Monday 27 August 2018

Nelson Annandale and his eponymous turtle

In my previous article I referred to Nelson Annandale’s survey of the ponds at Port Canning in India. But I had heard of the chelonian named in his honour many decades ago.

Thomas Nelson Annandale was an unlucky man. Not only did he die at the age of 47 but did so when has was about to be elected to the Royal Society in 1924. However, although not formally elected, the Royal Society did publish his obituary along with that of Fellows who died that year. Because this obituary is not widely known I am reproducing it verbatim since it describes fully his life and his achievements as well as being an illustration of its time. The writer was William Thomas Calman FRS, Keeper of Zoology at the Natural History Museum in London 1927-36:

The name of Nelson Annandale appeared on the list of those recommended by the Council for election to the Fellowship of the Society in the present year, but, before the formal election could, take place, his sudden and unexpected death, on 10th April, closed a career that had seemed to be in the full tide of achievement.
     Thomas Nelson Annandale was born on the 15th of June, 1876, and was the eldest son of Thomas Annandale, the distinguished Professor of Clinical Surgery in the University of Edinburgh. His mother was a daughter of William Nelson, the publisher. He was educated at Rugby, where his only recorded success appears to have been the winning of a prize for an essay on a natural history subject. The examiner on this occasion was surprised by the school­ boy’s acquaintance with anatomical terminology, which, however, was explained when he learned of the candidate’s parentage. From Rugby, Annandale went to Balliol College. He studied Zoology under Ray Lankester, and obtained second-class honours in that subject in 1898. His attention seems to have been directed also to anthropology under the guidance of E. B. Tylor. One of his Oxford teachers writes : “I found him an interesting, if rather strange, man, very keen in his way, and independent in developing his own particular tastes.”
     While an undergraduate, he spent several vacations in Iceland and the Faeroe Islands collecting the materials which, after some preliminary reports, in ‘Man,’ ‘Proceedings of the Royal Society of Edinburgh,’ and elsewhere, were dealt with in his book ‘The Faeroes and Iceland: a study in Island Life ’ (Clarendon Press, 1905). In 1899 he was a member of the Skeat expedi­tion to the Malay Peninsula, and he repeatedly travelled in that country in the years 1901 to 1903 in company with Mr. H. C. Robinson, with whom he published in 1903-1907 ‘Fasciculi Malayenses,’ a series of reports on the anthropological and zoological results of the expeditions. Annandale’s contributions to the ‘Fasciculi ’ were chiefly concerned with anthropology, and were worked out during his tenure of a research fellowship in anthropology in the University of Edinburgh (1902-1904), where he was awarded the degree of D.Sc, (1905).
     In 1904 Annandale went to India as Deputy-Superintendent of the Natural History Section of the Indian Museum, under Lt.-Col. Alcock, and, on Alcock s retirement in 1907, he succeeded him as Superintendent. Annandale threw himself with characteristic energy into the administration of the Museum, obtained considerable increases of staff and founded two series of publications, the ‘Records’ and ‘Memoirs’ of the Indian Museum. Of the former some 25 volumes, and of the latter 6 volumes, have now been published, con­taining very numerous contributions of the first importance in systematic and faunistic zoology and forming a worthy continuation of the series of catalogues begun by Alcock. From the first Annandale took up the position that the main object of the institution over which he presided should be research. He arranged that every member of the scientific staff should spend several months of each year in the field, and he obtained the help of practically every zoologist in India, as well as of many of the foremost systematists in other countries, in working out the rich collections so obtained.
     In 1916, after years of struggle with official apathy, Annandale achieved one of his principal aims in the establishment of the Zoological Survey of India, of which he became the first Director. By this measure, Zoology was for the first time placed on a footing of equality with Geology and Botany, and what had been merely the zoological section of the Indian Museum became recognized as one of the great scientific research departments of the Imperial Government.
Annandale would have been the first to acknowledge that the way for these reforms had been paved by the labours of his predecessors. Alcock, in parti­cular, had for years endeavoured to impress upon the Government of India the importance of zoological research and the need for its adequate support, and his premature resignation was a final protest against the discouraging conditions under which he had to work. There is reason to believe that the protest was not without effect, but with a less determined and enlightened successor the effect would have been only transient.
     Annandale was first and foremost a “field naturalist,” or, more precisely, a student of animal ecology. As the head of a great museum, he was perforce a taxonomist, but he was by no means of the type that deserves the contempt sometimes expressed for the “mere systematist” He constantly insisted that, even from a systematic point of view, the animal could not be understood apart from its environment and that museum study should be supplemented by field observations. His own systematic work ranged over an unusually wide field. Freshwater Sponges, Coelenterates, Polyzoa, Mollusca and Batracliia and the marine Cirripedia were the subjects of some of his most important work, but the long list of his published papers shows that very few of the larger groups of the animal kingdom had not, at one time or another, engaged his attention.
In recent years he had given much time and had travelled far and wide collecting material for a comparative faunistic study of the lakes of Asia. He himself visited the Sea of Galilee, the Hamun-i-Helmand in Seistan, Lake Chilka in Orissa, the Loktak in Manipur, the Inlé Lake in Burma, the Talé Sap in Siam, the Tai Hu in China, and Lake Biwa in Japan. Numerous systematic papers on these collections, many of them written by himself, have appeared in the publications of the Indian Museum and of the Asiatic Society of Bengal, but the final summary, in which the results of all these investigations were to have been brought together and compared, was still unwritten at the time of his death. An indication of what this summary might have given us is furnished by his paper on “The Evolution of the Shell-Sculpture in Fresh-Water Snails of the Family Viviparidse,” published in the ‘ Proceedings ’ of this Society last year (B, vol. 96, pp. 60-76). In this paper he compares the highly sculptured pond Snails occurring in the lakes of Yunnan and of the Shan States in Burma with those found fossil in the Pliocene lake-deposits of Slavonia, Dalmatia and the Levant. At first sight all these shells are very similar, but Annandale was able to trace each of the three series (Chinese, Burmese and European) back to a different smooth-shelled ancestral form, and to show that the similarity in the sculpture of the more specialized types was due to parallel or convergent evolution. “There is, or has been, some influence at work which has produced a similar collective peculiarity in the shells of the Viviparidae on diverse occasions and in different parts of the world.” Another instance of convergent evolution which he specially studied was that of the fish and the Batrachian tadpoles inhabiting mountain torrents. In this case the convergence relates to an adaptive character, an adhesive apparatus or sucker which enables the animals to cling to rocks and stones in a strong current. The details of this apparatus were shown to be remarkably similar in the two groups, and the degree of its elaboration in different species to be “ in direct correlation with the rapidity of the current in which the species habitually lives.”
     Annandale was of slight physique, with a highly-strung temperament and restless energy that must have made great demands on his strength. He travelled widely and his vacations were devoted to collecting expeditions. His impetuous methods and a caustic wit that seasoned his official corre­spondence did not contribute to popularity in the Government departments with which he had to deal. He was an outspoken critic of the administration of the Natural History departments of the British Museum, but this did not prevent him from maintaining up to the very end of his life, as the present writer can testify, the most friendly and helpful relations with many of the officials of that institution. To the junior members of his own staff he was unsparing of assistance and encouragement. He was always careful to give them full credit for their work and always ready to take up arms when he thought that others had not done so. He seemed to have few active interests outside his own work and that of his department, though his unusually wide reading was reflected in his literary style and in the unexpected turns of his conversation. He was an active member of the Indian Science Congress and of the Asiatic Society of Bengal, and he had been president of both bodies in recent years. For some years his health had been unsatisfactory, but it was only within the last few weeks of his life that duodenal ulceration was diagnosed, and within the last day or two that an unsuspected malarial infection, probably of old standing, revealed itself. Only nine days before his death he attended a garden party in the Museum grounds given by the three Indian members of the scientific staff to celebrate his selection for the Royal Society.
     The Indian Museum has been, for more than three-quarters of a century, an active centre of zoological research, and Annandale has enriched a tradition handed down from illustrious predecessors. Circumstances have combined to make the moment of his death particularly ominous for the institution thus suddenly deprived of its head; in the interests of Indian science it is to be hoped that the tradition will be maintained at the level at which he left it.
W. T. C
Having read that I have Annandale down as a good guy.

Annnandale had several species named after him. I had read about Annandale’s turtle or terrapin in the 1960s. It is now known as Heosemys annandalii (previously Hieremys annandalii). It was described and named by Boulenger in 1903. Like so many chelonians in south-east Asia it is endangered because of trade for human consumption. It is large, herbivorous and occurs in slow-moving rivers right down to the coast, as well as in swamps and bodies of freshwater. The common name it now goes under, Yellow-headed Temple Turtle, reflects its capture and then release for ‘merit’ in the grounds of Buddhist temples. It is said to be common in the waterways and parks of Bangkok, more so than in the wilder parts of its range. It is found in Cambodia, Laos, Vietnam, Thailand, Malaysia and possibly Burma.

Bangkok Zoo in 1968 had an extensive collection of native chelonians. Here is a photograph we took then:

Heosemys annandalii. Bangkok Zoo, May 1968

Calman WT. 1925. Thomas Nelson Annandale—1876-1924. Proceedings of the Royal Society B 97, xviii-xxi.

Annandale, Nelson (1876-1924). In, Contributions to the History of Herpetology, Volume 1 (revised and expanded), edited by Kraig Adler, pp78-79. 2014. Society for the Study of Amphibians and Reptiles. Note: Annandale's Royal Society obituary by W.T.C. is wrongly attributed.

Sunday 26 August 2018

Can the Common Asian Toad, Duttaphrynus melanostictus, live in saltwater?

In my previous post on tadpoles of this species I left the question of whether adult Common Asian Toads, Duttaphrynus melanostictus, can tolerate saltwater, and, if so, what strength of saltwater.

Before trying to answer that question I should point out that since the mid-1960s, the ability of a number of other amphibians to tolerate saltwater has been studied, the Green Toad (Bufotes or Bufo viridis) from Europe and the Middle East being one notable example.

In an epic of bibliographic research Gareth Hopkins and Edmund Brodie pulled together all the papers on the occurrence of amphibians in saline habitats beginning as far as I can see with Charles Darwin in 1834; their review was published in 2015. Useful though that exercise was (although I have since found at least one paper that was missed), I was less than impressed by the treatment of physiology in the review and the criteria used to extract meaningful data from individual papers, particularly those reporting observations rather than experimental evidence. I can illustrate these points by reference to a table in their review that includes Duttaphrynus melanostictus as a ‘well-studied salt-tolerant amphibian species’. Let’s look at the evidence from the papers they used to reach this conclusion.

Annandale’s paper from 1907

Quoting Nelson Annandale†, Hopkins and Brodie show a measured salinity of 12.87g/kg for where Annandale found this species in India. Please note for this article compared with the last, I am using g/kg (parts per thousand) rather than percentages for salinity in order to use the same units as Hopkins and Brodie. 12.7g/kg is equivalent to about 40% seawater and would be well above the point of isotonicity between body fluids and the external environment. In other words, there would be an osmotic flow of water out of the body. However, there are important caveats. Annandale’s paper describes the brackish ponds at Port Canning on the Matla river, about 60 miles from the sea. The ponds were dug as part of an ill-fated scheme to build a port to rival Calcutta and Singapore in the 1860s; judging by what I can see on Google Earth, they are still there. This is how Annandale describes the water and its salinity:

     An important factor in the environment is the nature of the water. I have described the ponds as brackish, but at some time of the year the water may contain the same proportion of soluble salts as the sea, at others it may even be more strongly saline, and again at others it is much more nearly fresh. As a rule the ponds are completely isolated both from one another and from the estuary. During the cold weather they are exposed to evaporation, which becomes intensified during the hot weather. During the rainy season, on the other hand, they become filled up with fresh water and probably often coalesce. They are also liable to be placed in temporary communication with the estuary occasionally, owing to a flood bursting the embankment but this does not occur by any means every year. When it does happen, it happens owing to the estuary being swollen with fresh water, which is flowing down from up-country so that the ponds, even under these conditions, are practically cut off from the sea. 
     Stoliczka*, in 1868 or 1869, had the water of the ponds analysed; but he does not say at what time of year his samples were obtained. He found that the proportion of soluble solids was 12.87 per thousand, sea-water containing from 32 to 39 per thousand. Mr. D. Hooper, Curator of the Industrial Section of the Indian Museum, has kindly examined samples taken by myself in December and March last. Two samples came from a pond in which the Hydrozoon Irene ceylonensis, as well as the Actinian, was reproducing its species, and in which the plant Naias was abundant. A sample taken from this pond at the beginning of December, a few weeks after the end of the rainy season, was found to contain 12.13 per thousand of soluble salts, while another taken on March 17th contained 20.22 per thousand. At the latter date water from the edge of the Matla at Port Canning contained 25.46 per thousand, and that from a second pond near the first 23.16… 
     Stoliczka noted that the water in the ponds was almost fresh during the rains…All that can be said, therefore, as regards the salinity of the water of the ponds, is that it varies considerably at different times of the year…

Listing the fauna of the ponds, Annandale had this to say:

     …and the only Amphibians the equally common Rana cyanophlyctis and R. tigrina. The Indian Toad, Bufo melanostictus, is abundant at the edge of the ponds, in which it possibly breeds.

So, equating measurements of the measured salinity of the ponds at a particular season to a presumed salt tolerance of the largely terrrestrial toad (it could breed in the ponds in the rainy season when the water is ‘more nearly fresh’) is perhaps unwise. The killer blow, though, to trying to relate measured salinity to salt tolerance of this toad is the occurrence of the two species of frog. The Indian Bullfrog, Rana tigrina, now reverted to its apparently correct specific name of tigerina in the genus Hoplobatrachus, was used by Gordon, Schmidt-Nielsen and Kelly in their 1961 paper as a freshwater species to compare with F. cancrivora. Their results were clear. Adult bullfrogs could tolerate a salinities up to 9 g/kg (25% seawater). Above that physiological point of isotonicity, the frogs died in 24-48 hours, even at 11 g/kg. Therefore, the salinity quoted by Annandale cannot be equated to the presence of the amphibian species in the water at the same season.

I therefore discount the evidence taken from Annandale’s paper on D. melanostictus.

George Chakko’s paper from 1968

The only direct study on the saltwater tolerance of D. melanostictus listed by Hopkins and Brodie is that by George Chakko of Madras Christian College published in 1968. He kept four local species in various concentrations of seawater. There was a difference between species. All survived for two weeks in 25% local seawater (salinity 32g/kg), i.e. 8 g/kg. These species were Jerdon’s Bullfrog (Hoplobatrachus, then Rana, crassus), Indian Green Frog (Euphlyctis, then Rana, hexadactylus), Marbled Narrow-mouth Frog (Uperodon or Ramanella variegatus*) and, of course, D. melanostictus. Only D. melanostictus survived in 35% seawater, i.e. 11.2 g/kg. These were the maximum tolerated concentrations.

What do we now know?

Looking at the experimentally-determined salinity tolerance of a number of anurans (frogs and toads, for brevity, although there are interesting examples in caudates, the newts and salamanders) listed by Hopkins and Brodie, there are a number that show a similar tolerance to D. melanostictus. I counted 7 species in the range 10-11.2 g/kg. Above that there are only 4: Xenopus laevis at 14g/kg (reminding us salinity can be high in inland waters); the possibly aptly named Marine or Cane Toad, Rhinella marina at 16; Green Toad, Bufotes viridis, at 20-26 and then the jump to F. cancrivora at 39.

In terms of mechanisms of adaptation, it is interesting that in the four species known to tolerate higher salinities as adults, an increase in urea as well as an increase in salt concentrations in blood has been found. Is that also true of those tolerant to only slightly hypertonic saltwater like D. melanostictus, or do they rely simply on a small increase in salt concentrations in their blood?

The take-home message is that D. melanostictus just enters the physiologically interesting, in terms of adaptations needed to survive, range of salinity, in that the 35% seawater (but not the 25% seawater) would be slightly hypertonic to the body fluids of toads in freshwater. In terms of physiological adaptation, there is a question still to be answered—an ideal project for an honours year student perhaps.

On the Common Asian Toad, taking the evidence discussed in this and the previous article, I can summarise:

  • Tadpoles must hatch in virtually fresh water
  • Older tadpoles must live in brackish water hypotonic to body fluids
  • Adults can tolerate salinities just but only just hypertonic to body fluids. The physiological mechanism has not been studied in this species.
  • The evidence matches observations that these toads can live near the sea, even breed in freshwater near the sea, where they may be exposed to brackish conditions.
  • D. melanostictus does not approach the capabilities of F. cancrivora in exploiting the far greater salinity of a mangrove swamp.

‡Tonicity is a measure of the effective osmotic pressure gradient, as defined by the water potential of two solutions separated by a semipermeable membrane. In other words, tonicity is the relative concentration of solutes dissolved in solution which determine the direction and extent of diffusion. It is commonly used when describing the response of cells immersed in an external solution. Unlike osmotic pressure, tonicity is influenced only by solutes that cannot cross the membrane, as only these exert an effective osmotic pressure. Solutes able to freely cross the membrane do not affect tonicity because they will always be in equal concentrations on both sides of the membrane. This Wikipedia definition is as good as any. An example of differences between isotonic and isosmotic would be the plasma composition of F. cancrivora in saltwater. Because of the impermeabilty of the membranes in question to urea, this isomotic concentration is isotonic. In a typical mammal, because urea passes rapidly across membranes, the isosmotic plasma would be hypotonic.

†Thomas Nelson Annandale (1876–1924), in 1907 Director of the Indian Museum in Calcutta.

*Ferdinand Stoliczka (1838 –1874), a Moravian (Czech) in the Geological Survey of India. An all-round palaeontology, geology and zoology, he described and named the frog Ramanella variegatus (studied by Chakko above) in 1872.

Annandale N. 1907. The fauna of brackish ponds at Port Canning, Lower Bengal. Records of the Indian Museum 1: 35–43. 
Chakko G. 1968. Salinity tolerances of some Indian anurans. Proceedings of the Indian Academy of Sciences - B 67, 233–236,
Hopkins GR, Brodie ED. 2015. Occurrence of amphibians in saline habitats: a review and evolutionary perspective. Herpetological Monographs 29, 1-27. DOI: 10.1655/HERPMONOGRAPHS-D-14-00006 

Friday 24 August 2018

Ronald Strahan in 1950s Hong Kong: Amphibians in saltwater

I had a surprise when researching the recent article on Ronald Strahan. I discovered that Strahan had done a small piece of research in Hong Kong that we unknowingly and in part repeated ten years later.

The background is that in the mid-1960s, the story of a frog and its survival in sea water had already become a classic of the heyday of comparative physiology. Again, Knut Schmidt-Nielsen (1915-2017) was one of the two main players. He describes the background in his autobiography:

     But I was curious about amphibians, such as frogs and salamanders. Normally frogs do not live in the sea; the high permeability of their skin would cause them serious problems in sea water because their blood and body fluids contain less than 1 percent salt, whereas sea water contains about 3.5 percent. A frog in sea water, it seems, should soon resemble a pickled herring. 
     I had come across a few reports of frogs and toads that live in brackish water, and even a mention of certain frogs that swim in full-strength sea water. This sounded incredible. If frogs living in the sea really existed, their physiological mechanisms certainly deserved a careful study. 
     In the spring of 1960 I planned to search for saltwater frogs where they had been reported, on the tropical coasts of Southeast Asia. Pete Scholander told me that Malcolm Gordon, a physiologist at the University of California at Los Angeles, was planning a similar study. “Why don’t you two work together?” he asked. 
     I was more than happy to collaborate with Malcolm, and we planned to work at the Oceanographic Institute at Nha Trang, a town north of Saigon in Viet Nam. At the beginning of the summer I flew from New York to Los Angeles, my first trip in a modem jet. From there, Malcolm, a student named Hamilton Kelly, and I took off for Viet Nam to search for saltwater frogs. After stops in Tokyo and Hong Kong, we arrived in Saigon.

Crab-eating Frog (Fejervarya cancrivora)**
After an unwelcome introduction to discomfort of a French bed and the horrors of the tapwater, the team failed to find any frogs of the species Rana cancrivora, now known as Fejervarya cancrivora. They decamped to Thailand where they not only found saltwater frogs but discovered how they could live in such conditions. The frogs could survive in seawater. The problem, as Knut Schmidt-Nielsen explained, is of water. The concentration of the salts in seawater is much higher than that in the blood of frogs in freshwater. Water would be lost osmotically, and the frogs would die. 

The adaptation of saltwater frogs is to match the concentration of solutes in their blood to that of seawater or whatever salinity of water they are accustomed to. They do this mainly by accumulating urea. In most animal tissues urea moves rapidly in and out of cells so that a concentration gradient for osmotic water flow is not formed. By contrast, the skin of saltwater frogs is impermeable to urea. Therefore, the sum of salts plus urea concentrations inside the body equals the salt concentration on the outside and the frog does not lose water. This is the same mechanism employed by sharks and rays living in the sea

This is the only photograph I can find of a Crab-eating Frog
in a mangrove swamp. From here

As a result of Gordon and Schmidt-Nielsen’s research in south-east Asia, frogs that occur in coastal mangrove swamps and the mechanism by which they survive became famous overnight. An 18-minute 16 mm film, which I saw at a ZSL meeting in the 1970s, of Malcolm Gordon working in Thailand on the frogs was released in 1967*.

With that knowledge from the early 1960s you can appreciate the feeling my wife and I had when one Sunday afternoon in spring 1966, wandering along the top of a beach on Hong Kong Island, we saw an amazing sight. In an isolated pool comprised entirely of sand were tadpoles, lots of tadpoles. It seemed inconceivable that these tadpoles were not exposed to a higher degree of salinity than those in a freshwater pool. Did we have another species that, like F. cancrivora, could live in marine conditions? We had nothing to transport any tadpoles back to the lab that day. However, Alan Wright the next day was similarly enthused and we set off that afternoon in his Volkswagen Beetle to collect some of the tadpoles plus a sample of the water they were living in.

Back in the lab, the tadpoles lived perfectly happily in tap water and concentrations of salt up to 0.9%. Above that they were in obvious distress until moved again to freshwater. I cannot remember the salt concentration of the water in the pool but I think it was around 0.2%, much lower than that of seawater and lower than we had expected it to be. We could only assume that freshwater flowed down the beach after rain and was trapped.

A modern view of Big Wave Bay on Hong Kong Island. The red circle
marks the approximate site where we found the tadpoles. The buildings
and beach paraphernalia there have all been erected since 1966

Physiologists reading this will realise that 0.9% saline is the approximate concentration of salt in the blood of vertebrates and only above this concentration would the tadpoles be resembling F. cancrivora. Oh well, worth a look but not very interesting physiologically. The tadpoles were released into one of the ponds in the university compound and my notes (long gone) filed under ‘Abandoned Experiments’. Over the years, as information on the amphibians of Hong Kong accumulated, I realised that the black tadpoles with relatively short tails tadpoles were of Bufo melanostictus (now Duttaphrynus melanostictus), the Common Asian Toad.

Ronald Strahan
Getting back to the first paragraph of this article, you can imagine my surprise to discover that ten years earlier (i.e. before the experiments Schmidt-Nielsen described) Ronald Strahan had done a similar, but more extensive, study—in the same lab. His short paper, published in Copeia in 1957, began:

The ability of some am­phibians to withstand brackish water is well-known and indicated, for example, in the name, Bufo boreas halophilus. The experiments described below give some information on the extent of this ability in the tadpoles of a toad normally found in fresh water. 

In short, he collected spawn and tested salt concentrations up to 1%. The ability to withstand greater than 0.25% appeared to develop with age after hatching. At 1%, even when transferred into that concentration at 8½ days, activity was reduced and metamorphosis was retarded. Our 1966 tadpoles were probably as old or older than 8 days older and, therefore, our quick look see produced the same conclusions as those of Strahan. An important point from Strahan’s experiments was that tadpoles from spawn laid in saltwater stronger than about 0.25% will not survive.

Duttaphrynus melanostictus
Common Asian Toad, Hong Kong 1966

Notice that we, and Strahan, had studied the tadpoles of Duttaphrynus melanostictus whereas Gordon and Schmidt-Nielsen had studied adult F. cancrivora. What about the tadpoles of cancrivora? Gordon, this time with Vance Tucker from Schmidt-Nielsen’s department at Duke University, made another trip to Thailand to study that question. Tadpoles were found to be ‘abundant in brackish ponds near high-tide marks in the mangrove swamps along the north shore of the Gulf of Thailand’. 

Relatively large tadpoles coped perfectly well in seawater, but used a different method compared to the adults. The osmotic concentration of the blood increased with increasing concentrations of external salt but the increase was in salts not urea. Like salmon and other teleost fish that move to salt water, tadpoles appeared to drink the water and then get rid of excess salt, possibly through the gills. However, young tadpoles could not cope with salt concentrations higher than about 0.6%, findings reminiscent of those of Strahan in D. melanostictus.

In addition, although older tadpoles could cope with, and grow to a large size in, seawater, they did not appear to metamorphose in those high concentrations.

Gordon and Tucker concluded:

     Our observations in the laboratory indicate that the initial stages of embryonic development and metamorphosis are interfered with by salinities greater than 20% sea water [i.e. approximately 0.6% salt]. These observations, together with field data, suggest that the torrential summer rains of Thailand play an important role in the developmental biology of this frog. Spawning may occur only during or soon after heavy rains when the salinity of the spawning pools is low. We spent several nights collecting dozens of frogs with ripe gonads at the edges of the spawning pools but never observed amplexus. Spawning could have been restricted to periods of heavy rainfall, when we did not collect. The general synchrony of developmental stages in a single pond suggests that spawning could have been synchronized by a period of heavy rain.  
     Tadpoles in the laboratory usually did not metamorphose if the medium was more concentrated than 20% sea water. In the field the largest immediately pre-metamorphic tadpoles were found in the saltiest ponds. These observations suggest that metamorphosis may be delayed as long as the pond salinity is high. 
     These requirements for dilute media, and the freshwater nature of all other ranids, make it seem probable that R. cancrivora has invaded the marine environment from fresh water in relatively recent times. The high temperatures of its spawning ponds would permit rapid embryonic development and metamorphosis when torrential monsoon thunderstorms temporarily dilute the ponds. Its otherwise great salinity tolerance permits this frog and its tadpoles to enter a rich environment closed to all other amphibians. 

Studies on other species that can tolerate concentrations of seawater in the physiologically interesting range have been done since the early 1960s. However, sticking with D. melanostictus for the moment, judging by a comment made by Strahan in an autobiographical note, he was interested in this species because in Hong Kong he found it on outlying islands. 

The Zoology Department, which had been sacked during the war, was not very well equipped, so I undertook research that needed little gear. I worked for a while on the water relations of a local toad, Bufo melanostictus, and its tolerance of saline water, which could explain its prevalence on offshore islands…

In the second article of this series I will discuss what is known about adults, rather than tadpoles, of this species and its occurrence in salty water and why what is known or assumed may be misleading. However, for the moment it is quite clear, from these early studies and later ones I have not described, that the water in which the spawn hatches must be freshwater or brackish water no stronger than about 0.2% salt, i.e. less than about 5% the concentration of salt in seawater.

†Hamilton Morgan Kelly, 1936-2006, became a psychiatrist in California.

*Adaptation to a Marine Environment. 18 min, colour and b/w, sound, 1967. Distributed by McGraw-Hill Text-Films. Produced by Lamont Geological Observatory of Columbia University with a grant from the National Science Foundation. I have not been able to find a digitised online version.

**By W.A. Djatmiko (Wie146) [GFDL (, CC-BY-SA-3.0 ( from Wikimedia Commons

Gordon MS, Schmidt-Nielsen K, Kelly HM. 1961. Osmotic regulation in the crab-eating frog (Rana cancrivora). Journal of Experimental Biology 38, 659-678.

Gordon MS, Tucker VA. 1965. Osmotic regulation in the tadpoles of the crab-eating frog (Rana cancrivora). Journal of Experimental Biology 42, 437-445.

Gordon MS, Tucker VA. 1968. Further observations on the physiology of salinity adaptation in the crab-eating frog (Rana cancrivora). Journal of Experimental Biology 49, 185-193.

Schmidt-Nielsen K. 1998. The Camel’s Nose. Memoirs of a Curious Scientist. Washington DC: Island Press.

Strahan R. 1957. The effect of salinity on the survival of larvae of Bufo melanostictus. Copeia (1957), 146-147.

Thursday 16 August 2018

70 Years on: the Platinum Anniversary of the Solution to Milk Ejection by the Mammary Gland. Part 3: What do Myoepithelial Cells do in other Exocrine Glands?

The German histologists of the 19th Century described myoepithelial cells in a number of exocrine glands including salivary, sweat and lacrymal glands. Later classical histologists and electron microscopists workers added to the list; snake venom glands are one example.

Jim Linzell, using the silver staining techniques that revealed the mammary myoepithelium, found myoepithelial cells in the sweat glands and submaxillary salivary gland. He remarked that up to the 1950s that the greatest interest in myoepithelial cells had been shown by pathologists, with respect both to the rare carcinomas they give rise to or their in metastasis. Looking at the more recent literature that generalisation still holds. The question is, though, what do the myoepithelial cells do in glands, other than in the mammary gland where they push milk stored in the alveoli into the duct system?

Top (8) Myoepithelial cells lying outside
cells of sweat gland in dermis. Cat.
Bottom (10) Submaxillary salivary gland
of cat showing smaller, less numerous
myoepithelial cells.
From Linzell, 1952.

The myoepithelial cells seem smaller in these other glands than in the mammary gland so the question arises: do they have any role in emitting secretion?

One function in the mammary gland I have not mentioned in the previous two articles is in relation to the mammary ducts. As well as the stellate or ‘basket’ cells which are arranged around the secretory alveoli, there are myoepithelial cells arranged longitudinally along the ducts. These, Linzell demonstrated, when stimulated to contract by oxytocin serve to shorten and widen the ducts. Obviously, milk will flow more rapidly in a shorter, wider vessel than in a longer, thinner one and so the two types of myoepithelial cell act in concert to move milk toward the sucking young.

There is evidence that the flow of saliva can be augmented under certain circumstances and that the contractile myoepithelial cells are be responsible. The nervous control of salivary secretion is complex and there remain many unanswered questions and anomalies in experiments dating from the 19th and first half of the 20th Centuries. Although written nearly 70 years ago, the best discussion I have found on this topic is that in the Physiological Society monograph, Physiology of the Salivary Glands, written by A.S.V (now Sir Arnold) Burgen and Nils Emmelin (1914-1997) published in 1961. Later in the 1960s, Nils Emmelin provided very strong evidence that the contractile myoepithelial cells, under nervous control (in contrast to the mammary gland), are involved in augmenting the rate of salivary secretion.

The strength of mammary myoepithelial contraction can have surprising consequences. Many infants—and passing adults—have been shot in the face by a stream of milk from their mother. Such streams formed the basis of Tintoretto’s famous painting The Origin of the Milky Way which can be seen in the National Gallery in London. Greek myths are all double Dutch to me but the illustration is of Hera who was suckling Heracles; he sucked* so strongly that Hera pushed him away. Her milk shot out in all directions (remember each opening in the human nipple is from a separate gland) across the heavens and formed the Milky Way.

The Origin of the Milky Way

S.J. Folley (see part 1 of this series) used that painting to illustrate the depiction of milk ejection in mythology and art in his Dale Medal Lecture to the Society of Endocrinology in 1969. Folley had not played any great part in discovering or delineating the milk-ejection reflex (although he had encouraged those who did) but it remained an abiding interest. I was in the audience at that lecture and it would not have been possible to guess that Folley was virtually completely blind and that he had committed to memory the whole script complete with hand gestures that pointed to the slides.

In some snake venom glands, ejection of stored secretion is achieved by external muscles pressing rapidly on the gland to eject the secretion at high pressure. Whether or not myoepithelial cells are involved in these front-fanged snakes or in the low-pressure ejection system of rear-fanged snakes appears not to have been studied. Obvious questions are: are the myoepithelial cells innervated? Do they respond to mechanical stimulation such that they contract as prey is being held tightly by the jaws and thereby help squeeze venom out of the gland of rear-fanged snakes?

I hope I have illustrated that 70 years after they hit the headlines there are many basic questions that remain on the rôle of myoepithelial cells and, indeed, on how exocrine glands work, and that in the Gadarene swine-like rush into molecular biology whole areas of biological organisation have been abandoned. 

*The entry in Wikipedia gets suckling wrong, as if often the case. Mothers suckle; babies suck is the accepted distinction. Mammals suckle their young. Suckle: to give suck to. Roast sucking pig not suckling pig etc etc.

†There was a famous exchange between Alec Bangham (1921-2010) and Jim Linzell (1921-1975) at Babraham. Alec Bangham then working on his later famous liposomes said to Jim who was working on lactation in goats, James, you will never get into the Royal Society on a ruminant’s back. Ah, said Jim, but there is always the Milky Way. It is an open secret that Jim was on the list for election in 1976 when he died in December 1975. Alec was elected in 1977.

#At a Gordon Conference in 1975, Jim Linzell was giving the main invited (Thursday Night) talk. Folley had recently. When Linzell came to a problem to which he didn’t have an answer, he looked towards the heavens and said, Perhaps Folley knows. Instantly, Howard Bern proclaimed loudly, He’s looking in the wrong direction!

Burgen ASV, Emmelin NG. 1961. Physiology of the Salivary Glands. London: Edward Arnold.

Folley SJ. 1969. The milk-ejection reflex: a neuroendocrine theme in biology, myth and art. Journal of Endocrinology 44, 476–90. 

Weinstein SA, Smith TL, Kardong KV. 2009. Reptile Venom Glands. Form, Function, and Future. In, Handbook of Venoms and Toxins of Reptiles, edited by Stephen P. Mackessy, pages 65-91. Boca Raton: CRC Press.

Tuesday 14 August 2018

70 Years on: the Platinum Anniversary of the Solution to Milk Ejection by the Mammary Gland. Part 2: James Lincoln Linzell

Jim Linzell, June 1974
Photographed at Babraham by
Alec Bangham
It was in May 1948 that Jim Linzell (1921-1975) realised he had, just like Keith Richardson, stained myoepithelial cells while looking for nerve fibres in the mammary gland.

Linzell had escaped veterinary practice and was working for a PhD in the University of Edinburgh’s physiology department. He had qualified as a vet in London but found veterinary practice frustrating and unrewarding in that he lacked the time to be thorough while having to undertake jobs that technicians were perfectly capable of doing. He changed direction by going to Edinburgh to work under Professor Ivan de Burgh Daly FRS (1893-1974). He was funded by an Agricultural Research Council (now morphed into the BBSRC) research traineeship. He told Daly that he would like to work on the uterus and placenta but Daly told him to find out everything about the anatomy and physiology of the mammary glands in as many species as he could; this he did until he died at the age of 54.

Linzell did not like Edinburgh. Cold, dank and unfriendly was how he described it. Jim and Audrey, together with their two sons, lived in a flat relatively near the university. He published a note in Veterinary Record which gave his address: 7 Blackwood Crescent. His trenchant views on that flat, the landlord, the neighbours and the inhabitants of Edinburgh were expressed many times in later years whenever Edinburgh was mentioned. He would be horrified by its current valuation of £210,000.

The Linzell family had
a flat in this house in
Google Street View
But Jim was not stuck in Edinburgh. A great deal was happening to Daly and the future of agricultural research. During the Second World War it was realised that the lack of progress in increasing food production was caused by lack of knowledge of how animals work. To this end, Daly was given the job of setting up a new research institute. He found a run-down stately home for sale near Cambridge and set about building laboratories, animal houses and housing for staff. Staff were appointed ahead of space becoming available and Linzell became one of the first members of staff of what is now the Babraham Institute on 1 October 1948, albeit based for a time in Edinburgh. Then, a temporary measure, Daly converted rooms in Babraham Hall into laboratories and a wash-house to hold Linzell’s goats. Linzell was one of the first scientists on site in 1950 as Daly eviscerated his old department in Edinburgh by appointing members of staff to Babraham posts.

It was in Edinburgh that Linzell found he had revealed myoepithelial cells in the mammary gland of cats. He was studying the role of nerves in controlling blood flow by classical physiological and histological techniques. In his first paper, published in Journal of Anatomy in 1952—Jim never rushed into print—he confirmed all the findings of Richardson. He also tried various methods of silver staining which were all notoriously capricious but extended his work beyond one species. He showed clearly the presence of myoepithelial cells in cat, dog, rabbit, rat, goat, and human mammary glands.

An important additional finding was that the myoepithelial cells have no connexions to the nervous system, thereby refuting one suggestion that the milk-ejection reflex was a pathway composed entirely of nerves.

He then went further. By observing the living gland he saw contraction of the myoepithelial cells in mouse, rat, guinea pig and rabbit and was able to study what caused them to contract and thereby expel milk. Oxytocin dropped onto the gland, of course, worked. So did direct electrical stimulation was one would expect with a muscle but stimulation of nerves in the region had no such effect. Sometimes, light mechanical stimulation was enough to do the trick.

Linzell’s observations provided the final link in the chain for the neuroendocrine milk-ejection reflex.

Two figures from Linzell's 1955 paper. On the left the alveoli can be seen
full of milk. When oxytocin was dropped on the gland (right) the alveoli
contracted and milk was driven into the duct (D)

Linzell’s observations on the living myoepithelium were presented to the Physiological Society at its meeting of 18-19 December 1953. That was his first appearance before the Society, a year after his election as a member. Presenting a paper was a daunting experience because right through the 70s and into he 80s there was a phalanx of Nobel prizewinners in the audience. Discussion was often fierce and every word in the circulated abstract had to be agreed before a vote was taken on whether or not the paper should be accepted for publication in Journal of Physiology. Because the paper was refereed by attendees it could be included in reviews that demanded reference only to refereed papers. For some reason I do not understand or agree with that system was dropped by the Physiological Society in the early years of the present century. Sometimes, if all the material had been published in the Proceedings, it was not necessary to clutter the literature with a full paper. However, Linzell had covered a lot of ground in his observations and a full paper appeared in Journal of Physiology in 1955. 

Neither Keith Richardson nor Jim Linzell took any further part in work on the milk-ejection reflex.

In the past I have used the history of the milk-ejection reflex to illustrate the fact that many advances in human physiology and medicine have come from fundamental research funded as part of agricultural research.

But there remain many unanswered questions about myoepithelial cells in exocrine glands—not in the mammary gland but elsewhere in the body.

Linzell JL. 1952. The silver staining of myoepithelial cells, particularly in the mammary gland, and their relation to the ejection of milk. Journal of Anatomy 86, 49-57.

Linzell JL. 1954. The contractility of the alveoli of the mammary gland. Journal of Physiology 123, 32P.

Linzell JL. 1955. Some observations on the contractile tissue of the mammary glands. Journal of Physiology 130, 257-267.