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.
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| 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
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| 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.
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| 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.
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| 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.
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| 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 (http://www.gnu.org/copyleft/fdl.html), CC-BY-SA-3.0 (http://creativecommons.org/licenses/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.
