication and tissue differentiation and growth, its understanding might throw interesting or important lights on some developmental biology problems. More attention should certainly be paid to this unsolved problem by the international scientific community.

But then things did start to happen. A group of Russian workers, later joined by the French, including Alain Dubois, found that Anomaly P is caused by the trematode Strigea robusta, i.e. a fluke. That work was mirrored by studies in North America which showed the devastating effects of trematode infection on some amphibian populations. 

The life cycle of S. robusta involves three hosts: planorbid (ramshorn) snails as the first intermediate, then the larvae of amphibians as the second intermediate, with anatid birds (ducks, geese and swans) as the definitive host. An important clue was the occurrence of Anomaly P in water frog tadpoles when raised in tanks containing a species of planorbid snail.

Current evidence is that some amphibians are affected by S. robusta while others are not. In Russia the limbs of other amphibians that live alongside P. ribibundus, the Marsh Frog, and are infected show no abnormalities. Those unaffected are the Smooth Newt (Lissotriton vulgaris)(but see below), Great Crested Newt (Triturus cristatus), Pallas’s Spadefooi Toad (Pelobates vespertinus), Red-bellied Toad (Bombina bombina) and Moor Frog (Rana arvalis). However, there is evidence that when S. robusta appeared in a pond in Germany, the population of Smooth Newts (of which 73% were infected) declined. By contrast, there was no effect on the population of Great Crested Newts, none of which became infected. More recent work indicates that Anomaly P can be induced in toads (Common Toad, Bufo bufo; Green Toad, Bufotes viridis; Batura Toad, Bufotes baturae) by the presence of S. robusta.

Rostand’s experiments in the late 1940s and 50s indicated the existence of a sensitive period for during the early stages of a tadpole’s development for an infectious agent to act. This was confirmed with the experiments in which tadpoles were exposed to S. robusta. Once the toes had formed, the tadpole was safe. There was also evidence that the severe forms of Anomaly P depend on the stage of exposure, the parasite load, the location of the parasites and the degree of immunological protection.

Given the complex nature of the lifecycle of the parasite, variations in the populations of planorbid snails and ducks in a particular pond at a particular time it is perhaps not surprising that the occurrence of Anomaly P at a particular locale varies greatly in intensity.

The mechanism by which the trematode exerts its effect on the tadpole is beyond the scope of this article. However, research on trematode infection on development of the limb in North American amphibians suggested the production by the cercariae stage of the trematode of an excess of a Vitamin A metabolite which affects gene expression adversely.

There are, of course, unanswered questions, many on the ecological consequences of infection with the parasite.  It also seems odd the parasite seems to be acting in a very unparasitical way—the selection pressure on parasites lies strongly against killing their hosts.

Rostand—the great ‘amateur’ developmental biologist—was right to conclude that the cause of Anomaly P is an infectious agent. However, it was not a virus but something much bigger, the fluke Strigea robusta.

From Svinin et al. 2020

From Svinin et al 2023

Jean Rostand
Animal Life No 2. October 1962

Dubois A. 2017. Rostand’s anomaly P in Palaearctic green frogs (Pelophylax) and similar anomalies in amphibians. Mertensiella 25, 49-56

Svinin A, Bashinskiy I, Ermakov O, Litvinchuk S. 2023. Effects of minimum Strigea robusta (Digenea: Strigeidae) cercariae doses and localization of cystson the anomaly P manifestation in Pelophylax lessonae (Anura: Ranidae) tadpoles. Parasitology Research 122, 889-894. doi: 10.1007/s00436-022-07778-z

Svinin AO, Bashinskiy IV, Litvinchuk SN, Ermakov OA, Ivanov AY, Neymark LA, Vedernikov AA, Osipov VV, Drobot GP, Dubois A. 2020. Strigea robusta causes polydactylyand severe forms of Rostand’s anomaly P in water frogs. Parasites & Vectors 13, 381 doi.org/10.1186s13071-020-04256-2

Svinin AO, Chikhlyaev IV, Bashinskiy IW, Osipov VV, Neymark LA, Ivanov AY, Stoyko TG,  Chernigova PI, Ibrogimova PK, Litvinchuk SN, Ermakov OA. 2023. Diversity of trematodes from the amphibian anomaly P hotspot: Role of planorbid snails. PLoS ONE 18(3): e0281740. doi.org/10.1371/journal.pone.0281740

Svinin A, Ermakov O, Litvinchuk S. 2022. The incidence of the anomaly P syndrome in water frogs (Anura, Ranidae, Pelophylax) from the Middle Volga River (Russia). Herpetozoa 35, 283–288 doi: 10.3897/herpetozoa.35.e95928

Svinin AO, Matushkina KA, Dedukh DV, Bashinskiy IV, Ermakov, OA, Litvinchuk SN. 2022. Strigea robusta (Digenea: Strigeidae) infection effects on the gonadal structure and limb malformation in toad early development. Journal of Experimental Zoology A 337, 675-686 doi.org/10.1002/jez.2599

Montezuma Oropendola. Honduras, November 2024

Gangs of Montezuma Oropendolas are common in the grounds of the Lodge at Pico Bonito on the Caribbean side of Honduras. The colonial, woven hanging nests we saw at Lancetilla Botanical Gardens were unoccipied because were were not there in the breeding season. Then the males defend large harems of much smaller females and produce a complex warbling song, seemingly quite out of keeping with the size of the bird.

Relationships between the various species of oropendola appear to be contentious, probably unsurprising given the use of only mitochondrial DNA to form the basis of the statistical analysis. Psarocolius montezuma seems in common use along with Gymnostinops montezuma. It is found from Mexico to Panama.