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.
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| 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 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.
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| 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:
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