A M . ZC.OLOC.IST, 6:213-219 (1966).
Neurosecretion and Salt and Water Balance in the Annelida and
Crustacea
FRED I. KAMEMOTO, KENNETH N. KATO, AND LOIS E. TUCKER
Department of Zoology, University of Hawaii
SYNOPSIS. The possible role of .the neurosecretory system in regulation of salt and
water has been studied in the annelids and crustaceans. In the earthworm, Lumbricus
terrestris, a brain factor influences 'the salt and osmotic concentration of the blood and
coelomic fluid. Removal of the brain results in the increase of water influx with a decrease in the salt and osmotic concentrations of the body fluids. The decreases in salt
and osmotic concentrations can be prevented by the implantation of the brain or the
injection of brain homogenates.
In the freshwater crayfish, Procambarus clarkii, a factor, presumably secreted in the
brain and released in the eyestalk, seems to maintain the normal permeability of the
body surfaces to water. Eyestalk removal, which eliminates the release site, results in
the increased influx of water with a decrease in the salt concentration of the blood. A
brain factor also seems to be involved in maintaining the sodium and osmotic concentrations of the blood.
In the semi-terrestrial grapsid crab, Metopograpsus messor, the thoracic ganglion,
under the control of an eyestalk element, secretes a factor- involved in increasing the
permeability of the body surfaces to water. The removal of the eyestalks, the implantation of the thoracic ganglion, or the injection of extracts of thoracic ganglia, results in
changes in the osmotic concentration of the blood tending toward that of the medium.
In all three species studied, the neuroendocrine factors seem to be involved primarily
in the regulation of the permeability of the body surfaces to water.
To study the possible role of the brain
on
the permeability of the body wall to
Salt and water balance in the various
invertebrate groups which have been stud- water, experiments were conducted in
ied appears to be under the regulation of which various parts of the central nervous
neurosecretory factors as they are in most system were removed. The removal of the
vertebrate groups. In the earthworm, a brain caused a marked increase in body
nervous control of permeability to water weight as compared to normal animals
has been suggested by Maluf (1939). He when the animals were placed in tap water.
observed increases in the weights of animals The removal of the subesophageal ganglion
either with the ligation of the anterior end or the severing of the circumesophageal
of the worm or the sectioning of the ventral connectives resulted in changes in weight
nerve cord. He suggested that this increase which were comparable to those of normal
in weight was the result of an increased animals. This suggested that only the brain
permeability of the body wall to water, and was necessary for maintaining the normal
that the stimuli which influence the perme- body weight of the animals placed in tap
ability of the body wall to water arise water, and that a nervous connection of
anteriorly, possibly in the brain. This view, the brain with the ventral nerve cord was
based on similar experiments, has been sup- not necessary.
ported by Chaucheprat and de Puytorac
The increase in weight caused by removal
(J961). Aros and Bodnar (1960) reported of the brain is accompanied by a decreased
histological changes in the neurosecretory sodium concentration in the coelomic fluid
cells of the brain with dehydration of the and blood. Changes in the sodium conearthworm, suggesting a neurosecretory centration in the body fluids can be precontrol of osmoregulation. This idea of the vented in brainless animals if a brain is
influence of the brain and possibly a neuro- replaced by either its implantation into
secretory factor on osmotic regulation in the coelom or the injection of brain homogthe earthworm, Lumbricus lerrestris, has enates. These results would suggest a presbeen explored further (Kamemoto, 19fi4). ence of a chemical factor or factors, perhaps
(213)
ANNELIDA
214
FKEU I. KAMEMOTO, KENNETH N. KATO, AND LOIS E. TUCKER
neurosecretory, in the brain of earthworms
which influence their salt and water balance. Histological examination of both
normal and implanted brains suggests a
normal functioning of the neurosecretory
cells in the implanted brain. The mechanism by which the postulated neurosecretory factor influences salt and water balance
is as yet unknown although a control of
permeability of the body wall to water is
suggested. Preliminary results from studies
of the concentrations of salt in the urine
of earthworms, collected according to the
method of Bahl (1947), indicate an increase
in chloride in the urine of brainless animals. The chloride concentration in the
urine of normal animals is approximately
1 meq/1, which is in good agreement with
the values reported by Bahl (1947), Ramsay (1949) and Boroffka (1965). Chloride
concentrations as high as 13 meq/1 have
been observed in the urine of brainless
animals after four days. This is a relatively
high value since the chloride concentration
of the coelomic fluid of L. terrestris is about
42 meq/1 (Kamemoto, el, al., 1962). The
possibility of the involvement of neurosecretory factors in the function of the
earthworm nephridium deserves more experimental attention.
Although considerable work has been
done on the osmotic relations in the polychaetes, very little is known about possible
neurosecretory relations with osmoregulation. Of the 26 nuclei described in the
brain of Nereis by Holmgren (1916), van
Damme (1962) considers only two nuclei,
the XVIFth and XXth, situated in the posterior part of the brain to be neurosecretory. We have studied the histological
changes in the aldehyde-fuchsin-positive
cells of these nuclei in Nereis virens collected at Woods Hole and exposed to dilute
salinities for 24 hours. Ar. virens, a volume
regulator, increases in weight when placed
in a dilute medium, then gradually decreases, approaching its initial weight in
time (Sayles, 1935). The anterior portions of
the worms kept in varying concentrations of
sea water were fixed in Bouin's fixative and
stained by the standard aldehyde-fuchsin
method. The numbers of aldehyde-fuchsin-
TABLE 1. Number of aldehyde-fuchsin-posU'we cells
in the brain of Nereis virens in varying salinities.
Medium
Normal SW
50% SW
25% SW
No.
animals Mean
SD
Range
30
103
423
22
34
214
9-75
66-193
231-804
—
0.001
0.001
positive cells in the two neurosecretory
nuclei are presented in Table 1. There is
a marked increase in the number of
aldehyde-fuchsin-positive cells in animals
exposed to dilute concentrations of sea
water. Interpretation of these results must
await further experimentation. It is interesting to note, however, that the exposure
of the animals to dilute media results in
an increased number of the so-called neurosecretory cells in the brain of N. virens.
Obviously, further studies are necessary in
order to elucidate the possible relationship
of neurosecretion and osmoregulation in
these polychaetes.
CRUSTACEA
Among the Crustacea, most of the work
on neurosecretion and osmoregulation has
been concerned with the changes in weights
and water content of the animal in relation
to the molt process. Guyselman (1953),
while studying the molt process in Uca
pugilalor, observed diurnal rhythmical fluctuations in the weights of the animals, presumably due to changes in their water content. He suggested a possible role of the
sinus gland hormone in regulating these
fluctuations. Scudamore (1947), while
studying the physiologic changes which accompany molting in the crayfish, reported
that removal of the eyestalk or sinus gland
resulted in increased weights with greater
water content, these changes being prevented by the implantation of the sinus
gland. Carlisle (1955) reported similar results in the crab, Carcinus maenas.
It occurred to us that, in addition to
changes in water metabolism accompanying
molt, there might be a neuroendocrine control of "homoiostasis" in salt and water balance in crustaceans as has been shown in the
vertebrates and suggested for the earthworm
(Kamemoto, 1964) and the cockroach (Wall
215
OSMOREGULATION IN ANNELIDA AND CRUSTACEA
FIG. 1. Weight changes in normal (N) and eyestalkless (D) Procambarus clarkii in tap water. Means
and standard deviations of 10 normal and 19 eyestalkless animals presented.
and Ralph, 1964). Perhaps the best indication for the existence of such a mechanism in the crayfish is the report of McWhinnie (1962). She injected extracts of
neurosecretory sites into normal crayfish
and observed significant changes in the
free calcium levels of the blood. Brain
extracts caused an increase in free calcium
while eyestalk extracts caused a decrease.
These results were obtained two hours after
injections. Such results are highly suggestive of the relatively rapid responses of
animals to environmental stresses which
are necessary for the maintenance of homoiostasis. With this "homoiostasis" in
mind, we have been studying the possible
neuroendocrine regulation of salt and water
balance in two species of decapod crustaceans, the freshwater crayfish, Procambarus
clarkii, and the semi-terrestrial grapsid crab,
Metopograpsus messor.
Procambarus clarkii, a species introduced
into Hawaii, is readily available in local
fresh waters. The animals were held in the
laboratory in aerated tap water for at least
a day before being used in any experiments.
All experiments were carried out at room
temperatures. To determine the effects of
eyestalk removal on the weights of these
animals, the animals were destalked either
by cutting off the eyestalks or by ligating
them at their bases. Animals are weighed,
replaced in tap water, and their weight
changes observed by subsequent weighings.
No difference was observed in the two methods of destalking. The results are presented
in Fig. 1. There is very little change in the
weights of normal animals over the fourday period of the experiment. In destalked
animals, there is initially a sharp drop in
weight followed by a recovery over the
four-day period. No explanation can be
offered for the initial drop in weight of
destalked animals. Except for this initial
decrease, there seems to be little difference
between them and the normal animals.
Weight changes were not followed further
because of the approach of the premolt
condition which influences the water content in crayfish (Scudamore, 1947). Changes
in the chloride concentration of the blood
were also studied in eyestalk-ligated animals. Samples of 10 jA of blood were collected from normal and destalked animals
at 6, 12, and 24 hours after eyestalk ligation.
Ten animals were used in each group.
Chloride concentrations were determined
with an automatic chloride titrator. The
results are presented in Table 2 in terms
of mean percent decrease in the chloride
concentration of the blood. There is_ a
significantly greater decrease in the chloride concentration after destalking as compared to the normal animals. Percent
changes are used because of the variability
in the chloride concentrations of individual
animals.
Although there is no increase in the
weight of destalked crayfish during the first
few days after eyestalk removal, there seems
to be a greater influx of water into the
animal which is possibly eliminated by the
nephridia as suggested by the results presented in Fig. 2. The nephropores of normal and destalked animals were plugged
with modeling clay, thereby preventing the
elimination of urine. The animals, kept
in tap water, were weighed over a 24-hour
TABLE 2. Mean percent change in chloride concentration of blood after eyestalk ligation in Procambarus clarkii. Normal chloride concentration = 1S7
meq Cl'/l blood.
Time
6hr
12 hr
24 hr
Normal
Mean SD
-0.7
-2.6
-3.7
1.26
1.80
1.34
Eyestal k-ligated
Mean SD
— 1.4
—4.3
—6.5
1.58
2.58
4.13
P
—
0.05
0.05
216
FRED I. KAMEMOTO, KENNETH N. KATO, AND LOIS E. TUCKER
FIG. 2. Weight changes in normal (N) and eyestalkless (D) Procambarus clarkii in tap water alter plugging of nephropores. Means and standard deviations of 16 normal and 9 eyestalkless animals
presented.
period. There is a significantly greater
increase in the weights of animals after destalking as compared to normal animals,
suggesting that there is a greater influx of
water, presumably through the gills or the
gut. The above studies would indicate that
there is an increased influx of water fol-
lowing the removal of the eyestalks accompanied by the increased elimination of the
urine and a decrease in the salt concentration of the blood. Preliminary studies indicate that there is no change in the salt
concentration of the urine. It may be possible that an increase in urine output,
although without change in the salt concentration, may be responsible for the decrease
in the chloride concentration of the blood.
Replacement experiments with aqueous
eyestalk extracts were unsuccessful. However, injection of extracts of the eyestalks
and brain into normal animals increased
the sodium concentration of the blood.
Sodium concentrations were estimated by
flame photometry while the osmotic concentrations were determined with the
Mechrolab vapor pressure osmometer. The
results are presented in Table 3 and Table
4. Each animal received two animalequivalents of the extracts in 0.1 ml of
physiological saline solution; physiological
saline injections were used as controls.
Maximal responses were obtained at six
hours after injections. Although the increases in sodium and osmotic concentrations of animals receiving eyestalk extracts
are not statistically significant, these results
seem to indicate the existence of a factor
or factors in the brain and eyestalk which
maintain the sodium and osmotic concentrations of the blood.
The grapsid crab, Metopograpsus messor,
was collected from mud flats which were
TABLE 3. Sodium and osmotic concentrations of blood of Procambarus darkii 6 hours after injection of eyestalk extract.
Na+ cone. (meq/1)
Normal
Injected
No.
animals
Mean
SD
16
16
184
193
15.9
16.1
'P
Osmotic cone. (meq NaCl/1)
No.
animals
Mean
SD
16
16
0.1
212
222
16.1
14.9
P
0.1
TABLE 4. Sodium and osmotic concentrations of blood of Procambarus clarkii 6 hours after i•njec-
tion of brain homogenate.
+
Na cone. (meq/1)
Normal
Injected
No.
animals
Mean
SD
P
16
16
175
196
15.9
19.8
0.01
Osmotic cone. (meq NaCl/1)
No.
animals
Mean
SD
16
16
202
224
15.7
17.4
P
0.001
217
OSMOREGULATION IN ANNELIDA AND CRUSTACEA
TABLE 5. Total osmotic concentration (meq NaCl/l) of blood of normal and destalked Metopograpsus messor placed in various salinities for 24 hours.
Medium
Normal SW
(560)
110% SW
(610)
25% SW
(140)
Normal
No.
animals
Mean
10
10
10
10
10
10
10
10
494
484
510
512
494
457
484
478
-Osmotic concentrationDestalked
No.
Mean
SD
animals
8.8
6.4
23.6
15.9
8.5
12.5
28.1
14.5
10
10
10
10
10
10
10
10
498
490
572
523
462
430
430
436
SD
11.4
16.8
34.7
18.0
11.3
10.2
17.5
14.4
.001
.001
.001
.001
.001
TABLE 6. Total osmotic concentration (meq NaCl/l) of blood of Metopograpsus messor 4 hours after
injection of thoracic ganglion extract.
Medium
Normal SW
110% SW
25% SW
Normal
No.
animals
Mean
SD
10
10
10
10
475
472
494
441
11.1
18.8
8.5
8.0
exposed during low tides. The animals are
readily found under rocks and debris or
can be collected from their burrows in the
mud. These animals were kept in the laboratory at least one day before use, and only
intermolt animals were used in the experiments. M. messor is an excellent osmoregulator, maintaining its blood osmotic
concentration to the equivalent of about
85-90% of normal sea water. In the experiments presented below, the animals were
kept in normal (100%) sea water, concentrated (110%) sea water or dilute (25%)
sea water for 24 hours. Under these conditions, M. messor is a hyporegulator in
normal and concentrated media and a hyperregulator in the dilute medium. When
destalked animals were placed in these
various media, there was a change in the
osmotic concentration of the blood tending
toward that of the medium (Table 5).
Thus, there was an increase in the osmotic
concentration of the blood in destalked
animals placed in normal and concentrated
sea water, while there was a decrease in
those placed in 25% sea water. It should
Thoracic ganglion injected
No.
animals
Mean
SD
P
9.1
9.3
6.4
7.1
0.001
0.001
0.001
0.001
10
10
10
10
515
515
476
425
be noted that there is a wide variation in
the osmotic concentrations of the blood
between the several groups of normal animals in 25% sea water, although there is
a lesser variability within a particular
group, as can be seen from the standard
deviations presented.
As found for the crayfish, replacement
experiments with the injection of eyestalk
extracts into destalked crabs produced negative results. On the other hand, the injection of extracts of the thoracic ganglia into
normal, unoperated animals resulted in
changes in the osmotic concentration of
the blood similar to those caused by eyestalk removal. Injection of extracts of
thoracic ganglia caused an increase in the
osmotic concentration in normal and concentrated sea water but caused a decrease
in animals placed in a dilute medium
(Table 6). Each experimental animal received two thoracic ganglion-equivalents
of extract in 0.05 ml crab saline. Control
animals received 0.05 ml of crab saline.
Maximal responses were obtained in four
hours. Similar results were obtained with
218
FRED I. KAMEMOTO, KENNETH N. KATO, AND LOIS E. TUCKER
TABLE 7. Total osmotic concentration (meq NaCl/l)
of blood of Metopograpsus messor after implantation of thoracic ganglion. Animals in 25% sea water.
Osmotic concentration
No.
animals
Mean
SD
Normal
Muscle implant
Thoracic ganglion implant
10
10
10
447
443
422
17.0
14.8
19.6
Normal vs. thoracic ganglion implant: p = 0.001.
Muscle implant vs. thoracic ganglion implant: p =
0.05.
the implantation of a single thoracic ganglion into normal animals and held in
25% sea water for 24 hours (Table 7).
Implants of muscle had no effect on the
osmotic concentration of the blood. These
results suggest the presence of a chemical
factor in the thoracic ganglion of the grapsid crab which may be effective in increasing the permeability of the body surface
to water, causing an efflux of water in a
hypertonic medium and an influx of water
in a hypotonic medium, resulting in corresponding changes in the osmotic concentration of the blood.
The results presented here on these two
crustacean species, however meager, suggest
a possible ncuroendocrine control of salt
and water balance. They further suggest
that there is a difference in the neuroendocrine pathway involved in the two species.
The following hypothetical schemes are
presented.
In the crayfish, a factor (or factors) is
secreted in the brain and transported to
the eyestalk for release, presumably by the
sinus gland. The factor maintains the normal permeability of the body surfaces to
water. Such a neurosecretory pathway has
been described by Bliss, et al. (1954). Eyestalk removal in these animals results in
the removal of the release site, causing an
influx of water and a decrease in the salt
concentration of the blood. The factor
also causes an increase in the salt and osmotic concentration of the blood as demonstrated by the increased concentrations
following the injection of brain homogenates.
In the grapsid crab, the removal of the
eyestalks causes a change in the blood os-
motic concentration toward that of the
medium. Similar results are obtained by
the injection or the implantation of the
thoracic ganglion. Perhaps a permeability
factor is produced in the thoracic ganglion
which regulates the permeability of the
body surface to water. Neurosecretory cells
in the thoracic ganglion of crabs have been
described by Matsumoto (1954, 1962) and
by Maynard (1961a, b). This permeability
factor may be under the control of an
eyestalk element, either nervous or humoral, with a normal inhibition of the
release of the factor when the animals are
exposed to hypo- or hypertonic conditions.
In any event, the results reported here suggest that a neuroendocrine phenomenon
exists for the maintenance of "homoiostasis" in salt and water balance in the crustacea.
ACKNOWLEDGMENTS
We arc greatly indebted to Dr. Robert L. Philibcrt, Southwest Missouri State College, for the collection and processing of the Nereis virens tissues
for histological study. The studies reported were
supported by grants GB-99 and GB-673 from the
National Science Foundation.
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