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Tuatara aims to stimulate and widen interest in the natural sciences in New Zealand, by publishing articles which, (a) review recent advances of broad interest; or (b), give clear, illustrated, and readily understood keys to the identification of New Zealand plants and animals; or (c), relate New Zealand biological problems to a broader Pacific or Southern Hemisphere context. Authors are asked to explain any special terminology required by their topic. Address for contributions: Editor of Tuatara, c/o Victoria University of Wellington, Box 196, Wellington, New Zealand. Enquiries about subscriptions should be sent to: Business Manager of Tuatara, c/o Victoria University of Wellington, Box 196, Wellington, New Zealand.
(This issue edited by
is the journal of the Biological Society, Victoria University of Wellington, New Zealand, and is published three times a year. Editor:
Since the beginning of this century the chromosomes of the shorthorned grasshoppers (Family Acrididae) have been used for a vast number of cytological studies. These chromosomes present a number of advantages to the cytologist.
They are large and relatively few in number.
The range of chromosome lengths in the complement is such that each bivalent formed at meiosis can usually be individually identified according to its length.
Chiasmata are very clear during diplotene and diakinesis thus allowing analyses of their structure, frequency, distribution and movement.
Often the position of the centromere is marked by relatively denser staining (precocious condensation) at early diplotene.
Besides these cytological advantages, the techniques involved in the preparation of slides of this material are quick and simple and therefore it is ideal for demonstrating the stages of meiosis to students.
The purpose of this article is to suggest suitable New Zealand species of grasshoppers, outline dissection and cytological techniques, and to give a brief description of the chromosomes and the stages of meiosis.
The family is represented in New Zealand by fifteen species (Bigelow, 1967) of which fourteen are endemic and one is the worldwide species. Locusta migratoria.
The most common species is Phaulacridium marginale which is found in dry grassland below 3000 feet over the entire country. Except for one South Island species which is found in a few restricted lowland localities, the rest of the endemic species are alpine. The only North Island alpine grasshopper is Sigaus piliferus which inhabits tussock in the Tararua Ranges, the East Coast ranges, the mountains of the Coromandel Peninsula, and Mounts Tongariro, Ngaruahoe and Ruapehu (but, surprisingly, not Mount Egmont). The other alpine species are found in tussock and on the screes of the South Island ranges. The distribution of the more common of these species is given in figure 1. A more detailed account of the taxonomy and
loc. cit.). Locusta migratoria, while also suitable, does not have a very wide distribution and is far less common than P. marginale. Also, since it is the only winged member of the New Zealand Acrididae, it is rather more difficult to catch.
From the point of view of availability, P. marginale is the best species to choose. However a comparison of the cytogenetics of several New Zealand species (Martin, 1970) showed the chromosomes of the alpine species to be slightly longer, with a higher number of chiasmata per cell, than P. marginale. For this reason the alpine grasshoppers are recommended if they are available.
If P. marginale is being used it can be distinguished from another lowland grasshopper, the long-horned grasshopper (Family Tettigoniidae), by the fact that the antennae of the former are only about 4 mm. in length whereas those of the Tettigoniidae are about 25 mm. long. The alpine short-horned species are the only grasshoppers found at high altitudes.
Young adult or last instar males (females are not suitable for studying meiosis) provide the best testis material. The males and females can be distinguished by the morphological differences shown in figure 2 and also by the fact that the males are smaller than the females. The grasshoppers can be caught by hand or with the aid of a small net. They are available from late November to early April during which time the spermatogonia are dividing rapidly.
The insects are chloroformed or etherised and then dissected in insect saline (see below). The testes lie in a dorsal position in the anterior half of the abdomen and can be easily located by making a dorsal, longitudinal, abdominal cut. They can be identified by the orange-yellow fatty tissue that cover them. Once this is removed with dissecting needles (while still in the insect saline) each testis can be seen to consist of many follicles.
The best fixative for these preparations is 1 : 3 acetic alcohol (see below). The testis material is left in the fixative for at least five minutes before one of the staining procedures described below is carried out. The material can also be stored before being stained either in the fixative or by transferring it to 70 per cent ethyl alcohol after fixation. It will keep satisfactorily for at least a year either at room temperature or, preferably, in a refrigerator.
Place a small drop (about 5 mm. in diameter) of the stain in the middle of a clean slide.
Take three or four testis follicles from the fixative (or alcohol), drain off excess moisture on a piece of blotting paper, and leave in the stain for three to five minutes.
After this time the follicles are broken up by firmly tapping them with a metal or glass rod until there is only a suspension of small particles in the stain.
After any remaining large pieces of material have been removed, a clean cover-slip can be applied.
Heating the slide gently over the flame of a spirit lamp at this stage will flatten and spread the chromosomes. The stain must not boil.
The slide should then be placed between two pieces of blotting paper which is firstly pressed down lightly so that excess stain around the edges of the cover-slip is absorbed, and then the preparation is squashed by firm, vertical, thumb pressure. Avoid any sideways movement.
If the cover-slip is ringed with petroleum jelly the temporary squash preparation will keep for at least ten days.
The material is transferred from the fixative or alcohol to the stain (in a small stoppered vial) for 24 hours.
After this time the material should be transferred to 70 per cent alcohol and stored in the refrigerator until needed.
Preparation of the slide is as above except that the material is squashed in a drop of 45 per cent acetic acid.
If desired, temporary squashes can be made permanent using a method similar to that of Conger and Fairchild (1953).
Freeze the preparation by inverting the slide on a flat piece of dry ice for 30 seconds to one minute.
Without letting the preparation thaw, prise the cover-slip off with a scalpel or razor blade. The material should remain attached to the slide but if some does remain on the cover-slip this can also be remounted on a clean slide by the same procedure.
Immerse the slide, while the material is still frozen, in absolute ethyl alcohol for ten minutes.
Place the slide in fresh absolute alcohol for at least another ten minutes.
Put a small drop of euparal in the middle of the squashed material and apply a clean cover-slip.
Heat the slide gently to remove any large air bubbles and then leave it in a dust-free place to harden.
Insect saline: 7.5 gms. NaCl per litre of water.
Fixative: 1 part glacial acetic acid: 3 parts absolute ethyl alcohol.
Acetic-orcein: 2 gms. synthetic orcein.
45 ml. glacial acetic acid.
15 ml. distilled water
Boil gently until the orcein has dissolved. Cool and then filter.
Snow's HCI carmine: 4 gms. carmine.
15 ml distilled water
1 ml. concentrated HC1.
Boil until the carmine dissolves. Cool, add 95 ml. 85 per cent ethyl alcohol, and then filter.
A recent investigation on the cytogenetics of some New Zealand grasshoppers (Martin, loc. cit.) showed that in five species (P. marginale, S. piliferus, Brachaspis collinus, B. nivalis and Alpinacris crassicauda) the male diploid chromosome number is 22 + X. L. migratoria and Paprides nitidus also have this number. This is typical of the majority of acridids and it is highly likely that the other New Zealand species also have the same number of chromosomes.
In the Acrididae the sex determining mechanism is an XO system. Therefore the females have two, and the males one X-chromosome. The X-chromosome (in the male) segregates randomly to either pole at anaphase I and at anaphase II its chromatids separate normally to opposite poles. Thus the meiotic division of one spermatogonium results in four spermatid nuclei, of which two have no X-chromosome (see figure 3). The XO system is basically similar to the XY system found in man except that in the latter, of the four spermatids resulting from meiosis, two have an X-chromosome each and two have a Y-chromosome each. Thus in figure 3 the Y-chromosome would be substituted for O.
Up to metaphase I the X-chromosome is densely stained, or heterochromatic, while the autosomes are lightly stained, or euchromatic (figures 4a-e). At metaphase I it becomes lightly stained but returns to its former state during meiosis II (figures 4f-l). If a chromosome has the ability to change from heterochromatic to euchromatic in this way it is described as being heteropycnotic. Thus the X-chromosome is positively heteropycnotic up to metaphase I when it becomes negatively heteropycnotic.
There are 22 autosomes all of which are telocentric (i.e. they have a terminal centromere). Therefore 11 bivalents can be recognised at the beginning of meiosis. The bivalents can be classified into three general size classes: long, which includes the three longest bivalents; medium, consisting of the next five longest bivalents; and short, which consists of the three shortest bivalents. The individual bivalents are enumerated as L1, L2, L3, M4, M5, etc. The S9 bivalent is usually easily recognised by the large heterochromatic blocks at the centromeric ends (figures 4c-e).
Some individuals of P. marginale possess one, and sometimes two extra chromosomes. These are supernumerary or B-chromosomes. The origin of these chromosomes is hard to determine but they have probably resulted from some form of mis-division of an autosome.
The B-chromosomes are very similar to X-chromosomes in appearance and behaviour. The type found in P. marginale are mainly heterochromatic but they also have a segment of euchromatin which is terminated by a very small segment of heterochromatin (figure 5a-b.) At anaphase I they segregate randomly, and independently of the X-chromosome and each other (if there are two), to either pole. At anaphase II the chromatids separate and segregate normally.
In grasshoppers these extra chromosomes do not appear to have any phenotypic effect but it is probable that they do influence other members of the chromosome complement e.g. by affecting the chiasma frequency of a nucleus.
I wish to thank Mr.
Deceased
Department of Zoology, University of New South Wales.
Pinniped skull remains from Cape Kidnappers, New Zealand were described by Berry (1928) as a new species of fossil Arctocephalus - A. caninus. In later years Berry himself appreciated the generic differences of Arctocephalus, Neophoca and Phocarctos and thought that as his specimen is considered to be the oldest fossil seal known from New Zealand it merited reassessment in the light of modern knowledge. This he did in a series of uncompleted papers and manuscripts that were still unpublished at his death in 1962. The second author has seen and examined the type specimen of A. caninus in the Dominion Museum, Wellington, (DM 532) and subsequently through the courtesy of the New Zealand Geological Survey, had access to Dr. Berry's manuscripts, and feels that our independently reached conclusions should be amalgamated.
The remains consist, (Plate 1) as Berry has already detailed them, of an incomplete left mandible with post canines 3, 4, and 5 still in position, but with the anterior part of the horizontal ramus broken in front of the level of pc 3, and the coronoid process and condyle abraded; the anterior portion of the left maxilla with the canine and post canines 1 and 2 in position; and nine separate teeth, one of which is the lower canine, and the others can almost certainly be given their correct positions in the jaws.
The bones were found about 1922 by the late Mr W. D. Southcott of Hastings. ‘Dr. Berry later went to great trouble to localise the point of collection and decided it was from the Opoitian sandstones immediately below the Black Reef Limestone, between Clifton and Cape Kidnappers, Hawke's Bay. It remains the geologically oldest fossil seal known from New Zealand’ (Fleming 1968). The Opoitian sandstones are considered to be Pliocene (Fleming 1962).
Berry (1928) refers to his specimen as Arctocephalus caninus, but it is quite obvious from his comparison with a specimen of ‘Arctocephalus hookeri’ that the modern generic name should in both instances be Phocarctos. He says that ‘the fragments obviously belonged to a young, and possibly a female animal’ and compared it with the skull of a female Phocarctos. The differences between the
A. caninus. Examination of large numbers of Phocarctos skulls shows well marked differences in the size of the canines and it is not difficult to determine the sex of the skulls from this character. The size of the fossil canine indicates without doubt that the animal was a young male (Plate 2), and this would explain why, when compared with a female, the fossil showed such obvious differences. Berry himself also referred to the specimen as a male in one of his manuscripts.
Berry's original estimated condylobasal length for the skull was about 140 mm, but it is considered that this is rather too small as a
Phocarctos skull of this length would be from a newborn pup with the milk canines still in position. A young male skull with canines about the size of those of the fossil would be about 200-220 mm condylobasal length.
Much of the manuscripts are taken up with very detailed descriptions of the teeth, but it is considered that these were adequately noted in the original paper. Berry does however compare his fossil with the skull of a young female Phocarctos found in Maori middens in the sandy dunes at Ocean Beach, a few miles south of Cape Kidnappers. The Ocean Beach skull although from a female is of the same degree of maturity as the fossil, and is in all respects very similar. It is in fact only in the details of the cusping of the post canines that Berry considers that the fossil differs enough from the Ocean Beach specimen and other available specimens of Phocarctos to be recognised as a new species. The characters he used are:
Upper pc 1 and 2 lack prominent accessory cusps — whereas in Phocarctos these are always present.
Upper pc 5 has a more prominent and sharper anterior accessory cusp than in Phocarctos, but is otherwise similar.
Lower pc 1 and 2 have smaller anterior accessory cusps than in Phocarctos.
Lower pc 3 has a posterior accessory cusp which is missing in Phocarctos.
Lower pc 3, 4 and 5 — the anterior accessory cusp is larger than in Phocarctos and is directed more horizontally forwards.
The accessory cusps on otariid post canines show a high degree of variability in detail and from inspection of a reasonable number of Phocarctos post canines it is felt that characters 2, 3 and 5 above come within the range of variability and are not useful as characters on which to recognise a new species. Similarly (character 1) the degree of prominence of anterior accessory cusps varies considerably and their complete absence occurs not infrequently. From the photograph (Plate 3) it can be seen that the posterior accessory cusp on lower pc 3 of the fossil is extremely small. Phocarctos usually has the posterior surface of this tooth, and also that of pc 4, without any accessory cusps, but occasionally such a small cusp does occur. A similar very small one is present on pc 4 of a single skull (1843.11.25.2) in the British Museum (Natural History) collection, so though it may occur only seldom, such a cusp may presumably also occur on pc 3. Plate 3 also illustrates the close similarity in shape between a recent Phocarctos jaw (DM 1359) and the Cape Kidnappers jaw.
It is thus felt that such a very variable character as the cusping detail is not sufficient to distinguish this fossil from modern specimens of Phocarctos hookeri and it is therefore suggested that Berry's name of Arctocephalus caninus be put in the synonymy of Phocarctos hookeri.
The present distribution of Phocarctos is on the Auckland Islands, Campbell Island and on the Snares, where the animals are to be found all the year round. As stragglers they reach Macquarie Island, Stewart Island and have been recorded as far north as Kaikoura, north of Christchurch (Mr. L. D. Bowring, Kaikoura, pers. comm.). But apparently as recently as 1863 these sealions used to breed on the west coast of the South Island, though during the next thirty years they became much rarer on the New Zealand mainland, and even on Stewart Island in 1874 had not been seen for some time, though their tracks through the bush were visible. (Thomson 1921).
Both the Ocean Beach and Cape Kidnappers specimens are from young animals, so either Phocarctos bred at least as far north as Hawke Bay, or close enough so that the young animals could swim there. The apparently very recent withdrawal of resident Phocarctos from the New Zealand mainland to the more distant islands, and the presence of both the Ocean Beach specimens and the Pliocene fossil from the same area suggest that the general distribution of Phocarctos was once considerably wider than it is now.
Arctocephalus caninus from Pliocene sandstone, Cape Kidnappers, Hawke Bay. New Zealand was described by Berry in 1928. The
Phocarctos hookeri from a Maori midden and also with recent specimens of P. hookeri indicate that this is the correct identification of the Cape Kidnappers fossil, thus extending the known range of Phocarctos.
The Seaweeds, marine members of the green, brown and red algae, are always larger and structurally more complex than the freshwater members of these groups. Maybe this is a reflection of the better suitability of the sea as an environment for evolution because of its greater stability to climatic change. Through geological time one can imagine the sea to have been a much more stable habitat for organisms — better able than the land to act as a buffer against climatic changes and extremes such as the Ice Ages must have presented.
Life as we know it is inconceivable without water. Not only is it the disperse phase of all cytoplasm but it is the universal biological solvent, the reaction medium and sometimes the catalyst of living processes. There is no evidence that the oceans have ever dried up. But this cannot be said of the land — some areas of which are prone to periodic desiccation and all areas susceptible to this fate. Organisms in such an environment must provide against this hazard or be wiped out of existence. It seems that freshwater algae have organised safeguards against hard times by evolving different types of asexual spores as a means of survival to tide over climatic adversity. Even the zygote of many — if not most — freshwater algae forms a zygospore before germination. But time spent in spore stages induced by desiccation or other unfavourable environmental conditions, although ensuring survival, decreases the turn - over rate of an organism's gene pool; and this must lessen the rate of selection of any genes which might produce a more complex thallus while still preserving features of good survival value in this risky environment. In the seaweeds we find neither aplanospores nor hypnospores — not even zygospores, for here the zygote germinates directly after formation. This is possibly a reflection of the stability of the sea, in that no provision need be made against importunate desiccation. Here, there must be a quicker turn-over of the gene pool and therefore exploitation of the genetical material available.
So maybe the uncertainties of the terrestrial habitat have not allowed freshwater algae to try out all the genetical possibilities open
Marchantia acquire such a thallus in an environment more hostile than that of a freshwater alga?
On the other hand, were the freshwater algae able to explore their genetical possibilities to the same extent as the seaweeds? It must be remembered that the dominant generation in many freshwater algae is haploid, and any haploid organism is limited in genotypic reaction to its environment. It is an interesting point to ponder that as far as the seaweeds are concerned the dominant generation is diploid, or if it is not dominant, the diploid phase is of a duration equal to the haploid phase. Furthermore, it is to be noted that no land plants — algae or bryophytes — with a dominant haploid generation ever evolved into a complex structure with much differentiation. Only when the sporophytic generation became dominant did evolution towards complexity seem to occur to any great extent. The mechanisms of meiosis are such that they ensure the greatest number of new combinations of characters that is possible for an organism to achieve. Every sporophyte has to undergo meiosis to produce haploid structures which will eventually give rise to gametes. An organism whose sporophyte is one-celled has the minimum possible potential for new combinations of genes which the environment can act upon. But in a multicellular sporophyte, meiosis occurs in many thousands — if not millions — of spore mother cells, as a result of which the number of possible recombinations for selection is beyond easy calculation. So a one-celled sporophyte such as the zygospore of Spirogyra is not endowed with a potential for great variation. This lack of a multicellular sporophyte in many freshwater algae must therefore have been a major rate-limiting factor in their evolution.
Many of these algae also exhibit another curiosity which must have considerably retarded their evolutionary progress. Irrespective of where meiosis occurs between zygote formation and germinating zygospore, the results of this division are four haploid nuclei — of which in many species (e.g. Spirogyra) three die. So the remaining nucleus carries only a quarter of the genetical material, and therefore only a quarter of the genetical possibilities which resulted from the previous gametic union; and the remaining three-quarters is dumped, so to speak. Little wonder their evolution has been slow!
Maybe, then, the uncertainties of an aquatic micro-environment in an otherwise terrestrial habitat coupled with the genetical limitations mentioned have militated against the evolution of the freshwater algae into something comparable in size with their marine counterparts.
It is really not surprising that seaweeds have come to be eaten. They are macroscopic, and their size would bring them to man's notice as something worth trying — particularly as keen observation could have revealed that some marine organisms were able to live on a
12 and of medicinal value in a number of ways — especially as a source of iodine to prevent goitre. Properties such as these have assured the seaweeds greater publicity than any other section of the algae when gastronomic appeal is the criterion — not to mention their potentiality as a food crop.
However, several instances are known where fresh and brackish water algae are eaten. An account of the location, circumstances of their growth and use forms the topic of this present article.
In his book ‘Our Oriental Heritage’, Durant crystallised for us in a most telling epigram one of the prime causes for the demise of numerous early civilisations — ‘The women were more fertile than the land’. Population pressure and famine have always been and still are forces which cause people to exploit most living things as possible sources of food as well as narcotic.
When the Conquistadores came upon Tenochtitlan, the old site of what is now the present-day Mexico City, they were apparently amazed at its size and population. Conservative estimates place this population at about 250,000 and therefore greater than that of the European towns at the time. Tenochtitlan was located on an island in Lake Texcoco whose water was brackish and undrinkable. Fresh water came to the city from the mainland via an aqueduct to supplement a modest supply from springs on the island. Concerning this city Farrar8 wrote: ‘How was such a large urban population fed, in a country of primitive farming, where all land transport was on the human back? There were fish in the lake, but there were no large edible domestic animals. The staple foodstuff was maize, but the varieties then cultivated were not high-yielding. From the descriptions by the Conquistadores of the wares offered for sale in the great market of Tenochtitlan, it would seem that the people, though by no means starving, were pressing hard on their resources. Nothing edible was neglected, not even snakes, ‘lice’, or the usually despised dog. It was no doubt the pressure of necessity which led to human sacrifice on a vast scale, followed by cannibalism; and to the invention of ‘chinampas’ or ‘floating gardens’, though the latter either fell out of use, or never existed except as an ingenious system of irrigation.’
Fortunately for us some of Cortez's retinue were more interested in chronicling than conquering and left accounts of all aspects of Aztec life. Mention is made by several of a food called Tecuitlatl, the name given to a scum that grew on the water of the lake. This was collected at a certain time of the year, dried in the sun in the form of cakes and then eaten — having a flavour and taste described
6, who thought it was a mineral, described the colour as purple or green.
Lake Texcoco was one of five that coalesced after the rains of the summer in to one large lake — the Lake of the Moon, situated in the Valley of Mexico. This valley is a natural landlocked drainage basin ‘entirely surrounded by mountains of volcanic origin’4 from which there is no external outlet. So, over geological time soluble salts have been washed down into this lake and concentrated by evaporation to produce ultimately a body of saline water. Humboldt gave a figure for the density of the water which corresponds to about 2.4-3.0% of dissolved salts, which were mainly sodium chloride and carbonate; sulphate was absent’8. Oddly enough, Humboldt does not mention the presence of nitrates among the soluble salts; but maybe he found none, and this would fit in with the fact that the surrounding mountains were volcanic — since nitrates are not usually present in rocks of volcanic origin. The pH of the lake water would be alkaline because of the presence of sodium carbonate.
While in retirement in Spain, Cortez recounted his experiences and observations to Lopez de Gomara, who assembled this information and produced the book ‘Conquest of Mexico’. Apart from giving details on the collection and preparation of Tecuitlatl, Gomara wrote ‘They make it into cakes like bricks, which they sell, not only in the market (of Tenochtitlan) but carry it to others outside the city, and far off. They eat this as we eat cheese, and it has rather a salty taste, which is delicious with chilmolli (a pungent sauce). They say that so many birds come to the lake for this food, that often in winter some parts are covered with them’.
Although we will never know for certain, it is the considered opinion of several who have examined this phenomenon that the scum was in fact a blue-green alga6. It is hardly likely to have been a mineral because few minerals have a specific gravity less than water, and this material was scooped off the surface; and one cannot imagine a mineral that would appear on the water's surface only at a specific time of the year. Neither can one imagine what kind of mineral would be so eagerly sought after as food by water-fowl. Farrar8 wrote ‘The Spaniards were evidently confused about the proper classification of tecuitlatl; they could not (lacking the microscope) identify it as a plant although it ‘bred’, but the breeding of minerals was still a common belief in the sixteenth century’. However the several references are highly suggestive.
Blue-green algae are very unusual in thir pigmentation because they contain one chlorophyll only — chlorophyll ‘a’, as well as one or both of two phycobilin pigments — the red phycoerythrin and the blue phycocyanin. There are two main chlorophylls, each having a distinctive colour; ‘a’ has a blue-green colour while ‘b’ is more yellow-green. So, if a particular species of this class of algae has
We cannot be certain of the kind of water the alga grew in; but we can piece sufficient information together to give us a very good idea of its possible nature. The famous chinampa gardens of Mexico are found in the southernmost of the five lakes, Xochimilco and Chalco, and of course relied on the availability of fresh water which came from springs on the southern boundaries of these two lakes. In the dry winter, evaporation of water from the Lake of the Moon reduced the level so much that the five independent lakes assumed their own identities. But there was always a constant fear that when the summer rains came and the five lakes coalesced, the salty waters of the eastern part of Lake Texcoco would flood the chinampa gardens and create havoc among them, thereby upsetting the economy of the whole valley. In the Aztec period, this problem of flooding became so acute4 that ‘in the fifteenth century Nezahualcoyotl, the poet-king of Texcoco, supervised for his relative Montezuma I the construction of an enormous dike of stones and earth enclosed by stockades interlaced with branches. The dike extended ten miles across the Lake of the Moon from Atzacoalco on the north to Ixtapalapa on the south. It sealed off the Aztec capital and the other chinampa towns from the rest of Lake Texcoco, leaving them in a freshwater lagoon.’ So the water of Texcoco must have been very saline to cause the people to go to these lengths to exclude it. The figure quoted by Humboldt of 2.5-3.0% dissolved salts gives us a good indication of the salinity particularly when we realise that the figure for open ocean salt-water ranges from 3.3 to 3.75%. A salinity such as that of Texcoco would be a barrier to the majority of algae found growing in bodies of water on land, especially as the pH of the water would be alkaline (conceivably about 10 or 11 due to the sodium carbonate present). No mention of the presence or absence of nitrate was made by Humboldt. Even if one assumes that none was present, this does not mean that algae would be automatically excluded through a lack of nitrogen, since numerous blue-greens are capable of fixing nitrogen. Because of this singular property, a few algae could live in this rather hostile environment providing they could tolerate the soluble salt level and the high pH. In fact, an amazingly similar phenomenon was discovered not many years ago in Central Africa which, when read about, immediately puts one in mind of this unusual food of the Aztecs and possibly gives us a clue to the identity of the organism which constituted tecuitlatl.
And so we move to Central Africa. First mention of an edible
5. A sample of a foodstuff called ‘die’ was sent back to France by a pharmacist attached to French Colonial Troops situated at Fort Lamy near Lake Chad. This sample was obtained in a local market at Massakori 100 kilometers east of the lake. On investigation it proved to be a mass of spiral filaments of a blue-green alga now known as Spirulina platensis. Dié was used for making soup and formed a jelly-like mass in water. Although this alga had previously been reported from the Rift Valley Lakes in Kenya and known to be eaten by flamingoes to the exclusion at certain times of all other forms of food, there had never been mention of its use as food for man.
Brandily14 wrote a popular article in 1959 highlighting this rather esoteric food of some of the tribes round Lake Chad. Unaware of Dangeard's identification of this alga, Brandily thought this organism to be Chlorella. However, it was not until the reports of a Belgian Expedition began to be published that we really found out the nature and value of this alga14.
There is still new terrestrial ground to cover and new terrestrial adventure to be sought even in these days of the now fashionable space flight and submarine exploration. Take for instance the trip made by the Belgian Trans-Saharan Expedition in 1964-65. Here was an itinerary that would make any potential explorers turgid with envy. While most of the personnel were military, a botanist joined the Expedition about two-thirds along the route. Immediately before his rendezvous at Faya-Largeau he spent several weeks at the Chad Research Station of the Office de la Recherche Scientifique et Technique Outre-mer at Fort Lamy, Chad. And this is where his story becomes of great interest to us.
In the markets of Fort Lamy, it is possible to buy a foodstuff called ‘Diné’ or ‘Douhé’ in the form of a flat greenish cake. These were subsequently found to consist almost exclusively of a blue-green alga Spirulina platensis. Dihé has a slight odour of dried fish and is slightly salty to the taste. The cakes are broken into small pieces and soaked in water, mixed with pimento, a little salt, and made into a nourishing sort of soup — sometimes with the addition of small pieces of meat. Or the dihé can be made into a thick gravy which is used as a seasoning on balls of millet. This food is eaten mainly by the Kanem tribe, north-east of Lake Chad.
The alga grows in the water of shallow ponds in wadis. When the water level drops, the algal mass concentrates and is collected in big baskets. The water is got rid of by decantation, and the remaining thick slush is spread out on the warm sand to dry in the sun in the form of large cakes about 1 cm thick. (Although collected in areas where bilharzia is prevalent, there seems little danger from this protozoan parasite since the dihé is dried and subsequently cooked).
The botanist of this Belgian Expedition also found a similar algal
Spirulina platensis. The inhabitants of the Ounianga Kebir area seem ignorant of the use and nutritive value of this alga. Ducks, however, feed extensively on this algal scum and were seen frequently on Lakes Yoan and Katam but were not seen on Lake Djobo which contains very little of this alga. It was assumed that the ducks ate the alga although no stomach content analyses were done to verify this observation.
Spirulina is rare or not abundant in the soft, iron or salty waters of Chad. But it seems to proliferate and become exceedingly abundant in soda waters rich in sodium sulphate or carbonate, whose pH varies from 9.5 to 11. In this apparently very particular but nevertheless ideal environment, Spirulina platensis is the dominant planktonic alga — often occurring in practically pure culture14.
Lake Yoan has a pH of 11, is highly charged with sodium salts and astonishingly green14. The shore of the lake is bordered by a white collar of crystallised salts to a width of 15-18 feet. The lake water itself has the following content:
According to people living in the near vicinity of the lake, the water has this green colour right throughout the year. The Spirulina can be found in small windrows on the shores of the lake. Lake Katam has a pH of 9.5.
Reports of the presence of Spirulina platensis in Africa are not new. Miss Jenkin, member of the Percy Sladen Expedition to East Africa in 1929, collected plankton from the Rift Valley Lakes in Kenya — Lakes Baringo, Naivasha, Nakuru, Elmenteita, and a small crater lake about 2° south of the Equator. An analysis of the last four lakes (which were examined more fully than Lake Baringo) is very interesting11.
She remarked that ‘in increasing concentrations alkalinity appeared to effect a marked reduction in quantity of fauna and flora’; and that the sodium ‘was derived from the surrounding alkaline lavas’. The increase in alkalinity raised the pH of the water from 9.0 to about 11.2. Analysis was made of the stomach contents of some of the flamingoes, when it was found that they had been feeding almost solely on Spirulina. Miss Rich18, who examined Miss Jenkin's plankton samples quoted Miss Jenkin as saying that the water of the last three lakes had the appearance of ‘green soup’.
Miss Rich also stated that Spirulina platensis had been described from specimens found in standing water in Uruguay; and that it had subsequently been recorded by W. and G. S. West24 in a collection taken from Lake Losuguta in Kenya. The Wests apparently commented on the remarkable habitat — ‘water with sulphides’ — but unfortunately gave no analysis of the water.
Later, in 1933, Miss Rich19 published an account of the phytoplankton collected by the Cambridge Expedition to the Lakes of Kenya and Uganda. The recordings of the occurrence of Spirulina are of interest. They were all found in association with Lake Rudolph, and only in the following places:
in crater lakes B and C on Central Island, in Lake Rudolph; in an enclosed alkaline pool on Ferguson sand spit — a part of Ferguson Gulf.
‘The alkalinity of these pieces of water was greater than that of Lake Rudolph itself, though less than that of Lake Elmenteita and Nakuru and Crater Lake’19.
Ross20 later gave the following pH values for some of the lakes mentioned above:
He described the water in the Ferguson Gulf of Lake Rudolph as having the appearance of green soup, due to a very thick phytoplankton of Spirulina and Anabaenopsis. The pH of the water of this Gulf was about 9.8 whereas the value for the Lake water was about 9.5. His remarks about Lakes Nakuru and Elmenteita are that they ‘are almost identical both in their physical and their chemical conditions. They are both very shallow, mostly less than 1 m deep, and very alkaline with pH c.11. When visited their water level was low and there were wide areas of dry soda flats around their margins. The water of both was a thick green soup of Spirulina and Anabaenopsis, on which large flocks of flamingoes were feeding’. These three lakes — Rudolph, Elmenteita and Nakuru — are in the Eastern Rift Valley and have no outlet.
Duvigneard and Symoens14 found this alga in a collection taken from Lake Mugunga, near Nzulu, in the Congo. This lake is south of the laval plain and immediately north of Lake Kivu. The water is alkaline with a pH of 8.5. Spirulina has been reported from Zambia, in a lagoon in the swamps of Lake Bangweulu; and its growth has also been studied in the open lakes of Ethiopia.
A condensation of information about Spirulina in Africa allows one to make the following statements.
Spirulina, a planktonic blue-green alga, is eaten by man as well as water fowl.
It is found in waters with a high pH caused by the presence of sodium carbonate, high salinity and high sodium content — a more specialised environment than the sea.
Places where standing water of alkaline pH seems to be found are those areas collecting drainage from rocks of volcanic origin high in sodium salts which include sodium carbonate or react with the atmosphere to produce this chemical.
How reminiscent this picture is of the conditions reported to occur in the old Aztec capital. Maybe tecuitlatl was Spirulina platensis! Evidence certainly points that way.
We will now shift scenes from the tropics to the cold temperate and sub-arctic areas of the globe — to the Far East and Asiatic Far North, and continue with an account of two more blue-green algae. The first is Nostoc. Despairing of being able to base a good classification on cell details, the Russian algologist Elenkin7 resorted to using morphological features of the Nostoc colony as the basis of a workable system of classification. We will follow suit and apply his system to help us correlate various accounts about this genus.
Elenkin divided edible Nostoc mainly into two groups; one where the colonies are often encountered in great quantities, the other where they are rather rare. The latter group is not significant and will not concern us further. The former group he divides into three main
Sphaeronostoc — characterised by its colonies being invariably spherical; it occurs submerged but not attached to a substratum.
Stratonostoc — whose colonies are laminate; it is mainly terrestrial in habitat.
Nematonostoc — those occurring in thread-like colonies.
Let us consider the first — Sphaeronostoc pruniforme, which he reckons to be synonymous with Nostoc edule. It does not occur free-floating on the surface but can be found in large masses at the bottom of rivers, lakes and swamps. It is very common in the Northern parts of U.S.S.R. where it is found sometimes in enormous quantities. Elenkin quotes from the accounts of Middendorf who in 1860 wrote of his travels through Siberia. At one stage the food supply of his party must have been getting perilously short. Discussing this predicament later, he lamented not knowing at the time that Nostoc was edible because he recalled that in one stream his party could have collected about 1000 cubic feet of colonies, which would have been most useful in alleviating their problem of food shortage. Meyer, in a review article ‘The Algae of the Lake Baikal’, reported that Sphaeronostoc has been found in unbelievable quantity in various bays around this lake, where it has been known to occur as a solid layer 10-20 metres thick over the bottom of the bay. It has also been found from
Dr. Hooker, in a paper read before the Linnean Society of London, January 20, 1852, mentioned that N. edule was found abundantly in streams in Tartary. It was highly esteemed as an ingredient in soups. This form of Nostoc was well known and eaten in Mongolia and China in which countries it was used extensively as an article of commerce, generally sold in dried form. Although common in northern China, its use as food has been recorded by the Frenchman, Ivan, in such southern places as Canton and Macao. Apparently it was highly esteemed as a dish to be eaten on feast days and other occasions for celebration.
Sphaeronostoc pruniforme appears to be wholly aquatic, Undoubtedly this type of habitat permits the colonies to develop their spherical shape. Another characteristic seems to be that it is submerged but not attached to a substratum. These properties clearly delineate this species from the next one we will talk about — Stratonostoc, which is described by Elenkin as occurring in laminate colonies, found mainly on the soils of steppes and semi-desert areas.
Stratonostoc commune (the Nostoc commune of other folk) is found free-lying on the soil as convoluted small plates which can grow to many centimetres both in length and width. These very dark-coloured plates are brittle when dry but become leathery when wet. It occurs all over U.S.S.R., Tibet, Mongolia, and from Pamir down to central China: also in numerous west European and non-European
Nostoc esculentum. He subdivides the species into four forms, and reports this about the form ‘crispum’. This is found in enormous quantities in Mongolia where it is eaten after boiling with meat and other additives. Harvey 9 quotes from the journal of Dr. Sutherland describing travels in the Arctic regions of Canada. ‘It grows,’ says Dr. Sutherland, ‘upon the soft and almost boggy slopes around Assistance Bay; and where these slopes become frozen at the close of the season, the plant lying upon the surface in irregularly plicated masses becomes loosened, and if it is not at once covered with snow, which is not always the case, the wind carries it about in all directions. Sometimes it is blown out to sea, where one can pick it up on the surface of the ice, over a depth of probably one hundred fathoms. It has been found at a distance of two miles from land, where the wind had carried it. At this distance from the land it was infested with Podurae, and I accounted for this fact by presuming that the insects of the previous year had deposited their ova in the plant upon the land, where also the same species could be seen in myriads upon the little purling rivulets, at the side of which Nostoc was very abundant.’ Sutherland later mentions having tried it as an article of food, and found it more nutritious than the ‘Tripe de Roche’ of the arctic hunters and perhaps not inferior to Iceland Moss. Harvey also mentions that Dr. Thomson noticed a very similar plant growing in Tibet up to a height of 17,000 feet, floating in large masses on the surface of pools and lakes in soils impregnated with carbonate of soda, and drifted in heaps by the winds along their banks.
Large amounts of Stratonostoc are found in saltpans where the temperature is high in summer and low in winter. In a wet spring or autumn the alga may grow on the soil to such an extent that the soil is seen only through an algal film. In summer, the layer dries out and becomes brittle and black in colour. It was reported9 that ‘a similar species has been seen in Australia, after a shower of rain, to cover what had seemed previously to be a bare hillside, with such a thick coating of jelly as to render it impossible to walk over it without sliding’.
Elenkin also mentions another species of Stratonostoc, S. verrucosum. This is an aquatic underwater form found attached to stones and growing up to about 10 cms. diameter. It is very soft, often crenulated but sometimes spherical and smooth. Here is a report by Smith21 taken from the Journal of the Siam Society which seems to refer to this species.
‘Under the names of dok hin (rock flower) and kai hin (rock egg) the people of the Chiengmai region in North Siam designate small dark green spheroidal plants which grow in abundance in clear, cool streams attached to the top, sides, and under-surfaces of stones and boulders. The plants when apparently full-grown are 10 to 15 mm. in diameter, and have a bladder-like form, a gelatinous consistency, and
‘These plants are rather extensively eaten by the local people. On the Mekhan, a mountain stream southwest of Chiengmai, on February 8, 1932, four men from the nearest village were observed scraping or pulling the plants from the rocks with their fingers and holding them in baskets and loose-mesh bags attached to their waists, their combined product at the time of observation being over two liters. The plants are prepared for use by boiling, and are eaten with sugar, salt, or dried prawns.’
The next species of Nostoc to be considered is the one Elenkin calls Nematonostoc flagelliforme. This appears to be what the Chinese refer to as earth-hairs’ or Fah Tsai: and in the Eurasian Continental mass. Northern China seems to be the centre of distribution although it is found in all semi-arid zones of the U.S.S.R. as far west as Astrakan. It is also reported from Texas, Mexico and Montana in America, and Morocco in Africa. It is a highly prized food in China and was traded widely. Like Sphaeronostoc, it finds special use at times of celebration. Skvortzow22 has this to say: ‘In China the forms living on the surface of the ground are used as food …’
‘Nostoc in the Shantung province appears in summer rainy time on the clayey ground and on humid soil, but when the ground dries the alga contracts and begins to be imperceptible. The local population eat the Nostoc not for lack of food, but simply for the same reason as mushrooms, and wild vegetables are used.’
‘Nostoc has no particular flavour. They eat it roasted with different seasonings, which give it taste. Indubitably Nostoc is used in other places in China, seeing that here on account of a damp climate, this alga is very common. In masses it is found in June and in July near Shanghai and in South China.’
According to Prescott17, at one time Nostoc commune was eaten in Ecuador where it was called Yuyucho; and Tiffany23 comments that it was boiled with garden vegetables to add flavour. Wood25 also reported that a form of Nostoc has been eaten in Fiji: and the same alga has been eaten in Okinawa.
We move to Japan to consider the next blue-green alga which is still eaten by man. This is Phylloderma sacrum or suizenji-nori. It was collected mainly in the mountainous regions of the provinces of Higo and Chikugen in the southern-most island of Japan — Kyushu. It was gathered with nets all the year round but mainly in the summer months, and cleaned of other adhering algae. The mass of colonies were cut into small pieces, spread on bricks and dried in the sun — forming thin sheets15. In present times it can still be bought but is regarded as a somewhat expensive delicacy.
The last group of edible freshwater algae we will deal with all belong to the Chlorophyceae, and include the filamentous Spirogyra
Oedogonium collected and eaten in some sub-tropical areas and the laminate Prasiola from high altitude cold water streams. Biswas1 mentions that a thick coarse species of Spirogyra is widely eaten in the Northern Shan States of Burma. The algal filaments are dried in bundles, packed in boxes and sold in this form in the markets. Spirogyra is also mentioned by Bourrelly14 as being eaten in Vietnam where it is sold fresh. Tiffany23 says that both dried Spirogyra and Oedogonium could be bought in packets in Indian markets.
Prasiola is the remaining alga to discuss. Jao10 reported that Prasiola yunnanica was collected and sold in dried form in the local markets of Yunnan in West China. Unlike the blue-greens and the green algae Spirogyra and Oedogonium already described, Prasiola is macroscopic and quite large — growing up to 8 inches long and 1½ inches wide. Apparently it is very abundant in mountain streams at altitudes of about 8000 feet. Reference is also made by Bourrelly14 to the edibility of this genus, who mentions that it was eaten in the Himalayan region by various of the local population. Prasiola japonica is eaten in Japan; and again comes from reasonably high altitudes — about 6,000 feet or more. (Prescott17 mentioned that some species are confined to swiftly flowing cold water such as is found in the Andes and the Rocky Mountains.) Namikawa15 reported that it was collected mainly at Nikko, in central Japan — hence one of its names, Nikko-nori. It was also known as Daiyagawa-nori; in recent times, Kawa-nori or Fuji-nori. It is made into sheets like asakusa-nori — but is much more expensive.
Now that we have reviewed the edible fresh and brackish water algae, let us see what published chemical analyses reveal about the nutritive value of these forms of food. All figures are expressed on a dry weight basis.
Unfortunately, analytical figures for fresh and brackish water algae are sparse and incomplete. The main point to notice is the high crude protein levels of Spirulina and Prasiola. It is also surprising that Nostoc, a known nitrogen-fixer, has such a low crude protein content compared with Spirulina. A figure of 20.6% quoted for Nostoc commune13 is of the same order as the one quoted in the above table for N. flagelliforme. Since protein is much more important than carbohydrate, discussion will be confined to a few aspects concerning the protein contents. If Spirulina fixes nitrogen, then its high protein figure can be accounted for; but one is at a loss to explain why Prasiola should be so high. However, Spirulina is the only alga of this group to be eaten in large quantities and is thus the one which merits much comment. Considerable work has been done on its chemical and nutritional analysis and several points should be brought to focus more sharply.
As far as man is concerned, not only is it necessary to ingest protein, but it must be of the right kind. He has inherited a number of physiological inabilities, not the least being his incapacity to synthesize certain amino acids. These are — threonine, valine, methionine, leucine, isoleucine, lysine, phenylalanine, tryptophane, histidine. It is critical therefore to analyse for these amino acids. No matter how rich a foodstuff is in protein, it is of little use if any one of these is missing, particularly in places where protein intake is low. Spirulina comes out well in terms of these acids with the exception of methionine which is 62% of the value defined in the provisional pattern of necessary amino acids laid down by Food and Agriculture Organisation of the United Nations. For all the other required acids, Spirulina is a good source. Methionine is one of the sulphur-containing amino acids. FAO also set a minimum level on sulphur-containing acids in their provisional pattern; and here Spirulina does not fair well, reaching about 43%. But this is its only limitation in terms of essential metabolites for man. This comes as a surprise in a way when one considers the sodium sulphate figure quoted earlier for Lake Yoan at Ounianga Kebir in the Sahara — 22.3gms/litre. One would have expected Spirulina to be well stocked with these sulphur amino acids. Tryptophane and lysine which are deficient in many foods, are present in sufficient quantity (Clément and others3). They also go on to say: ‘A relatively good correlation was found between the chemical score calculated from standard amino acid analyses and the net protein utilisation determined on rats. The limiting amino acid assessed analytically is methionine; however, another essential amino acid, present but not in an available form, might contribute to reducing the protein value.’
Referring to the use of Spirulina in Chad, Clément and his co-authors wrote3: ‘According to local opinion, ‘dié’ advantageously replaces meat sauce and largely contributes to maintaining the nutritional value of the diet when meat is scarce. At one time, ‘dié’
Spirulina grows luxuriously and the local tribes appear to be ignorant of its use as a wonderful source of protein. Who in Africa can afford to neglect a foodstuff which can partially if not completely prevent the pall of kwashiorkor from enshrouding them?
At the beginning of this article we mentioned the fact that freshwater members of the green, brown and red algae are always smaller and much less complex than the marine members of these groups. In terms of a large and well-developed thallus, which algae among the freshwater greens can compare with Ulva, Enteromorpha, Monostroma or even the coenocytic Codium or Caulerpa? An immediate answer to this is Prasiola, having a thallus 8 or so inches long and 1½ inches wide.10. One is then required to seek a reason to explain this apparent exception.
Prasiola is a genus whose species range over the whole gamut of algal habitats, except thermal hot-pools. It is found in salt-water as well as fresh, and it is known to live in the spray-zone above high-tide level as well as in pools which can vary from full salinity, through brackish to freshwater. Prescott reports it as being even subaerial. Because Prasiola shows such tolerance it is easy to believe that the freshwater species could be readily derived from their saline counterparts merely by shifting from one habitat to another. A similar migration can be envisaged for Enteromorpha and Cladophora, both of which show a somewhat similar range of tolerance except that they are not reported from subaerial situations. But Prasiola and Enteromorpha are of more interest because they have an expanse of thallus and are not filamentous like Cladophora; they are thus more complex in structure than the latter.
Altogether Prasiola is a most unusual alga. Boney2 reports that the cells of the thallus are double-walled; the inner-most being cellulose impregnated with pectin, and the outer-most layers forming a continuous ‘coating lamella’. However, the really unusual feature is to be found in its reproduction and the cytological character of its thallus. In at least two species investigated so far (Prasiola stipitata and P. meridionalis), it appears that the lower half of the thallus is always sporophytic, whereas the upper half may be sporophytic or gametophytic. Therefore the basal vegetative half is always diploid. The species mentioned are both marine. Thus this structurally complex, sporophytic vegetative state must have evolved in the sea, and not on the land.
So, the opening statement of this article still stands — that the marine members of the green, brown and red algae are always larger and structurally more complex than the freshwater members of these groups. Although freshwater species of Prasiola appear to be exceptional, they are in all likelihood marine ones transposed — with a diploid thallus already dominant before they moved from the sea.
Prasiola with other freshwater algae since those with a haploid-dominant generation must have migrated from the sea into their terrestrial aquatic habitats in the haploid condition, or evolved from earlier haploid ancestors. We must therefore regard Prasiola as something quite different from all other algae which share a freshwater environment.
I wish to thank Professor
‘This Small Particle acts as the digestive tract of the living cell. Its enzymes dissolve the substances ingested by the cell and under certain circumstances can dissolve the cell itself.’
Such was the definition given by de Duve in 1963 of the role of the lysosome. All future work stemmed from this definition and it is now agreed that the lysosomes are tiny bags filled with a powerful digestive juice capable of breaking down most of the constituents of living matter.
Most of the work done on lysosomes has been on rat liver cells, although the evidence that is accumulating supports the hypothesis that they are universal in animal cells.
Only a few plant tissues have been investigated for lysosomes.
Cytological and biochemical studies with the aid of the electron microscope have shown the lysosome to be a cytoplasmic organelle, spherical in shape with a mean diameter of about 0.4 microns and an average density of about 1.15, and a single, limiting membrane, but no definite internal structure. Associated with the lysosome are a number of easily soluble acid hydrolases with acid pH optima but there are no oxidative enzymes as one finds in mitochondria. These lysosomal enzymes are released by treatment of the lysosome with agents known to affect the binding of lipin to protein.
The latency of lysosomal enzymes is considered to be due to the presence of this limiting membrane barrier of lipid-protein which restricts the accessibility of the internal hydrolases to any external substrate. The presence of such a membrane was deduced from experiments concerning the action of lecithinase and proteolytic enzymes on the particles. After treatment with these enzymes all hydrolase activities were released simultaneously and in a fully active form, from the particles. One of the biochemical characters of the lysosome is this structure - linked enzyme latency. In studying acid phosphatase activity, de Duve found that the substrate beta-glycerophosphate did not readily penetrate the membrane of the lysosome unless the particle had been subjected to an agent such as acid pH or high temperature (37°C.). Presumably these treatments increased the permeability of the membrane by altering its structure.
Whereas enzyme action is not confined to lysosomal particles, the activation of an acid hydrolase at a particular cytoplasmic site may be considered indicative of the presence of a lysosome, especially if the
‘It has been discussed that the histochemical identification of a lysosome in a particular tissue ideally should rest upon the evidence demonstrating the presence of two or more acid hydrolases contained within a particle limited by a single membrane’.
While the presence of lysosomes in animal tissues has been well established, the same is not true for lysosomes in plant tissues. Using the presence of acid phosphatase located in particles as a criterion, lysosomes have been recorded in the embryo of Triticum vulgare (single membrane observed), root meristem of Vicia faba, root meristem and epidermis of Allium cepa, and pollen grain of Tradescantia bracteata. Cytochemical demonstrations of the activity of acid phosphatase have also been made in the fungus Botrytis cinerea.
All the major classes of biologically active compounds, including proteins, nucleic acids and polysaccharides, were shown to be susceptible to action by the enzymes contained in lysosomes. The number of acid hydrolases discovered stands at ten: acid phosphatase, cathepsin, acid deoxy ribonuclease, acid ribonuclease, beta-glucuronidase, arylsulfatases A and B, phosphoprotein phosphatase, beta - galactosidase, beta - N - acetylglucosaminidase, and alpha - mannosidase. But all are in different proportions.
Considered as a group, the enzymes present in the lysosome have one major function: a lytic, or digestive one. Hence the name ‘lysosome’ (meaning lytic body) that de Duve gave to the body.
De Duve first concluded that the membrane must act as a shield between the enzymes and the rest of the cell. The digestive processes, he deduced, must be confined within the limits of the membrane, and the substances to be digested must somehow be taken up in the particles. He then endeavoured to find out how this took place and then to look for those conditions that might lead to the release of the enzymes inside the cell and the dissolution of the cell.
De Duve noticed that lysosomes are not readily distinguishable in any type of cell and cannot be identified solely on the basis of their appearance. They come in a bewildering assortment of shapes and sizes, even in a single type of cell. This polymorphism is now understandable: their digestive activities cause them to be filled with a variety of substances and objects in an advanced state of disintegration and it is their contents that determine their shape, size and density.
To discuss de Duve's work, one must explain the phenomenon of pinocytosis. Not all of the substances that nurture a cell require digestion by lysosomes. In higher animals tissue cells receive most of
De Duve distinguished four conditions of lysosome states: ‘storage granules’, digestive vacuoles, residual bodies and ‘autophagic vacuoles’. The first three are directly involved in the main digestive process (refer Fig. 1). The storage granule is the original form of the lysosome. De Duve postulated that the enzymes in the granule presumably were produced by the ribosomes associated with the endoplasmic reticulum, but he did not comment on the origin of the lysosome membrane. A storage granule fuses with a phagosome to form a digestive vacuole. Digestion products diffuse through the membrane into the cell. The digestive vacuole can continue its digestive activity, gradually accumulating indigestable material until it becomes a residual body, which may then be eliminated by fusion with the cell membrane. The distinguishing feature of the autophagic vacuole is the material digested: parts of the cell itself, such as mitochondria and portions of the endoplasmic reticulum.
Another mode of action of the lysosome involves the actual rupture of the lysosome membrane inside the cell and the digestion of the cell as a whole by the released enzymes. Such ruptures take place fairly quickly in dead cells, e.g. cells suddenly deprived of oxygen or exposed to cell poisons of certain kinds.
There is much speculation concerning the formation of lysosomes. It is known that protein synthesis in the cell cytoplasm occurs at ribosomal sites, and the assumption that this is the site of hydrolytic enzymes is also certain.
It is thought that the hydrolases may pass from the ribosomal sites of synthesis to the lysosomes either by the formation of lysosomes directly from the endoplasmic reticulum or via the Golgi apparatus.
Wolman and Weiner (1963) suggested that lysosomes are derived from the endoplasmic reticulum by a change in its structure in which the membrane is folded into a globular body surrounded by polar lipids, with the hydrophilic groups pointed inwards. By this means, the enzymes normally situated on the outer surface of the membrane are enclosed in vesicles formed by the inversion of the membranes.
Dalton (1962) found that small vesicles appeared to arise from
Benedetti and Leplus (1958) working with erythroblasts of chickens infected with erythroblastosis virus found granules like lysosomes in appearance, in, or adjacent to the highly developed Golgi zone. There is, according to them, some indication of continuity between Golgi membrane and membrane of the granule, i.e. the granule appears to be like an enlarged Golgi vacuole. They suggest the Golgi apparatus is a site for segregation of lysosomal hydrolases.
It is unlikely that all of the lysosomal hydrolases are contained within each lysosome since, e.g. rat liver lysosomes do not appear to behave as enzymically homogeneous particles (de Duve, 1963).
Constituent enzymes seem to show a slightly different distribution pattern when the particles are analysed for enzyme content.
Novikoff (1964) considered that it was difficult to distinguish between secretion-transporting vesicles and those vacuoles separating from the Golgi, which he feels are likely to be lysosomes. He demonstrated an experimental procedure which changes the distribution of the Golgi apparatus which correspondingly changes the lysosome distribution. Novikoff even suggested that lysosomal hydrolases may be transported to larger lysosomes by Golgi vesicles. This was based on studies of the uptake of exogenous proteins (horse-radish peroxidase) by rat kidney cells. He reported that proteins gained access by pinocytosis into the cell, thereby forming pinocytotic vacuoles and migrating into the centre of the cell and acquiring acid hydrolases on passing close to the Golgi region. Novikoff did stress, however, that this hypothesis was not adequately supported by experimental data.
Brandes (1965), working on Euglena gracilis, showed hydrolase activities demonstrable at the Golgi sites. This supported the idea that enzyme-rich vesicles, presumed to be lysosomes, originated as terminal dilations of the Golgi cisternae, which in many instances also possessed intense enzyme activity.
On the other hand Ogawa and Shinonaga (1962) found acid phosphatase activity to be associated intimately with the smooth membrane system of the cell. They considered the lysosomes to be specially differentiated organelles originating from the smooth endoplasmic reticulum.
Further evidence interpreted to indicate the formation of lysosomes from the Golgi complex is given in the study of sebaceous glands from adult male rats (Brandes, 1965). In an electron microscope study of the localization of glycerophophatase and esterases, Brandes found that in undifferentiated cells the Golgi apparatus was poorly developed There was no evidence of secretory activity nor any reaction for hydrolases. In differentiating cells secretory activity was indicated by the formation of vacuoles and a highly developed Golgi apparatus which was positive for acid hydrolase activity.
The two hydrolases appeared limited to small granules similar to Golgi vesicles, and to larger bodies which could have been lysosomes. On the basis of this evidence, it was suggested that the Golgi apparatus plays an important part in the formation of lysosomes in sebaceous cells.
In review of this section, the available evidence would suggest that lysosomes may be formed directly from the endoplasmic reticulum, perhaps by invagination of the membrane, or by the production of vesicles from the Golgi cisternae. Whether either or both of these possibilities in fact operates is argumentative at this stage.
The difficulty in assessing evidence concerning the role of lysosomes in cell death is that although the cells may give the appearance of dying and there is free hydrolase activity present in the cells, it is necessary to determine whether
the cell has died and this has resulted in a release of the lysosomal enzymes, or
the lysosomal enzymes have been released into the cells, to cause the death of the particular cell.
De Duve coined the term ‘suicide-bags’ for lysosomes with respect to cell death.
Brandes (1965) using electron microscope studies of esterase and glycerophosphatase on the sebaceous cells of the adult rat, showed that in fully mature cells about to disintegrate, the lysosomes enlarge and appear to become ruptured. When the stage of actual cellular lysis occurs, the lysosomes are no longer visible and scattered glycerophosphatase-rich particles are seen in all parts of the cell.
Of the several cytological changes described as correlated with ageing the most widely accepted is the accumulation of lipofuscin granules. Novikoff found the appearance of lipofuscin granules in the Golgi zone and suggested that these granules may be altered lysosomes. He noticed that in aged cells the Golgi apparatus fragmented and disappeared whilst lipofuscin accumulated.
Lipofuscin granules have been seen to accumulate with age in human myocardium. These lipofuscin-rich granules show high acid phosphatase and cathepsin activity and so may be conceived as being altered lysosomes in which metabolic waste materials have accumulated.
Sulkin and Kuntz (1952) noted that as lipofuscin accumulated in the autonomic ganglia of ageing men and dogs the Golgi apparatus fragmented and largely disappeared.
Dalton and Felix (1957) reported granules adjacent to Golgi membranes and vacuoles in human neurons. They postulate they are altered Golgi vesicles, lysosomes, in transition to lipofuscin granules.
Jarrett and Spearman (1962), basing their consideration on the role of lysosomes in the autolysis of the contents of the epidermal cells prior to normal keratinisation, suggested that abnormal keratinisation found in the condition of psoriasis may result from the failure of the lysosomes to release their enzymes. That the lysosomes may not rupture in the usual way has been shown by the electron microscope.
The problem of the possible role of lysosomes in cell death has also been considered in respect of plant tissues. It is known that on differentiation of meristematic cells into primary xylem cells, the differentiating cells gradually lose their cell contents, a change accompanied by a markedly increased impermeability of the cell wall. Using glycerophosphatase as a marker for lysosome-like particles in the roots of Vicia faba, Gahan (1967) found that in undifferentiated meristematic cells the glycerophosphatase activity was confined to particular sites, but with the onset of differentiation, as demonstrated by the elongation of the cells accompanied by changes in the structure of the cell wall, the acid phosphatase activity was no longer solely particulate, but was also diffuse. On further differentiation, the loss of cytoplasmic contents was accompanied by the presence of acid phosphatase activity right throughout the cell.
The author observed a similar situation in the cells of the root cap of Allium cepa where the innermost cell adjacent to the meristematic region showed a particulate reaction for acid phosphatase, whereas the outermost cells of the root cap showed a diffuse reaction.
The above observations may support the concept of the self-digestion of the cell contents by the release of the hydrolytic enzymes from the lysosomes.
From the accumulated data it is not possible to make a definitive statement concerning the role of lysosomes in cell death. It seems
Robbins and Gonatas (1964), when studying mitosis in Hela cells, found the acid phosphatase - containing granules aggregated circumferentially in packets during prophase and metaphase. This was contrary to the polarised distribution of individual granules during interphase.
In a study of the effects of spindle inhibitors, colchicine and vinblastine sulfate on Hela cells, Robbins and Gonatas found marked changes in the nature and behaviour of the lysosomal particles.
After treatment for fifteen hours with the inhibitors, the lysosomes were circumferentially dispersed instead of in their normally polarised, juxtanuclear position. Furthermore, they appeared to be larger than their normal counterparts in untreated cells due, apparently, to their being clustered and not individually resolvable in the optical microscope. The general behaviour of these lysosomes after treatment with the spindle inhibitors is identical to that normally observed in the untreated mitotic cell.
Holt (1968) also found changes in the distribution of lysosomes in rat liver cells during mitosis. At interphase the lysosomes occupied their typical position, but moved to a juxtanuclear position at the beginning of prophase. During metaphase they became uniformly distributed throughout the spindle. At anaphase, as the chromosomes moved to opposite poles, so a cluster of lysosomes preceded each chromosome set to the poles where they formed a ‘cap’. The lysosomes remained in the ‘cap’ until the end of telophase, after which they returned to their normal location in the cytoplasm.
The behaviour of lysosomes during mitosis has been considered also by Maggi (1966) in her studies upon Hela cells. Lysosomes were identified by the reaction for acid phosphatase when it was found that after incubating the cells for five minutes the number of granules varied from ten to thirteen per cell. The number of particles increased to nineteen for prophase, twenty-one for metaphase, seventeen for anaphase, and twenty-nine for telophase. It was concluded that during division the permeability of the lysosomal membranes to the substrate employed was increased, so enabling a more rapid penetration by the substrate. Thus, there would seem to be some involvement of the lysosomal particles during mitosis, possibly relating to the dissolution of the nuclear membrane and of the spindle.
A further point arising from the observations of Maggi (1966) was that at telophase an approximately equal number of particles passed to each of the two daughter cells, the sum of the two
Allison and Mallucci (1964) considered that the lysosomes were involved in the initiation of mitosis due to a release of an unspecified activator from the lysosomes or to one of the released hydrolases inactivating a repressor. However it has also been suggested that lysosomes may be responsible for chromosome breakage. The results of Allison and Paton (1965) showed that treatment of human embryonic lung cells in tissue culture with photosensitising agents allowed high frequency of chromosome breaks on irradiating the cells with light from a high intensity tungsten source. It was claimed that the effects of the photosensitising agents (neutral red and acridine), were observed only at lysosomal sites, from which it was concluded that a cytoplasmic event was giving rise to a structural alteration in the chromosomes. It was suggested that deoxyribonuclease from the lysosomes might be the agent responsible for the chromosome breaks. This is difficult to explain in the light of results of Chevremont et al., (1960), who grew mammalian cells in tissue culture in the presence of acid deoxyribonuclease. This treatment inhibited cell division, but allowed DNA synthesis to proceed and did not appear to alter the nuclear DNA. Thus, while it is tempting to implicate lysosomal activity in mitosis and chromosome behaviour, via the release of lysosomal enzymes, some caution must be observed in evaluating this field, which at present is rather limited.
Little work has been done from this aspect, although Cohen et al., (1964) studying the ovary of the rat, observed the localisation of acid phosphatase and noted a variability of lysosomal activity depending on the particular stage of the oestrus cycle. During oestrus the number of small, enzymically active particles increased, with the epithelial cells containing numerous large lysosomes. As the cycle continued the number of particles decreased, until they disappeared with the beginning of the new cycle. Although this study was performed at the level of the optical microscope only, Cohen suggested that the large lysosomes may be equivalent to cytolysomes.
The work of Scheib (1963) would indicate a possible involvement of the sex hormones during regression of the Mullerian ducts of male chick embryos where he claims both androgen and estrogen caused the release of lysosomal hydrolases.
When a new cell particle is discovered, attempts are made to link its activities with a range of functions, and the lysosome has proved no exception.
Cytological and chemical studies have contributed to extending the lysosome concept from one of ‘a bag of hydrolytic enzymes’ to one in which the lysosome has assumed a physiological importance. Despite the wide range of studies, it is still too early to be definite about the role of the lysosome. The nature of the enzymes associated with it led de Duve (1963) to postulate a role in degradation rather than in biosynthesis and histochemical findings would point strongly to a role in intracellular digestion. This, in fact, may prove to be the essence of all lysosomal functions, for while there are changes in the behaviour pattern of the particles under a wide range of cellular activities, such as mitosis, virus infection and physiological cycles, the underlying function of the lysosomes is still likely to be one of digestion. However, more data is necessary concerning the behaviour of lysosomes in conditions such as cell death and cell division, in cellular metabolism and ageing, before a final answer can be obtained to the question, ‘What is the role of the lysosome?’
The brown tree, or Whistling, Frog (Hyla Ewingi) though limited in its New Zealand distribution, is reasonably numerous in the town of Foxton. I have found it in several gardens, where it survives well in large clumps of Agapanthus.
In October, 1969, the Foxton Borough Council chlorinated its water supply, and this appears to have been detrimental to the existence of the frog. During the breeding season (spring), I noted that several horse troughs in the town no longer contained tadpoles. In previous years I had collected many from the same spots, and their disappearance was obviously caused by the new water supply. I also visited a neighbour's goldfish pond to observe the hatching frog spawn. The adult frogs that lived in the plants surrounding the water were still there. The pond, however, had been filled with chlorinated water, and the goldfish had died. The frogs, though they entered the water themselves, had found the pond unsuitable for spawning and no eggs were to be seen.
Nearby there stood an open concrete tank that had not been used for months. It contained about 2½ inches of rainwater, and into this the frogs had deposited their eggs. Soon the tadpoles hatched by their hundreds, and, as the food supply was virtually negligible, I scooped them up and released them into an unpolluted pond. This was fortunate, for two weeks later, the tank dried up during a particularly dry spell of weather.
Perhaps the frogs will become chlorine-resistant, or possibly the colonies near fresh water will keep up the numbers, for it would indeed be sad if Hyla Ewingi were to disappear from Foxton.
Hawaii, like New Zealand, is a group of oceanic islands with a pauperate indigenous mammal fauna and a lengthy history of introduced exotic species that have brought with them problems ranging from the careful conservation of game stocks to the control of animal pests. The appearance of this book is therefore of particular interest to New Zealand biologists as well as to students of mammalian introductions and of mammals generally. It also comes as a timely reference book in view of the proposed International Biological Programme investigations of the Hawaiian fauna and flora.
Following the introductory section, the book is divided into four parts. The first of these is a check list which is presented clearly and simply, using only the ordinal, family, generic and specific names based on Simpson's classification of mammals. Appended to each group is a common name and we are told whether each species is ‘indigenous’, ‘immigrant’, or ‘introduced’. A table showing the distribution of mammals separates the check list from the individual accounts of each species. These latter are each headed by brief notes on the original description of the species, type locality, native range and a more general section that includes the history of introduction or immigration (except in the case of the endemic bat), notes on control or management (where relevant), ecology, and references to the works of other authors. A miscellany entitled ‘Perspectives in Hawaiian Mammalogy’ precedes a substantial annotated bibliography.
The general layout, as described above, is good and orderly, although for the reader not familiar with the Hawaiian geography I feel that the sole map would have been more useful placed next to the table of distribution, earlier in the book, rather than in the chapter on ‘Perspectives’ near the end. Another minor fault in layout can be found in the occasional mixing of information from Hawaiian and overseas sources (e.g. as in the section on parasites and diseases). There would be no risk of confusing the reader if, where possible, overseas comparisons were kept separate or consistently discussed at the end of each section.
Turning now to content, I wonder whether in a book of this kind it is necessary to give a detailed account of the Linnaean system of classification. Working biologists should already be familiar with the system, and if a wider audience is envisaged for the book, then a short note (with appropriate references) should suffice for the serious amateur naturalist. I would also question the value of short, anecdotal accounts of aberrant or other behaviour patterns observed in some of the species examined by the author. These observations seem to be
The text is illustrated with a number of photographs. Most of these are good, although some (e.g. the photographs of feral cattle) are not of particularly high quality. Bearing in mind the difficulties encountered in taking good photographs of such animals, and the author's desire to be consistent and illustrate all the species he discusses, I wonder if it might have been better to omit such examples.
In going through the book, one is aware that the author has combed through a substantial literature, and nowhere is this better reflected than in the excellent annotated bibliography. This presents a vast range of material, from newspaper articles to formal scientific papers, all concisely annotated. This is a most valuable source of references for any worker in the field.
I can sum up by saying that this book is an interesting and valuable addition to the literature on Pacific mammals and that the minor criticisms above do not detract from the fact that this is a worthy publication in observation of the eightieth anniversary of the founding of the Bishop Museum.
The Identity of the Pliocene Seal from Cape Kidnappers, previously known as Artocephalus caninus pp. 13-18.
The Optical Coincidence System of Indexing Information. pp. 97-102.
Rain Forests and Gondwanaland. pp. 94-95.
Observations on the Effect of Chlorinated Town Supply Water on a Colony of Brown Tree Frogs in Foxton. p. 45.
Botany of the Southern Zone. Exploration, 1847-1891. pp. 49-93.
GRegg, D. R.
The Selection of Lectotypes in Palaeobotany. (Letter). p. 96.
Circadian Rhythms. pp. 124-131.
The Biological and Economic Importance of the Algae, Part 3. Edible Algae of Fresh and Brackish Waters. pp. 19-35.
The Use of Grasshopper Chromosomes to Demonstrate Meiosis. pp. 1-12.
Palaeoclimatic Change in the Last 1000 Years. pp. 114-123.
Concepts in Vegetation/Soil Dynamics. pp. 132-144.
Rowland, R. E.
The Lysosome. pp. 36-44.
The application of Electron Microscopy to the Study of some interesting Spiral Micro-organisms found in Pond Water Collected at Otari Plant Museum, Wellington. pp. 103-113.
Algae —
The Biological and Economic Importance of the Algae, Part 3, Edible Algae of Fresh and Brackish Waters, by
Artocephalus Caninus — see Seals Circadian Rhythms —
Circadian Rhythms, by
Ecology —
Concepts in Vegetation/Soil Dynamics by
Frogs —
Observations on the Effect of Chlorinated Town Supply Water on a Colony of Brown Tree Frogs in Foxton, by
Gondwanaland —
Rain Forests and Gondwanaland, by
Grasshoppers —
The Use of Grasshopper Chromosomes to Demonstrate Meiosis, by
Indexing —
The Optical Coincidence System of Indexing Information, by
Lysosomes —
The Lysosome, by R. E. Rowland. pp. 36-44.
Micro-Organisms —
The Application of Electron Microscopy to the Study of Some Interesting Spiral Micro-organisms found in Pond Water collected at Otari Plant Museum, Wellington, by
Palaeobotany —
The Selection of Lectotypes in Palaeobotany (Letter) by D. R. Gregg. p. 96.
Palaeoclimate —
Palaeoclimatic Change in the last 1000 Years, by
Rain Forests. see Gondwanaland.
Reviews —
Of P. Quentin Tomich's “Mammals in Hawaii: A Synopsis and Notational Bibliography”, by
Of A. L. Lehninger's “Biochemistry”, by
Of S. F. Singer's “Global Effects of Environmental Pollution”, by G. Stephenson. pp. 147-148.
Seals —
The Identity of the Pliocene Seal from Cape Kidnappers, previously known as Artocephalus caninus, by
Southern Zone —
Botany of the Southern Zone. Exploration, 1847-1891, by