Monday, 31 August 2015

Giant Panda: life in the slow lane. But how?

Most papers published in Science attract media attention. A number of articles picked up the story of studies on the metabolic rate of the Giant Panda (Ailuropoda melanoleuca) which confirm what has been long suspected—it is a ‘slow’ mammal in that its metabolic rate is much lower than expected for an animal of its body weight. Daily energy expenditure was, in five captive and three wild panda, only 38% of that predicted from a modern version of the ‘mouse to elephant’ line in a log-log plot.

Science 2015 349, 171-174
The Giant Panda is a bear equipped with a short digestive tract, typical of the carnivores, that eats pretty well only bamboo, a diet that would even give a herbivore a problem. The efficiency of digestion is, therefore, low and the panda must feed for many hours a day and consume large quantities of bamboo. Even then, as the latest work confirms, a low metabolic rate is a key adaptation of this species that enables energy balance to be achieved.

In part the low metabolic rate can be accounted for by relatively low levels of activity and speed of movement compared with other bears. However, what I found particularly intriguing is the manner in which resting metabolic rate is kept low. Organs of high energy demand—brain, liver and kidneys—were found to be relatively small but there must be a generally low basal metabolic rate in each tissue. In his connexion it was found that plasma or serum concentrations (which is not stated) of thyroid hormones (thyroxine and triiodothyronine) of captive pandas were also low. So the Giant Panda seems to be using a conventional control of metabolic rate by thyroid hormones. The authors then went on to compare the genome of the panda with that of other mammals in terms of genes controlling thyroid hormone signalling and synthesis. One apparently key difference was found: a unique variant of the DUOX2 gene which would appear to lead to loss of function. This gene is involved in the final step of thyroid hormone synthesis and in mice and man, loss-of-function mutations lead to hypothyroidism. However, this is where I see both a problem and a possibly incomplete explanation.

Loss of function of DUOX2 has been shown to have severe effects on a wide range of functions in addition to to virtually preventing the production of thyroid hormones. But even if the variant found in the panda were shown to have an incomplete loss of function that cannot be the entire explanation for the maintenance of low concentrations in plasma. If the pituitary-thyroid axis were operating as in a ‘normal’ mammal, the low thyroid hormone concentrations in plasma would lead to an increase in TSH from the pituitary with growth of the thyroid until sufficient thyroid hormone had been produced to reduce TSH production by negative feedback. In short, unless the plasma concentration at which TSH production is suppressed is lower in the panda, it matters not at all whether or not the synthesis of thyroid hormones is partially impaired.

That then raises another question: how is the concentration of thyroid hormones set for the negative feedback operating between those hormones and TSH release from the pituitary in different mammals? Could it be that they are set during ontogeny to the prevailing concentration of thyroid hormones, low in the case of the Giant Panda because of a reduced rate of synthesis? This is where comparative endocrinology, as a part of comparative physiology, needs to be brought in. These areas of knowledge have been grossly neglected in the last couple of decades as the world has leapt aboard the genome bandwagon, leaving important questions unanswered at those levels of biological organisation between the whole animal in its environment and the gene.


Tuesday, 18 August 2015

Dorothy Sladden (1907-1937): Ernest W. MacBride, Evolution and Eugenics. Part 4. At London Zoo

We know from the council minutes of the Zoological Society that when she died, Dorothy Sladden was working on Proteus anguinus, the cave-dwelling Olm. She must have been trying to see if she could repeat Kammerer’s experiments in which he claimed eyes developed when they were kept under alternating red light and daylight, or, possibly, at  the colour changes when they are kept in the light, but I have no further information.

E.W. MacBride had retired in 1934, presumably at the end of the year when he reached 65. He was still active on the Zoo council and must have played a part in obtaining a grant from the old Department of Scientific and Industrial Research (DSIR*) to support her work. It is also clear that she was continuing her work on stick insects up to the time of her death in June 1937, presumably at Imperial College. My estimate is that she moved to the Zoo (where, MacBride reported, she worked in the aquarium) sometime in 1935 since her observations on breeding Angel Fish were made in June1936.

The Zoo aquarium must have been an interesting place. The curator was Edward George Boulenger (1888-1946); he and his father George Albert Boulenger FRS (1858-1937) had examined aspects of Kammerer’s claims and were known not to believe them. E.G. had shown that Kammerer’s salamanders with different colour patterns, which were said to be the result of being kept on substrates of different colour, were more likely to be from different geographical races. So we have Boulenger, clearly not a convinced Lamarckian, an employee at the Zoo, Julian Huxley (not quite then but soon-and-desperate-to-be FRS), as Secretary, and a Darwinian, another employee but as chief executive, at the Zoo and we have Ernest MacBride FRS, arch-Lamarckist and Kammerer’s vicar on earth, a powerful figure on the Zoo Council. In the middle somewhere was Dorothy Sladden.

Angel Fish (from Wikipedia)
From her brief time at the Zoo Dorothy Sladden wrote one scientific paper, The breeding habits of the Angel Fish, Pterophyllum scalare, published in Proceedings of the Zoological Society  (A107, 187-189) published shortly after her death in 1937. She also started to write for the weekly magazine Water Life, first published in 1936. The articles appeared between April and June 1937 and can be seen in full here. Her first article was a reprise of her paper on breeding Angel Fish. It began:

Although the Angel Fish (Pterophyllum scalare) is bred in large quantities un Germany, so far little success has attended the efforts of the experts in this country. In view of this fact, the following notes on the conditions under which these fish were bred at the London Zoo may be of some interest.

Her next article, A Fantastic Toad at the Zoo, described the Escuerzo (Ceratophrys cornuta) given to the Zoo by a Mr J.J. Morris. Then she described British Adders at the Zoo:

The first warm days of spring see the re-stocking of the the Zoo’s outdoor reptiliary. Apart from the hardier species of reptiles imported from Southern Europe and obtained from a dealer, the Zoo relies on the efforts of the snake catcher of the New Forest, who annually catches several hundreds of British adders (Vipera berus) for the Society. Mr George Wateridge, successor to the New Forest’s former snake catcher, the well-known Brush Mills, has already sent thirty Adders to the Zoo…

Finally, she drew attention to A “New” Disease in Tortoises:

Among the many enquiries from pet keepers which have reached the Zoo’s Reptile House recently a large proportion deal with a very infectious disease among Tortoises which leads to blindness. The disease was first noticed in this country in 1935, among some newly imported Greek Tortoises.

I started this story because I came across her name while looking into Burgess Barnett’s departure from the Zoo in 1937. Therefore, while copying the articles she wrote in Water Life, it was fascinating to find that two photographs used to illustrate the articles, both shown here, were taken by Joan Barnett. This must be Burgess Barnett’s daughter, Joan, born in 1917, and then aged 20. My story had come full circle.



That article from the issue of 1 June 1937 was followed in the issue of 29 June by the announcement of her death as the result of a road accident. She had died two days before the date of issue of the magazine. The editor, Margery Elwin, must have been informed very quickly to get the news into print that quickly. Did she know Dorothy Sladden well? They were both about the same age, born in 1907 and 1908, both zoology graduates and both keen animal keepers with connexions to the aquarium at the Zoo. The reason I wonder is that Margery Elwin was, in her articles, sympathetic to Lamarckism but her membership of the Communist Party of Great Britain and the espousal of Lysenko in the years after the War is, perhaps, another explanation.

I have been unable to find any further information on Dorothy Sladden’s fatal accident. There must have been a report on the inquest into her death in a local newspaper but progress at scanning is now being done at such a very slow rate by British Newspaper Archive that I suspect it will be some time until I can find out what happened.

We also do not know whether Dorothy Sladden was a follower of MacBride or, like so many at Imperial College, even those involved with the work itself, openly or quietly dismissive of his attachment to Lamarck and Kammerer.

Finally, a plea for more information. I have been unable to find a photograph of Dorothy Sladden; if anybody has one or can point me in the direction of where I might find one I would be most grateful. I have found her mother and father (but not her) on a public family tree which covers her maternal line. I shall contact the owner, with these posts now complete, to see if there are any surviving cousins who might know more of her history.

As I said at the beginning of Part 1, this is a sad story. I hope I have done her justice in describing her contributions to the debate on how evolution happens that was raging in the early decades of the 20th Century, and have drawn attention to her skills as a zoologist who actually knew a lot about animals, real live animals.

This is her full bibliography (including the articles described above):

Dorothy Ena Sladden
1907-1937

Sladden, D.E. 1930. Experimental distortion of development in amphibian tadpoles. Proceedings of the Royal Society B 106, 318-325
Sladden, D.E. 1932. Experimental distortion of development in amphibian tadpoles. Part II. Proceedings of the Royal Society B 112, 1-12
Sladden, D.E. 1934. Transference of induced food-habit from parent to offspring. Part I. Proceedings of the Royal Society B 114, 441-449
Sladden, D.E. 1935. Transference of induced food-habit from parent to offspring -II. Proceedings of the Royal Society B 119, 31-46
Sladden, D.E. & Hewer, H.R.1938. Transference of induced food-habit from parent to offspring. III. Proceedings of the Royal Society B 126, 30-40
Sladden, D.E. 1937. Angel fish bred at the zoo. Water Life  (13 April 1937) 2, 172
Sladden, D.E. 1937. A fantastic toad at the zoo. Water Life (4 May 1937) 2, 211
Sladden, D.E. 1937. British adders at the zoo. Water Life (11 May 1937) 2, 217
Sladden, D.E. 1937. A “new” disease in tortoises. Water Life (1 June 1937) 2, 269.
Sladden, D.E. 1937. The breeding habits of the Angel Fish, Pterophyllum scalare. Proceedings of the Zoological Society of London  A107, 187-189

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*DSIR was abolished in 1965, its major function of supporting research in UK being taken over by the Science Research Council (SRC) under the Science and Technology Act. I applied to DSIR in 1965 and my grant to go to Hong Kong came from SRC.

Friday, 14 August 2015

Dorothy Sladden (1907-1937): Ernest W. MacBride, Evolution and Eugenics. Part 3. ...and Lamarck. Transference of Induced Food-Habit from Parent to Offspring in Stick Insects

This series of three papers all published in Proceedings of the Royal Society and communicated by MacBride established that food preference to match the prevailing source could be passed from parent to offspring. The background to the work is best left to Dorothy Sladden’s own words in the introduction to the first paper.



     The question of the possibility of the transference of an acquired habit from parent to offspring is of fundamental biological importance. That a series of experiments of this nature could be devised to produce a.conclusive answer to this question was originally suggested to me by Professor E. W. MacBride.
     In 1912 Professor MacBride working in conjunction with Miss Jackson (1915) began an investigation into the inheritance of colour in the stick-insect Carausius (Dixippus) morosus. During this investigation, which extended over a period of three years, as Professor MacBride has informed me, great difficulty was experienced in obtaining adequate supplies of privet (the food on which the insects were being reared) during the winter months. On account of this the insects were induced, not without difficulty and practically by a process of starvation, to feed on ivy. In this way a number of insects were lost, a few, however, reached maturity and eggs were deposited. When the young emerged from these eggs they took readily to the ivy. This seemed to indicate the inheritance of an induced habit, and it was felt that it would be of interest to conduct further investigations on these lines. Four years later Ling Roth (1916) who made a study of the growth and habits of the stick-insect, experienced the same difficulty in procuring privet during the winter and rediscovered the fact that under these circumstances ivy could be substituted.
     In attempting to induce a new food-habit it is necessary to employ an insect which is not rigidly monophagous and the stick-insect complies with this requirement in that it is polyphagous as shown by both MacBride and Roth. The usual food-plant employed in this country is privet.
The next difficulty encountered in any such experiments is the effect of inter-breeding which may introduce concealed characters from one or other parent into the offspring. The stick-insect is for long periods parthenogenetic, the males which are smaller than the females appear only at intervals. so that no crossing can occur during the duration of the experiment. The young when hatched from the egg closely resembles the adult except for size and colour. As in the lower Insecta there is no complete metamorphoses such as leads to the intervention of pupal stages, and the changes conditional on the growth of wings, which occur in Insecta generally, are absent, since the adult Carausius is wingless.
     In a problem of this nature it is necessary to employ large numbers of offspring and to extend the work over a number of generations. The life-cycle should consequently be reasonably short, Carausius morosus having a life-cycle of 9 to 10 months and producing an average of 513 eggs per parent individual (Roth, 1916) fulfils these requirements.

The stock of stick-insects she used had been kept in Britain for at least 20 years and had been fed exclusively on privet. Two types of experiment were done. The first, ‘presentation tests’, involved offering the novel food, ivy, over a period of 1-3 days after the insect was hatched. If the ivy was untouched, it was withdrawn, privet given for 1-2 days and the test with ivy repeated. This sequence was repeated until the insect ate ivy. For the first paper alone, she tested 1075 individual stick-insects; only 12 died. In the second type, ‘preference tests’, an individual on hatching was offered both ivy and privet over a period of several days until it showed a preference; this test was repeated twice more. Some individuals preferred privet, some ivy and others showed no preference.

The experiments began with stick-insects laying their eggs in autumn 1931 and continued until June 1937, the month of Miss Sladden’s death. The final of three papers was completed by H.R. Hewer who appears as co-author. MacBride added a footnote to the final paper which I cannot help but construe as patronising to both authors:

The tragic death of Miss Sladden occurred on 20 June 1937 when the final figures included in this paper were on the point of completion. The preparation of the manuscript is therefore solely the responsibility of Mr Hewer. It ought to be added that Mr Hewer deserves a considerable portion of the credit for Miss Sladden’s previous papers as he devised the type of experiment which she carried out.

H.R. Hewer
Hewer was Humphrey Robert Hewer (1903-1974); a student at Imperial College he was Lecturer and Reader there from 1926 until 1964 when he was given a personal chair. From 1941 until 1945 he was Chief Rodent Officer at the Ministry of Food (there was a huge effort to control rats during the war). From 1967 until 1974 he was Chairman of the Farm Animals Welfare Committee for what was then the Ministry of Agriculture, Fisheries and Food (now DEFRA); in that role he had a public spat with W.H. Thorpe (see below) on the housing of poultry in battery cages. He is best known for his work on seals.

The first paper contains the results that are the most convincing since the experiments on the offspring of some of the privet-fed and all of the ivy-fed adults were done at the same time of year. I am using a more limited range of results (those from the second generation on privet only and from the second generation given ivy only) than those shown in the summary of the paper since it was shown subsequently that the season of the year had an effect on the tests done on privet-fed individuals.

31% of hatchlings of the privet-fed adults took ivy at the first presentation, 46% at the second. By contrast, 78% of those from ivy-fed adults ate ivy at the first presentation, 19% at the second. That was a clear demonstration of the food being eaten by the adult having an effect on the acceptability of food by the offspring.

Differences were also evident in the preference tests in that of the offspring of privet-fed parents, 44% preferred privet, 35% were neutral (no-preference) and 21% preferred ivy. By contrast, of the offspring of ivy-fed parents, 28% preferred privet, 35% were neutral and 35% preferred ivy.

Studies were continued into succeeding generations of privet- and ivy-fed stick-insects. Dorothy Sladden did an enormous amount of tedious organisational and observational work through six generations. With hindsight, it is easy to criticise an over-complicated experimental design and the fact that the various groupings of animals are not very well explained. More seriously, although she used large numbers of animals for the tests (50 or 100), some of the groups were derived from eggs produced by very few adults, those that had survived or been kept to adulthood, opening the possibility not of genetic selection but of bias in the epigenetic (the term used widely) variability of individuals. So instead of n=50, say, the true n was sometimes 3, or 4, or 6, or 8.

Later results were confounded by seasonal changes in the acceptability of ivy versus privet which could have been related to the physical and chemical composition of the leaves at different times of year. At first sight there is a curious finding. Even those fed privet throughout accepted ivy to a greater extent in each succeeding generation to an extent such that in the 6th generation, privet-fed and ivy-fed mothers were producing offspring that were virtually identical in their acceptance of ivy; it was only in the generations before this that there were differences between the ivy-fed and privet-fed. Hewer’s calculations in the final paper suggest that the seasonal changes were seen in the willingness of privet-fed but not in the ivy-fed adults to accept ivy.

The claim in the summary of second paper that experiments in which the insects for several generations were forced to accept a new food-plant (ivy) and then reared to maturity on it, have shown that the offspring of each subsequent generation accepted the ivy more readily than did their parents, is not supported by the data. From the second generation (the first from ivy-fed parents) the percentage of those hatchlings accepting ivy on the first presentation was 78; in succeeding generations it was 94, 90, 100 and 82. Because of the confounding effect of season and the marked changes in the acceptability of ivy to the offspring of privet-fed parents, it is perhaps better to interpret the results of a step-change in a single generation to the acceptability of the food plant the parents were eating.

A ‘reversal test’ was done in which the offspring of parents fed on ivy for three generations were offered privet; 95% accepted it on the first occasion. It is a pity that the persistence of the effect was not studied. For example, if the offspring of ivy-fed parents were fed privet, would the young from that generation readily accept ivy? Is there a critical period for the feeding of ivy, during egg-laying, for example. But these questions are with hindsight; in the climate the the day, I can see why Miss Sladden pressed on with just more generations and a reversal test.

Enquiries were also made in India on what might be the natural food plant of the species of stick-insect and of the preference for other species of plant. Poplar was consistently refused but rose, lilac and blackberry were more readily eaten than ivy or privet, while fuchsia was on a par with ivy and privet.

Hewer, who wrote the last paper, ended with:

It is therefore difficult to escape the conclusion that the forced ivy-feeding has induced the increased ability to accept ivy in succeeding generations in this parthenogenetic insect…

So that was it, an apparent Lamarckian effect and one that was seized on in no uncertain terms by MacBride, who could never be accused of understatement, when he wrote a memoir in The Times newspaper on 28 June 1937, the day after Dorothy Sladden’s death.

MISS DOROTHY SLADDEN
THE PROBLEM OF FUNCTIONAL INHERITANCE
Professor E. W. MacBride, F.R.S., writes:—
     The recent death of Miss Dorothy Sladdcn in consequence of a motor accident was an irreparable loss to the science of experimental zoology. She was a trained investigator working in the aquarium of the Zoological Society, and supported by the Department of Scientific and Industrial Research. But the research on which she was engaged was no ordinary scientific problem; it was an attack on that most fundamental problem of life, the nature of inheritance.
    Long ago Herbert Spencer said that no biological question was of greater importance than the problem whether the effects of acquired habit were, or were not, carried over to the next generation. Darwin's theory that deviations from the normal occur in all directions, and that those which happen to fit the environment survive, for a time carried all before it. But as the years revolved the whole basis of Darwinism was destroyed by the "pure line" experiments of Johannsen, Agar, and Jennings: these showed that though such deviations occurred, they were not inherited; the deviating individual had exactly the same hereditary powers as the type. As further studies of extinct types and existing races were made in detail, the conclusion that the formation of species was due to the handing on of acquired habits became irresistible, and was adopted by all the leading palaeontologists and systematists. This view was exceedingly unwelcome to a certain school of biologists, and they refused to accept it unless actual experimental proof was furnished.
     This proof Miss Sladden set herself to provide.  With indomitable perseverance and
marvellous skill she pursued this end: her ability to keep animals in good health and to induce them to breed in captivity was extraordinary. She loved and studied them all her short life; this power was one of those inborn gifts of genius which are bestowed on few. After years of patient study she triumphed, and two of her papers were published by the Royal Society; a third and finally conclusive one was nearly finished when a cruel fate snatched her away.
     When the history of zoology comes to be written she will rank with McDougal [sic*] as one of the few who supplied the principle of functional inheritance with a basis of incontrovertible fact.

MacBride also referred to Dorothy Sladden in a letter to The Times of 7 May 1938 in a spat with R.A. Fisher. His customary—and completely erroneous—arguments were deployed and he ends:

The reaction to environment by constant repetition becomes slowly ingrained in the constitution of the stock was proved by my pupil, Miss Sladden, who was killed by a motor accident nearly two years ago, and to whose memory you devoted such a generous obituary notice in your columns. I suggest that Professor Fisher should study her papers in the Proceedings of the Royal Society…

MacBride was wrong; Dorothy Sladden’s work was not remembered ‘as one of the few who supplied the principle of functional inheritance with the basis of incontrovertible fact’. It has received the occasional mention in books on evolution and the epigenetic revolution.

When searching for places in which her work my have been cited I came across an online copy of the book written in 1952 by Philip Gilbert Fothergill (1908-1967), Historical Aspects of Organic Evolution; Fothergill was Senior Lecturer in Botany in what is now the University of Newcastle, then a constituent college of the University of Durham; it contained a description of Dorothy Sladden’s work although there was an error of interpretation and an incorrect table. I found the book a very strange read for 1952; it was written as if the modern synthesis had never happened. When I delved more, I found that a number of professional biologists had also not been impressed, although it has to be borne in mind that there was still a dichotomy of world views: Darwinian selectionist versus Lamarckist. Extracts from these reviews are in Wikipedia. A few were encouraging “the author has kept his personal opinions in the background and has considered the data without bias”; but others were damning: "there is no evidence presented [that] the author has understood or digested the great advances made in evolutionary theory during the past twenty years”; "biased by both the author's inclination to Lamarckism, and by his religious views”;”too uncritically accepting of Lamarckism, orthogenesis and John Christopher Willis' age and area hypothesis whilst ignoring evidence of their refutation”. Ernst Mayr wrote that the "book suffers from an overzealous belief in the inheritance of acquired characters [but] is most useful for a sympathetic account of neo-Lamarckians.” Fothergill, a native of Dumfries was elected to the Royal Society of Edinburgh in 1955. He wrote a number of articles for catholic magazines and newspapers in which he tried to explain bible stories in biological terms and how evolution was compatible with catholic beliefs. Fothergill was clearly not my sort of guy.

Dorothy Sladden’s work has been confirmed in insects, in the butterfly Pieris rapae**, for example.

Even soon after publication of her work, suggestions as the the non-genetic mechanism that might be involved in what would clearly fit into the category of maternal effect were appearing. For example, W.H. Thorpe in Cambridge suggested†:

But it seems worth considering whether the mechanism of pre-imaginal conditioning, such as has here been demonstrated, might not explain some of the results of Sladden and Hewer on the transference of the induced food habit of the stick insect Carausius morosus. If we assume that the new food plant, i.e. ivy, contains a chemical substance of particularly persistent and penetrating qualities, it might be possible to imagine enough of this substance being contained in the eggs to produce positive conditioning in the newly emerged nymph. This might be particularly the case in those insects in which the first food consists of the empty egg shell.

Julian Huxley initially had difficulties with the Sladden experiments in Evolution: The Modern Synthesis, first published in 1942:

There remain one or two results, such as that of Metalnikov (1924) on immunity in waxmoths, and of Sladden and Hewer (1938) on food-preferences in stick insects which seem prima facie to demand a lamarckian explanation (but see p. 303 n.). However, in view of the fate of other claims, and of the theoretical difficulties we shall discuss below, too much weight must not be attached to such isolated cases.

I have the third edition (1974) and I do not know whether the footnote on page 303 appeared in the first edition. It reads:

As Thorpe (1939) suggests, these results may also explain the interesting results obtained by Sladden and Hewer (1938) on the food-preferences of stick insects, for which, prior to Thorpe’s work, a lamarckian interpretation seemed almost inevitable. It will be of great interest to test Sladden’s results in the light of Thorpe’s methods, and with a species capable of sexual reproduction.

Huxley’s when he first wrote of Sladden’s results, was falling into that terrible trap of dismissing anything that did not fit what he interpreted as the explanation for the mechanism of evolution. Such a statement as, ‘too much weight must not be attached to such isolated cases’, diminishes, in my mind, Huxley as a scientist; the consensus of scientists is x; therefore, any other evidence can be ignored, was Huxley’s stance. Now where have I heard before?

Huxley does though demonstrate again the complete split in biological thought at the time: if there was a hint of transmission of a change in character of the parent to the offspring, it must be Lamarckian since only one pathway of  heredity was known or considered, that of alteration to the germline.

However, by the time he wrote the footnote, the penny must have been dropping on the notion of transmission of useful information to the offspring by the mother that does not involve alterations to the germline. It is odd that in the main text, he deals with plasticity in relation to olfactory conditioning, and the limits to plasticity ‘hedged about by genetic safeguards’, but only later adds Thorpe’s (1939) interpretation of the Sladden results as, in different words, an epigenetic phenomenon, in a footnote.

Miss Sladden was working at the Zoo when Huxley was Secretary and he must surely have discussed the results with her before her death with or without the presence of MacBride who was on the Zoological Society’s Council but who had retired from Imperial College. But clearly, he had not considered any explanation of her findings other than a classic Lamarckian one when he wrote his book. 

I know insufficient insect physiology to know whether the mechanism has now been elucidated but going back to the increased acceptance of ivy with succeeding generations of privet-fed parents, which Hewer ascribes to a seasonal effect, could it be that at the outset—a time when the stick-insects were reluctant to accept ivy—there was no ivy in the laboratory whereas if the privet- and ivy-fed stock were kept in close proximity could it be possible that the privet-fed could have been ‘conditioned’ by the odour of ivy?

The sort of maternal effect that Dorothy Sladden found is analogous to the findings in human infants where the diet of the pregnant mother has been shown to affect the food preferences of the young. There was a general article in Science (345, 750-751) in 2014 on the topic although it failed to give credit to workers other than those mentioned.

Whether one calls such phenomena as adaptive parental epigenetic effects Lamarckian is open to debate. Some people do. I do not because to me Lamarckism was viewed as such an alternative to the modern synthesis and espoused with such wrong-headedness that it is a term which reflects a concept from a bygone age that has been eliminated by natural selection; the fittest concept has survived and can easily accommodate additions such as epigenetic mechanisms that have been uncovered (although some clues, like the one Dorothy Sladden discovered, were there) since the genesis of the modern synthesis more than 70 years ago.

These are the three papers:

Sladden, D.E. 1934. Transference of induced food-habit from parent to offspring. Part I. Proceedings of the Royal Society B 114, 441-449.

Sladden, D.E. 1935. Transference of induced food-habit from parent to offspring -II. Proceedings of the Royal Society B 119, 31-46.

Sladden, D.E. & Hewer, H.R.1938. Transference of induced food-habit from parent to offspring. III. Proceedings of the Royal Society B 126, 30-40.


The fourth and final part of this series will cover Dorothy Sladden’s brief period at London Zoo.


--------------

*He was referring to William MacDougall FRS (1871-1938)
**Hovanitz, W. & Chang, V.C. 1965. The alteration of host plant specificity in larvae of Pieris rapae by induction. Journal of Research on the Lepidoptera 4, 13-21.
†Thorpe, W.H. 1939. Further studies on pre-imaginal olfactory conditioning in insects. Proceedings of the Royal Society B 127, 424-433.


Tuesday, 11 August 2015

Dorothy Sladden (1907-1937): Ernest W. MacBride, Evolution and Eugenics. Part 2. Frogs’ eggs, sports and monstrosities

As well as for the inheritance of acquired characters, MacBride was also looking for environmental factors that caused mutations, in order, it seems, to provide an explanation for the findings of the Mendelian geneticists. The basis is explained by the opening paragraph of his article, The work of Tornier as affording a possible explanation of the causes of mutations, written in 1924 for Eugenics Review (15, 545-555):



Gustav
Tornier
In a previous communication to the Society I gave an outline of recent discoveries which tended to show that the effects of habits acquired during the lifetime of the individual were transmitted to posterity. Since that time more and more evidence pointing in the same direction has come in, and finally the recent experiments of Pavlov may be regarded as decisive on the question. The inheritance of environmental effect is therefore a vera causa, and has probably been the chief, if not the only cause, of the evolution of the plant and animal kingdoms, but it is certainly not the only cause of variation. The conspicuous deviations from the normal, known as sports or monstrosities, which appear suddenly, which are inherited strongly, and which "mendelize" when crossed with the type, still demand an explanation of the cause of their occurrence. It is on characters such as these that the breeder seizes when he wishes to produce a new strain; and it is the offspring of similar "sports" in the human race that fill the slums of our great cities.
     To say that these sports owe their origin to "mutations" in the chromosome-complex of the type is merely playing with the question; it is, in the words of Darwin, to substitute a form of words for an explanation. Granted that if the hereditary potentiality of a stock is changed, the nuclei of the germ cells have undergone a change; to assert this is merely to push the difficulty one step further back. What has changed the nuclei? Till we can answer that question all talk of an explanation of mutations is futile.
     …In view of these facts it occurred to Gustav Tornier [1858-1938; he worked in Berlin] that there must be some general underlying cause for the production of these abnormalities. It seemed to him futile to attribute each one to the separate appearance by chance of a '*factor." He was thus led to formulate what Milewski calls an “epoch-making theory” to the confirmation of which Milewski has himself largely contributed. Like the Mendelian theory itself the Tornierian hypothesis did not at first attract the notice to which it was entitled. This was due to two causes—first because the theory conflicts with all preconceived ideas as to the origin of mutations, and secondly because Tornier has published his results for the most part in journals which have not a sufficiently wide circulation to reach zoologists in general. Tornier's theory is as follows:—Every embryo is endowed at the beginning of its existence with a certain quantity of protoplasmic energy. This energy manifests itself in two ways: (1) by the early beginning and vigorous character of the movements of the embryo; (2) by the ability of the embryo to resist the tendency of all its tissues, especially those of a less active growth, to absorb an excess of water—in a word to regulate the intake of water. In practically all eggs the portion of less active growth is that in which the yolk globules are stored, and hence Tornier speaks rather too loosely of an absorption of water by "the yolk" and its consequent swelling. The swelling is not confined to yolk-containing cells, nor strictly speaking is it the yolk itself which swells, since this consists chiefly of globules of a lecithin-like substance insoluble in water. The very same swelling takes place in mammalian embryos in which there is no yolk. Davenport and Parker showed long ago that a large part of the increase in bulk which embryos undergo during the earlier period of their development is due to the imbibition of water.
     Now according to Tornier, when an embryo is exposed in the earliest period of existence to scarcity of oxygen, the protoplasm becomes weakened and is not able to prevent the overswelling of the less active portions of the body by water. This abnormal swelling— since the embryo is confined within a relatively inextensible membrane—leads to pressure on the actively growing parts which impedes their growth, distorts their shape, and in extreme cases, prevents their growth altogether. If we examine in detail its effects in goldfish embryos we find that all the natural cavities of the body, such as the buccal cavity, the gill cavity (beneath the operculum), and the body cavity become enlarged beyond their natural sizes, whilst strong pressure is exerted by the swollen yolk-sac against the growing embryo in an antero-posterior direction, so as to impede the natural tendency of the embryo to grow in length.
     …The interest in all studies of heredity to the Eugenics Education Society is, of course, their   applicability to human conditions.
     Tornier's work suggests strongly that bad conditions during the period of conception and the early phases of development may be the original cause of the degenerative mutants in man, including under that head not only mental defect, but haemophilia, night-blindness, colour-blindness, &c. But the important fact is that, however caused, the plasma-weakness is handed on to posterity. It is the object of negative eugenics to prevent these plasma-weak stocks from adding their quota to the race, by discouraging their reproduction. In the future we may hope that eugenics, in conjunction with medical research, may perhaps detect and isolate the specific causes of plasma-weakness, and thus diminish, if not entirely prevent, the appearance of plasma-weakness in the race.

It is difficult, when looked at from 2015, to believe that this was written by anything other than a crank. It is also difficult to believe, again when looked at from 2015, that the work in the first two papers, on the development of frog embryos after an environmental insult, were other than fanciful and difficult to not believe that they were thought as fanciful at the time by those not involved directly with MacBride. The introduction to Dorothy Sladden’s first paper summarises the thoughts of Tornier as expressed in MacBride’s article and explains the line of reasoning for the experiments she did:

In 1908, Tornier published a paper on the probable causes of the formation of the abnormal "fancy" races of goldfish. All these races originated in China where the wild ancestor (Carausius [sic] auratus*) still abounds in the streams, It had been supposed that these races were produced by a secret process known only to the Chinese breeders. The fish during winter were kept crowded in earthenware pots, on shelves in dark and insanitary huts; in summer they were transferred to small and filthy tanks overgrown with weeds. In these tanks they spawned and much of the spawn perished; amongst the fraction which survived, however, all sorts of abnormalities were found. By selecting the most striking of these, the breeders secured the parents of their “fancy" breeds, which showed in every succeeding generation a strong tendency to revert to the normal; only by the most rigid selection was anything like a "pure" race obtained.
    Tomier drew the conclusion that, the abnormalities were due to the effects of lack of oxygen in very early stages of development. This lack induced what he called "plasma-weakness" in different parts of the formative area of the very young embryo. In consequence of these localised areas of weakness the protoplasmic part of the egg was liable to mechanical distortion, induced by abnormal pressure from the yolk ; the latter absorbed water and swelled, thus crushing and destroying the protoplasmic structures from which the future organs of the adult arc formed.



The practicalities of the initial experiments involved keeping frog eggs in a 10% sucrose solution for four hours and seeing how the eggs and tadpoles developed. the sucrose solution was used in the belief—as in Tornier’s experiments outlined in MacBride’s article—that it would reduce the concentration of oxygen in the water sufficiently to affect the developing embryo. This is the summary of that paper:

Eggs of Rana temporaria were exposed, at the end of segmentation, 24 hours after fertilisation, to a 10 per cent, solution of sugar in tap-water for 4 hours; they were then transferred to normal aerated water. The resulting larvae exhibited marked structural abnormalities, although these might not be obvious for a prolonged period, e.g., 3 to 4 months after fertilisation. These abnormalities have, been described as (a) distention of body-cavity; (b) rupture of gut and extrusion of yolk; (c) flexure of tail; (d) distortion of sacral region; (e) non-appearance of limb.

However, in the second paper it was realised that the effect of immersion in sucrose was probably caused by an osmotic effect rather than lack of oxygen (I calculate the oxygen concentration in the water would have been reduced by only 9%):

Summarising these somewhat inconclusive results, it would seem that the effect of sugar is not the removal of oxygen as previously assumed, but very possibly osmotic action and is responsible for the early abnormalities in particular, while pH decrease may be responsible for the later ones…It now seems possible, however, that overcrowding may have seriously affected all the previous results.



The experiments show nothing other than when frogs’ eggs are damaged, the tadpoles do not develop normally, although I am sure that MacBride really fancied the notion that dirty conditions lead to ‘mutations’ (as proposed by Tornier for the genesis of the domestic strains of fancy goldfish) which are then embedded forever in the working class unless expunged by negative eugenics, and that is the reason why the newly-graduated Miss Sladden was given the project.

Dorothy Sladden’s skills in rearing frogs through metamorphosis are evident and were obviously put to good use by others in in the department. For example, Peter Gray in a paper on development of the development of the amphibian kidney also published, like her first paper, in 1930, thanks her profusely: ‘indeed, it is to her skill in the rearing of animals under artificial conditions that I am indebted for the greater part of my material.”

The studies described here have nothing of importance that I can see. By contrast the next set of experiments she did produced biologically significant results and should be seen in a different light to those on frogs' eggs. I will deal with them in my next post, Part 3.



The two papers are:

Sladden, D.E. 1930. Experimental distortion of development in amphibian tadpoles. Proceedings of the Royal Society B 106, 318-325


Sladden, D.E. 1932. Experimental distortion of development in amphibian tadpoles. Part II. Proceedings of the Royal Society B 112, 1-12

*She was confusing the name of the goldfish (Carassius auratus) with the organism she wroekd on next, the stick insect (Carausius morosus)

Dorothy Sladden (1907-1937): Ernest W. MacBride, Evolution and Eugenics. Part 1

This is a sad story. It is also a much longer story than I thought at the outset and several posts will be needed to cover it; this is the first.

On 25 January on my other site dealing with the history of keeping reptiles and amphibians I noted that I had found a short series of articles on fishes, reptiles and amphibians at London Zoo in volume 2 of Water Life magazine published in 1937, written by a Miss D.E. Sladden, and that the final issue of the volume contained the news that Miss Sladden D.I.C., C.M.Z.S. had died as the result of a road accident. The report in Water Life continued:

She had been engaged in research work at the London Zoological Gardens, and was particularly interested in tropical fish. Shortly before she died, she finished a paper on breeding Angel Fish, which gives fuller details than any previously written on this subject, and is a valuable contribution to fish-keeping knowledge…

I then found that Dorothy Ena Sladden of The Poplars,  2 Somerset Road, Brentford, Middlesex died on 27 June 1937, aged 29, in Hounslow Hospital, leaving £858.4s.11d. At that stage I could not find a record of her birth in England or an entry in the 1911 Census. This, together with the fact that she was described by Water Life as a C.M.Z.S. (Corresponding Member of the Zoological Society of London) suggested to me that she was born overseas and came back to Britain. However this was not the case since by searching for the person granted administration of her estate, Edward Cuthbert Sladden, tailor and outfitter, I found in the 1911 Census an Edward Sladden, Gents outfitter with a daughter, Dorothy, living at 17 Clifden Road, Brentford, Middlesex. From there it was easy to determine that Dorothy Sladden was born in the fourth quarter of 1907 in the Dartford Registration District of Kent, the daughter of Edward Cuthbert and Annie Isabella (née Oudney). I cannot find the birth of any siblings. One child of the marriage (five years earlier) is recorded in the 1911 Census. After 1912, the mother’s surname was recorded in the registers and there are no Sladden births recorded with an Oudney as mother.

Dorothy Sladden’s mother died on 9 February 1939, aged 62; her father died on 13 November 1961, aged 83, leaving £8784; probate was granted to Barclays Bank as executors of his will.

I looked up the additional genealogical details because I came across an intriguing item in the minutes of the Council of the Zoological Society when I was searching for information on Burgess Barnett. At the meeting on 21 July 1937, Council noted with regret the death of Miss Sladden and passed condolences to her parents. But Professor E.W. MacBride then said that Miss Sladden had been working on Proteus and that it was important that a replacement be recruited to carry on the work. At this time MacBride had taken up the cudgels on behalf of Burgess Barnett and was attempting to get him reinstated as Curator of Reptiles.

Proteus could only refer to Proteus anguinus, the Olm, Knowing that MacBride was a Lamarckian supporter of Kammerer and his memory and that Kammerer had worked on P. anguinus as well as on the Midwife Toad, Alytes obstetricans, I guessed that Miss Sladden was working on an attempt to replicate Kammerer’s experiments and that she was some way working with MacBride. A Google search linking Sladden with MacBride came up trumps. Before describing the work she did, it is worth considering the state of evolutionary theory and research in the early decades of the 20th Century, especially in Britain.

While evolution was on firm ground, natural selection was not. With hindsight—and this area has been covered extensively by historians, often not that well—progress was blocked by individuals in positions of power and influence defending fixed positions to the bitter end and refusing to accept that evidence could come from the emerging field of genetics; equally the geneticists were split between those who could envisage evolution occurring gradually or in sudden steps. Not until the ‘modern synthesis’ (the term invented by Julian Huxley in his book of 1942) was it realised that Darwin, Mendel and the advances in population genetics could be brought under the one all-embracing concept of how evolution happens.

Proteus anguinus - the Olm          View image | gettyimages.com
                                                  Proteus anguinus

Ernest William MacBride FRS was from 1913 until he retired in 1934, Professor of Zoology at Imperial College, occupying T.H. Huxley’s old chair. I am using Imperial College here as a convenience because papers from his department give the Royal College of Science as the address. The Royal College of Science was a constituent of Imperial College from 1907.

E.W. MacBride
MacBride not only defended Lamarckian evolution; he used his interpretation of it to promote extreme eugenic views. The picture that emerges is of a publicly combative but personally supportive individual. This is what Lancelot Hogben FRS (1895-1975) had to say about him (Lancelot Hogben. Scientific Humanist, edited by Adrian and Anne Hogben, Merlin Press, 1998):

While I was at Imperial College, the head of my department was Professor E. W MacBride. He had long since passed from the dynamic to the prostatic phase of the life cycle of a professional man. Till the beginning of World War I, he had carried out research on the embryonic development of sea urchins, starfishes and other marine forms. In the course of the war, he had relapsed into mental hibernation during the colder part of the year and aestivation during the warmer half. By birth he was an Ulsterman, and in a precocious dotage, he had exchanged the Calvinism of his forefathers for eugenics and had become a pillar of the Eugenics Society. This was a circus of snobs and racist cranks.
     In my first term at Imperial College, MacBride invited me to attend a lecture he gave on behalf of the Society. To improve the race, he urged on his audience the desirability of legislation to make sterilisation by vasectomy obligatory for males earning less than £400 a year. At that date my own modest salary was £350 and I had already committed paternity—and quite legitimately. On the whole though we got on well together. My output of published work from his department was prolific. It gave him something new to talk about at lunch in the Athenaeum Club, one of the geriatric wards of the Establishment.

Hogben omits to mention, by the way, that the ranks of the Eugenics Society were filled by men of the left as well as those of the right.

William Thomas Calman FRS (1871-1952) wrote MacBride’s obituary notice for the Royal Society. Unlike the many hagiographic efforts characteristic of the time, Calman clearly tried to achieve a balanced view:

Already, in a paper published in 1895 on 'Sedgwick's theory of the embryonic phase of Ontogeny as an aid to phylogenetic theory', MacBride had declared his adhesion to Lamarck's views on the causation of evolution—changes in the environment of organisms leading to changes in habits, and these, in turn, through the inherited effects of use and disuse, to changes of structure. In later years, the support of the Lamarckian theory became one of the dominant interests in his life. He championed it unceasingly, uncompromisingly. and, it must he confessed, sometimes uncritically, and expounded with characteristic vigour what he believed to be its social implications. He welcomed with an interest that came dangerously neat credulity any experimental results tending to demonstrate inheritance of impressed modifications, brushing aside as irrelevant the objection that ‘the more unequivocal the experiments devised to demonstrate its reality the more clearly do they show it to be of so fugitive a kind as to have no significance in evolution'. He also glossed over what still seem to some the chief flaws in the Lamarckian theory, the very limited structural changes that can he produced by use and disuse and the fact that they do not seem to be, of necessity, adaptive  in  nature. But, as sometimes  happens with ardent controversialists, he was much less extreme in private conversations than when writing or on the platform. And, after all, over-statement is not confined lo the defenders of Lamarckism. Has it not been stigmatized as 'a doctrine supported by far less positive evidence than exists for the reality of witchcraft’?
     There is no space here, neither is there any need, to do more than mention the many diverse fields other than zoology over which MacBride's activities extended, philosophy, sociology, eugenics, even theology. Sometimes, it is to he feared, he ventured into regions where his reach exceeded his grasp. He had the type of mind that can never rest content with the narrow outlook of the specialist but must always try to see even the larva of a starfish in its relation to the universe. It is an attitude that will never be understood by those biologists, be they systematists or geneticists or what not, who arc content to spend happy and laborious lives exploring the details of their specialities and are 'not interested in evolution'. Even the champions of lost causes and forsaken beliefs fulfil a useful function if they keep youth from forgetting that there are some questions on which the last word has still to be said.

In an earlier paragraph Calman writes of MacBride’s style of management:

One distinguished pupil who has had unusually wide experience writes; 'MacBride was a most admirable director of a research department and took a greater personal interest in the work of his students than any professor I have ever known—although, in practice it was sometimes a little wearing, as I felt that one was expected to have spectacular results to report every morning at 11.30 a.m. However, MacBride cultivated a most friendly atmosphere in which all the research students were interested in each other's work and were ready to discuss it and to give and receive help all round in a way that I have not met with anywhere else.' One who served under him as a lecturer says: 'One thing that can be said about MacBride is that he was an ideal chief. Even those of his subordinates, who like myself disagreed most strongly with his ideas always entertained the most sincere appreciation of his kindness and consideration in that capacity.’

My guess is that the lecturer referred to by Calman was Harold Munro Fox FRS (1889-1967). In his biographical memoir, Sir James Eric Smith FRS (1909-1990) wrote:

In 1913 when Fox was a lecturer at the Royal College of Science he brought to Professor MacBride’s notice the work of Paul Kammerer which purported to show from his experiments on salamanders, frogs and the ascidian Ciona that characters acquired in response to the environmental conditions in which they were kept or as a result of certain kinds of operative treatment were transmitted to their offspring. MacBride's stubborn advocacy of the inheritance of acquired characters is well remembered, and he was not disposed to think well of young men who strongly opposed his views on the subject. It says much for Fox's courage therefore that, while still in MacBride's department, he drew attention to the essential weakness of Kammerer's experiments which had taken little account of the need to rear the offspring under conditions which eliminated the possibility of their acquiring their characteristics as environmental adaptations in the same way as had their parents. After the war Fox repeated Kammerer's experiments on Ciona. This involved the amputation of the branchial siphons, after which (Kammerer had said) the siphons regenerate to a greater length, the animals thereafter transmitting this change to their offspring. Fox showed, however, that if the animals were kept in similar culture conditions before and after the amputation of the siphons there was no increase in length of the regenerated structures. However, an increase in the food supply induced the growth of long siphons and this happened both in siphon-amputated and in uninjured animals.

MacBride is still remembered for his opposition to the then new science of genetics. The history of the John Innes Centre notes the founding of the Journal of Genetics by William Bateson FRS (1861-1926) who also coined the word genetics:

The Journal was an important focus for British geneticists who faced powerful opposition from EW MacBride, Professor of Zoology at Imperial College, London, and a member of the governing body of John Innes (from 1919)[Bateson was its first Director] and Karl Pearson, Professor of Applied Mathematics and Mechanics at University College. ‘They stood at the entrance of the Royal Society like the leographs which guard the portals of a Burmese Buddhist temple’ (Crew, 1969).

Bateson was a leading opponent of Paul Kammerer and had been present at a private meeting with Kammerer in 1923. Cock and Forsdyke in their book, Treasure Your Exceptions: The Science and Life of William Bateson (Springer, 2008) wrote:

H Graham Cannon wrote in 1959 that there had been a private meeting in MacBride’s room at the Royal College of Science, with just four people present: MacBride, Kammerer, Cannon and Bateson. Kammerer produced his Alytes [Midwife Toad] specimen in a glass tube and MacBride examined it “literally for a couple of seconds” (those who are already converts are easily convinced!) before passing it on to Cannon, and in turn to Bateson, who examined it with a hand-lens. Cannon then alleges that Bateson then said to him, sotto voce “It looks to me like a spot of black ink.” If this is correct, then Bateson’s comment showed a quite remarkable prescience, for three years later the nuptial pad on the same specimen was shown to have been injected with Indian ink. However, it seems likely that this part of Cannon’s account owes more to his own creative hindsight than to Bateson’s prophetic powers.

I do not know how Cock and Forsdyke came to make the last comment. Herbert Graham Cannon FRS (1897-1963), in MacBride’s department in 1923, much later turned to re-interpreting Lamarck’s work, also believing the ‘modern synthesis’ based on Mendelian genetics was an inadequate explanation for evolution and that a correctly interpreted account of what Lamarck actually said—which I think in modern terms would be extreme phenotypic plasticity rather than inheritance of acquired characters—was more likely, with characters appearing in any generation to respond to a change in environment.

The 1920s and 1930s saw MacBride fiercely defending Lamarckism but also looking for evidence of the sort that he thought Kammerer had obtained for the inheritance of acquired characteristics.

That is where Dorothy Sladden came into the picture. She seems to have worked for and/or under MacBride since 1928 (when she would have graduated at the age of 21). At some time before 1933 she was awarded the Diploma of Imperial College (D.I.C.), a qualification approximately equivalent to that of a Master’s by research.

Lamarckists like MacBride would interpret any environmentally-induced change in the adult which appeared in the offspring, however small, as evidence in their favour. And this is where some of the experiments done, dismissed or ignored by the Neo-Darwinians, become interesting. The distinction could not, or was not, then be drawn between the inheritance of an acquired characteristic by alterations in the genome (the Lamarckian interpretation) or by non-genomic, or epigenetic mechanisms. Only in the past twenty years has the extent of epigenetic inheritance become well known and indeed fashionable, largely, I would argue because of David Barker’s work indicating, from historical human epidemiological evidence, the relation of differences in fetal growth to differences in the incidence of diseases in later life. The evidence for maternal, intergenerational effects in plants and animals had been accumulating for decades and had really been brought to more general notice by the work of plant and animal breeders—the quantitative geneticists—who needed to distinguish between the effects of genes and the non-genetic effects that were interfering with their experiments. Some acquired characteristics were inherited but not through the genes.

But in the 1930s, any putative demonstration of a maternal effect—‘where the phenotype of an organism is determined not only by the environment it experiences and its genotype, but also by the environment and genotype of its mother’—would be seized on by the Lamarckists as evidence in their favour but dismissed by the Darwinians as irrelevant or simply wrong. That adaptive maternal effects, where mothers sense environmental conditions and transmit signals to their offspring through materials in their eggs or through the placenta or through milk in order to enable them to adapt more quickly to the environmental conditions in which they are born or hatched would be discovered, for example, or through affecting the activity but not the underlying DNA sequence, would simply have been beyond the ken of either faction at the time.

So it was work on alterations in the environment that brought about changes in the offspring that Dorothy Sladden was looking for. I found five papers by Dorothy Sladden on work she had done in MacBride’s department at Imperial College from 1928 when she would have been 21, before moving to the Zoo, I presume in 1936. It is clear from the papers that the original and general ideas for the experiments were generated by MacBride. This is what she had to say on one of her papers on frogs’ eggs:

I would like to express my deep gratitude to Prof. E. W. MacBride not only for the great encouragement and assistance which he has given me, while I have been working under his supervision, but also for his original suggestion that this line of research could be profitably followed.

In Part Two of this long story, I will consider the first topic of Dorothy Sladden’s research.