THE EXTERNAL FACTORS OF REGENERATION IN ANIMALS
There is a constant interchange of material and of energy that takes
place between a plant or an animal and its surroundings, and this
interchange may be influenced by such physical conditions as
temperature, light, gravity, etc., or by such chemical conditions as the
composition of the atmosphere or of the water surrounding the organism.
We can study the process of regeneration either by keeping the
regenerating organism under the same conditions that it is subject to in
its natural environment, or else we can change the surrounding physical
or chemical conditions. In this way we can determine how far the
regeneration is affected by external changes, and how far it is
independent of them. If a change in the external conditions produces a
definite change in the regeneration, then the new condition is called an
external factor of regeneration.
_TEMPERATURE_
That the rate at which regeneration takes place can be influenced by
temperature has been shown by Trembley, Spallanzani, Bonnet, and by many
more recent writers. In fact, so familiar is the process to every one
who has studied regeneration, that it is usually taken for granted that
such is the case.
In general it may be stated that the limits of temperature under which
normal growth may take place represent also the limits of temperature
for regeneration. Lillie and Knowlton (’97) have determined the limits
of temperature within which regeneration takes place in _Planaria
torva_. The worm was cut in two transversely through the pharynx, and
the time required at different temperatures to produce a new head on the
posterior piece was recorded. The lowest temperature at which
regeneration was found to take place was 3°C. Of six individuals kept at
this temperature only one regenerated at all, and in this one the eyes
and brain were still incomplete after six months. The optimum
temperature, or at least that at which regeneration takes place most
rapidly, was found to be 29.7°C.; a new head developed in 46 days at
this temperature. At 31.5°C. regeneration was slower, requiring 8.5
days to make a new head. At 32°C. incomplete regeneration sometimes took
place, but death occurred in about six days. At 33°C. regeneration was
very slight, and the animals died within three days. At 34°C., and above
this point, no regeneration took place, and death soon occurred.
In _Hydra viridis_, Peebles (’98) has found that regeneration is quicker
at 26°-27°C. than at 28°-30°C. At the former temperature regeneration
takes place in 48 hours. If kept at 12°C. pieces may regenerate in 96
hours, but not all the pieces had regenerated in this case until 168
hours.
_INFLUENCE OF FOOD ON REGENERATION_
While the growth of an animal or of a plant is, in most cases, and, of
course, within certain limits, directly connected with the amount of
food that is obtainable, nevertheless extensive regeneration may take
place in an animal, or part of an animal, entirely deprived of food. In
this case the material for the new part is derived from the excess of
material in the old part, and not only surplus food material, but even
the protoplasm itself appears to be drawn upon to furnish material to
the new part. The relation between regeneration and the amount of food
present in the old part is well shown by experiments with planarians. If
a planarian is kept for several months without food, it will decrease
very much in size. In fact, the volume of a starved worm of _Planaria
lugubris_ compared with that of a fully fed individual may be only
one-thirteenth of the latter (Fig. 13, _A_, _B_). If a starved worm is
cut in two pieces, each piece will regenerate, although less quickly
than in a well-fed worm. The new part will continue to increase in size
at the expense of the old piece that is already in a starved condition.
On the other hand, an excess of food does not necessarily produce a
hastening of the regeneration, for, as Bardeen (’01) has shown, worms
that have been for several days without food may regenerate more quickly
than worms that have been fed just before they were cut into pieces.
[Illustration: FIG. 13.--Drawn by N. M. Stevens. _A._ Large well-fed
individual of _Planaria lugubris_. _B._ Same after being kept without
food for 4 mos. 13 days. Both drawn to same scale.]
The growth of the new part at the expense of the old tissues is a
phenomenon of the greatest importance, an explanation of which will
involve, I think, the most fundamental questions pertaining to
[Illustration: FIG. 13½.--_Planaria lugubris._ Dotted line indicates
where the worm was cut in two lengthwise. Upper three figures show how a
half, that is being fed, regenerates. Lower three figures show other
half kept without food.]
growth. The results show that growth is connected with a structural
factor, and is not simply a physiological phenomenon, although no doubt
physiological factors are involved. But the physiological factors that
are here at work seem to be different from what is ordinarily
understood; for the fact that a tissue that is slowly starving to death
should be reduced still further, and at a more rapid rate, in order to
supply material to a new part, is certainly a remarkable phenomenon. At
present we are not in a position to offer any explanation that rests on
observation, or experiment, as to how the transfer of material takes
place, or as to how the new tissue manages to get hold of the material
from other parts. It is possible to protect the old part to a large
extent by keeping the regenerating piece well supplied with food. If a
well-fed planarian is cut in two along the middle line of the body as
indicated in Fig. 13½, _A_, there develops, in the course of five or
six days after the operation, new material along the cut-side of each
piece, and a new pharynx appears at the border between the old and the
new parts. If one of the pieces is fed at intervals, it is found that
the new part grows more rapidly than does the new part in the piece
without food. The old tissue in both pieces has shortened somewhat after
the operation, and has also decreased somewhat in size as the first new
material developed along the cut-side, but in the piece that is fed the
old half begins to increase again until it reaches its former size, and
may even surpass the latter. A large full-sized worm is produced from
this piece, as shown in Fig. 13½, _B_, _C_, _D_. In the starved piece
the old part continues to grow small, due to the lack of food and also
to the increase in the new side. This increase takes place very slowly,
but ultimately a small symmetrical worm may be produced, as shown in
Fig. 13½, _E_, _F_, _G_. It will be seen that the starved piece needs
to produce relatively less and less new material in order to become
symmetrical, because as the old material diminishes, the pharynx comes
to lie nearer to the middle line.
_EFFECT OF LIGHT ON REGENERATION_
Although few experiments have been made to test the effect of light on
regeneration, it is certain that in many cases light has no effect on
the process, neither as to the quality nor the quantity of the result.
In one form, a tubularian hydroid, _Eudendrium racemosum_, it has been
shown by Loeb that the regeneration of the hydranth takes place only
when the animal is exposed to light. When a colony of eudendrium is
brought into the laboratory and placed in an aquarium, the hydranths
soon die; but if the colony is kept in a lighted aquarium, new hydranths
are regenerated in a few days. If, on the other hand, the colony is kept
in the dark, new hydranths do not appear; but if it is brought back
again into the light the hydranths appear. In one experiment one lot of
pieces was kept in diffuse daylight, and another lot in the dark. The
former produced fifty new hydranths in a few days; those in the dark had
not made any hydranths after seventeen days. They were then brought into
the light, and in a few days several hydranths had developed on each
piece.
Loeb also tried the effect of different colored light on the
regeneration of eudendrium. Dishes containing pieces of the hydroid were
put into a box that was covered by colored glass plates. Pieces
subjected to dark red and to dark blue light gave the following results.
The old hydranths, as is generally the case, were absorbed in the course
of three days. The first new hydranths appeared in the blue light on the
fourth day, and during the following days the hydranths in this lot
steadily increased. Eight days after the beginning of the experiment
there were eighty hydranths under the blue glass, but not one had
developed in the red light. On the ninth day the red glass was replaced
by a dark blue one. Two days later hydranths began to appear, and on the
following day thirty-two hydranths had appeared, and in a few days more
as many as sixty had developed.[19] Loeb concluded that only in the more
refrangible (blue) rays does the regeneration of the hydranth take
place, while the less refrangible (red) rays act as darkness does.[20]
This hydroid is the only animal yet found that shows the effect of light
on regeneration, and it is interesting to find that it is one of the few
animals known in which light has an influence on the growth, if the
heliotropism, or turning towards the light, of the hydranth is looked
upon as a phenomenon of growth.
There is another series of experiments made to test the effect of light
on regeneration, which gave, however, negative results. Herbst observed
that when the eye of certain crustacea[21] is cut off, sometimes an eye
and sometimes an antenna is regenerated. A number of individuals from
which the eyes had been removed were kept in the light, and others in
the dark, in order to see if the presence or absence of light is a
factor in determining the kind of regeneration that takes place. It was
found that as many individuals regenerated eyes in the dark as in the
light. It was discovered later by Herbst and myself, independently,
that, when the end only of the eye-stalk is cut off, an eye regenerates,
but when the eye-stalk is cut off at the base, an antenna regenerates.
The difference in the result has therefore no connection with the
presence or absence of light.
_GRAVITY_
The only case known amongst animals, in which regeneration is influenced
by the action of gravity,[22] is that of the hydroid _Antennularia
antennina_. This hydroid lives attached to the bottom of the sea several
metres below the surface. The hydroid consists of a single, vertical,
central stem, or axis, with two or four series of lateral branches along
which the hydranths arise (Fig. 14, _A_). The stem is attached by
so-called stolons, or roots. In its normal growth at the free end the
hydroid has been shown by Loeb to exhibit marked geotropic changes. If,
for instance, the stem is bent over to one side the new growth that
takes place at the apex of the stem directs the new part upwards in a
vertical direction.
If pieces are cut from the stem of antennularia and suspended in
[Illustration: FIG. 14.--After Loeb. Normal stalk of _Antennularia
antennina_. _B._ Piece regenerating in vertical, normal position. _C._
Piece regenerating in inverted position. _D._ Piece regenerating in
inclined, vertical position. _E._ Piece regenerating in inclined,
inverted position. _F._ Piece regenerating in horizontal position.]
the water, regeneration takes place at the cut-ends. If a piece is
suspended with its apical end upwards (Fig. 14, _B_), a new stem
develops at the upper cut-end, and new roots from the lower cut-end. If
a piece is suspended with its basal end upwards (Fig. 14, _C_), there is
formed at its upper (basal) end a new stem with its branches also
slanting upwards as shown in the figure. Roots appear at the lower
(apical) end. Since gravity is the only force that acts in a vertical
direction under the conditions of the experiment, Loeb concluded that it
plays an important rôle in determining the kind of regeneration that
takes place. Its action is of such a nature that a new stem develops
from the upper cut-end, and roots from the lower end, regardless of
whether the upper end is the basal or the apical end of the piece.
Similar results are also obtained, according to Loeb, if the pieces are
suspended obliquely. In a piece of this sort, it is found that new stems
arise along the upper surface of the old stem, and roots from the lower
surface as well as from the lower cut-end (Fig. 14, _D_, _E_). If a
piece of the stem is placed horizontally on the bottom of an aquarium,
the branches that come off from the under surface of the stem begin to
grow downwards at their ends, and where they come in contact with a
solid body they fasten themselves to it, thus showing that they are true
roots (Fig. 14, _F_). One or more stems may arise from the upper side of
the main stem. These stems grow vertically upwards, and produce lateral
branches. Only in one case did a new stem, or stem-like structure, arise
from one of the vertical branches, as shown to the left in Fig. 14, _F_.
Loeb found it also possible to change the character of the growth of the
apex of the normal stem and to transform it into a root. A long piece of
the hydroid was cut off and suspended vertically with the basal end
upwards. From the upper end a new stem began to grow, and then the
entire piece was reversed, so that the new stem pointed downwards. Under
these circumstances the young stem did not bend around and begin to grow
upwards, as a young plant might have done, but it ceased to grow as a
stem, and at its apex one or more roots developed. Loeb concludes: “I
cannot imagine by what means the place of the formation of organs in
antennularia is determined in connection with the orientation of the
animal except by means of gravity.”
The response of antennularia to the action of gravity is, I think,
conclusively demonstrated by Loeb’s results, but that the phenomenon may
be complicated by other factors is shown, I think, by the following
experiments. Driesch found that if pieces of antennularia are cut off
and placed between horizontal plates, so that both ends are free, roots
are produced by the basal end.[23] If the basal end with its new roots
is cut off, new roots may appear, but sometimes a thin stem also. If the
end is again cut off, a larger stem, and also one or two roots, may
appear, and if the operation is repeated again only a stem is formed.
The factor that brings about this change is not shown by the experiment.
The piece had been kept in a horizontal position throughout the whole
time. The apical end died in most cases without producing roots, but it
is not stated whether or not roots appear on the stem between the plates
of glass. If they develop they may affect the result, as certain
experiments that I have made seem to show.
In my experiments, made at a different time of year from that at which
Loeb’s experiments were made, pieces of the stem were suspended
vertically,--some with the apical end upwards, others with the basal end
upwards. In nearly all cases roots were formed by both the upper and
lower ends. In a few cases, in which the apical end was upwards, a new
stem developed at that end. Pieces suspended in a horizontal position
also produced roots at both ends. After removing the ends with their new
roots from the pieces suspended vertically, I found that roots again
appeared at both ends in nearly every case. The difference between these
results and those of Loeb may be due to the time of the year at which
the experiments were made, or possibly to some other difference, but the
results show that the response to gravity is not always so constant as
Loeb’s results indicate.
In a few cases in my experiments the basal end of the hydroid was left
attached to the stem on which it had grown, and the piece was put into
the same aquarium used for the preceding experiments. In those pieces
that lay on the bottom of the aquarium, with the stem standing
vertically, a new shoot, and not new roots, appeared on the upper end.
Other pieces were hung at the top of the water of the aquarium with the
stem turned downwards, and the basal, attached end of the piece upwards.
These pieces produced neither a stem nor roots from the apical end. The
results show that the presence of roots at one end has an influence on
the regeneration at the other end. The same thing was shown in one case
in which a short piece sank to the bottom of the dish and, developing
roots at its basal end, became fixed: a stem grew out of the apical end.
A number of other experiments that I made, in which pieces of
antennularia were fixed to a rotating wheel, gave negative results,
since neither roots nor stems appeared on the pieces. The rubbing of the
ends of the piece against the water as the wheel turned round, or else
the agitation of the water, prevented, most probably, the regeneration
from taking place.
How gravity acts on antennularia has not as yet been determined. The
only suggestion that we can offer at present is that it brings about a
rearrangement of the lighter and heavier parts of the tissues. A
rearrangement of this sort has been demonstrated when the egg of the
frog is inverted, and in consequence certain changes are brought about
in the development that will be described in another chapter.
_EFFECT OF CONTACT_
The contact of a newly forming part with a solid body has been shown by
Loeb in a few cases, at least, to be a factor in regeneration. If a
piece is cut from the stem of the tubularian hydroid _Tubularia
mesembryanthemum_, and the piece held so that its basal end comes in
contact with a solid body, a root develops at that end. If a piece is
held in a similar position, but with its apical end in contact with a
solid body, a root does not develop from this end. Evidently the
development of a root in this form is also connected with an internal
factor; but that there is in reality a reaction in this case, and not
simply the development of a root at the basal end, is shown by the
following experiment: If a piece is cut from the stem and suspended so
that both ends are surrounded by water--it makes no difference whether
the piece is vertical or horizontal--a hydranth develops first on the
apical end, and then another on the basal end (Fig. 15, _B_). When the
apical end of a piece is stuck in the sand, leaving the basal end free,
a hydranth develops on the latter, but not on the end in the sand.
[Illustration: FIG. 15.--After Loeb. _A._ A piece of the stem of
margelis placed in a dish. Roots come off where stem touches dish, and
polyps at other points. _B._ Piece of the stem of tubularia producing a
hydranth at each end. _C._ _Cerianthus membranaceus._ Piece cut from
side producing tentacles only on oral side of cut.]
In another hydroid, _Margelis carolinensis_, studied by Loeb, the effect
of contact is more easily demonstrated. If a branch of margelis is put
into a dish of water and is kept from all motion, the parts that come in
contact with the dish produce roots that attach themselves. Even the
apical end of the stem may grow out as a root, as shown in Fig. 15, _A_.
Those parts of the branch that are not in contact with any solid object
give rise to new hydranths. Another hydroid, _Pennaria tiarella_, also
shows, according to Loeb, the same response to contact. In this
connection it is interesting to find that a growing hydranth of
pennaria, if brought in contact with a solid body, turns away from the
region of contact and bends at right angles to the body which it
touches. We find, once more, that a factor having an influence on the
growth of the animal has also a similar influence on the regeneration.
Loeb has found that if pieces of the hydroid _Campanularia_ are cut off
and placed in a dish filled with sea water, all the hydranths that touch
the bottom of the dish are absorbed and transformed into the substance
of the stem. The cœnosarc may creep out of the stem wherever it comes in
contact with the glass, and produce stolons that give rise to new polyps
on their upper surfaces. Loeb shows that growth takes place at the end
of the stolon that pushes out of the perisarc, and this growing region
draws the rest of the cœnosarc after it. If a new hydranth appears along
the old piece, the cœnosarc is drawn towards the hydranth.
_EFFECT OF CHEMICAL CHANGES IN THE ENVIRONMENT_
Temperature, light, gravity, and contact are the most familiar kinds of
external physical agencies that have a direct influence upon the growth
of organisms. Food, though coming from the outside, yet acts only after
it has entered the body. Organisms that live in water may be affected by
the quantity and the kinds of the salts contained in the water, and also
by the dissolved gases. The only experiments that have been made to show
the influence of this last class of agents on animals are those made by
Loeb. He placed pieces of the stem of tubularia in sea water of
different degrees of concentration. After eight days the pieces, that
had meanwhile produced hydranths, were measured. It was found that the
maximum growth in length takes place, not in normal sea water, but in a
much diluted solution. Loeb interprets this result to mean that the
cells of tubularia must have a certain amount of turgidity in order to
grow, and this is possible so long as the concentration does not pass a
certain limit. This limit is reached by the addition of 1.6 grams of
sodium chloride to each 100 c.c. of sea water. With a decrease in the
concentration, the cells become more turgid, the maximum point
corresponding to the maximum amount of growth. Below this point the
solution is supposed to act as a poison. The most important result of
this experiment is to show that the maximum growth does not take place
in sea water in which the animal is accustomed to live, but in a much
more dilute solution. Normal sea water contains about 3.8 per cent of
salts; the maximum growth takes place in a solution containing only 2.2
per cent. Not only is the length of the stem greater in the latter
solution, but the thickness of the stem is also greater. The stem is
smaller in a solution containing more salt than that contained in
ordinary sea water.
There is another variant in these solutions which Loeb takes into
account. With the increase in concentration of the solution its power of
absorbing oxygen decreases, but the difference is too slight to affect
the main result.
Not only does the amount of salts in solution affect the osmotic
condition of the cells, but the salts also play a part in the metabolism
of the animal. As the result of a series of experiments, the details of
which may be here omitted, Loeb has shown that the regeneration of
tubularia takes place only when the salts of potassium and of magnesium
are present. A very little of the potassium salt is necessary, too much
retards, and still more prevents regeneration.
There must be also a certain amount of oxygen dissolved in sea water in
order that regeneration may take place. If a piece of the stem of
tubularia is cut off and one end pushed into a small tube that fits the
stem closely, and if the tube is then stuck into the sand at the bottom
of an aquarium, a hydranth develops only at the free end of the piece,
and none at the end in the tube. The result appears to be due to the
lack of oxygen. If the piece is then taken from the tube, a hydranth may
appear at the end that has been in the tube.
Another experiment shows the same result even more clearly. If a piece
of the stem is suspended freely in the water, so that its lower end is
almost in contact with the surface of the sand, but does not quite touch
it, no regeneration takes place at the lower end. This result is
interpreted by Loeb as due to the lack of oxygen in the water near the
surface of the sand.[24]
_GENERAL CONSIDERATIONS_
In connection with the action of external factors on regeneration it is
evident that in some cases they may not be in themselves necessary for
the growth of a new part, yet when growth takes place they may determine
what sort of a part is produced. For instance, if gravity determines
the kind of regeneration in antennularia, it is possible that if the
regenerating piece were placed on a rotating wheel, the piece might
still produce a new stem at the apical end, and roots at the lower end.
In an experiment of this sort that I made, the pieces did not, it is
true, regenerate at all, but this was probably due not to the change of
position in regard to gravity, but to agitation of the water, or to the
rubbing of the cut-end against the water. It is also possible that in
this form the attachment of the piece at one end may be a factor that
may counterbalance the action of gravity. Other factors, such as food,
or temperature, or oxygen, appear not to determine the kind of product
that results, but only the rapidity with which the change takes place.
The salts in solution seem also to act on the rate and extent of the new
growth, but possibly other cases may be found in which the kind of
regeneration may also be affected by the salts.
It is important to find that those animals whose growth and regeneration
are influenced by such external factors as light, gravity, and contact
are attached animals that stand in a constant relation to these physical
agents. They form only a very small part of the entire number of animals
in which regeneration takes place. Animals that constantly move about
are not, as a rule, influenced during their growth and regeneration by
gravity and contact, and under natural circumstances they are always
changing their position in regard to these agents. Temperature, and
food, and substances in solution act alike on fixed and free forms, and
they are, it appears, both influenced in the same way by these agents.
The most significant fact that has been discovered in connection with
the influence of external factors on regeneration is that the same
factors that influence the normal growth of the organism also affect in
the same way the regeneration.
As yet an analysis of the external factors that influence growth has not
been made out as completely for animals as for plants, especially in
those cases in which the result is determined by several factors at the
same time. An examination of the factors that influence regeneration in
plants will be made in a later chapter. First, however, the internal
factors of regeneration in animals will be considered.