WLLIS, Richard Eugene, 1936- NEDROENOOCRINE CONfROL OF WATER BALANCE IN TIlE DECAPOD CRUSTACEAN ~ 'THALAM!TA' CRENATA. University of Hawaii, Ph.D., 1972 Zoology
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\ THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED. I f F
NEUROENDOCRINE CONTROL OF
WATER BALANCE IN THE DECAPOD CRUSTACEAN,
THALAMITA CRENATA
A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAII IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
IN ZOOLOGY
AUGUST 1972
By
Richard E. Tullis
Dissertation Committee:
Fred I. Kamemoto, Chairman Sidney J. Townsley Pieter B. van Weel Frederick C. Greenwood Richard D. Allen
I. PLEASE NOTE:
Some pages may have
indistinct print. Filmed as received.
University Microfilms, A Xerox Education Company Abstract
The involvement of the central nervous system in hydromineral regulation of decapod crustaceans has been demonstrated in the past, yet the control of this regulation has not been fully explored. It has been postulated that a neuroendocrine mechanism may control water and mineral movements in steady state animals and that neurosecretory products produced and secreted from the central nervous system may be the factors responsible for this control.
Two factors contained in the ventral ganglion and brain of the brackish water crab Thalamita crenata Latreille were separated into water soluble and acetone soluble fractions. When these fractions were bioassayed in crayfish and crabs, it was found that the acetone soluble fraction contained a biologically active compound which, when injected into the assay animal, caused an increased influx of tritiated water into the animal; the water soluble fraction produced a decrease in water influx after injection. Neither the acetone soluble nor the water soluble fractions had an effect on sodium-22 influx.
This increase and decrease of tritium influx was also demonstrated by perfusing the gill chambers of the crab with the isotopic medium; the acetone soluble fraction increased the influx while the water soluble fraction caused a decrease. By isolating the intestine of the crayfish, it was observed that the acetone soluble fraction increased the lumen to haemolymph tritiated water flux; the water soluble frac tion did not cause a change in the flux rate.
The water soluble fraction which produced a decrease in water influx in whole animals was partially purified by column chromatography. iv
Semi-pure fractions were obtained by separation with a Sephadex G-25 to G-IO gel series. Further purification was obtained by eluting the
Sephadex G-IO activity-containing fraction through CM Sephadex ion exchange gel utilizing a continuous ionic strength concentration gra dient. The active material was a small molecular weight polypeptide.
Substances other than these central nervous system factors were bioassayed for their effect on water and sodium influx. Acetylcholine, norepinephrine, glutamic acid, and vertebrate neurohypophyseal octa peptides did not alter the rate of the influx of water or sodium.
5-hydroxytryptamine and pericardial organ extract increased sodium-22 influx while ecdysone appeared to increase the influx of tritiated water.
It is proposed that these two factors found in the central nervous system and which affect the influx of tritiated water are important in the maintenance of the osmotic steady state of decapod crustaceans.
These factors modify the permeability to water of the gills and intes tine to maintain the osmotic balance of these animals. Table of Contents
Page
Abstract •• •••• • • •• • •• • •••• • •• • •• • • • iii List of Tables •••• • • • • •• • • • • • •• • • •• •• • vi List of Illustrations • • •• • •• •• ••• • • •• • • •• • viii Introduction •••• • • •• • ••• • • • · •• • · •• • • 1 Material and Methods • • • · • • · • •• · • • •• • · • ••• 8 Bioassay • · • • • ••• • • • • • •• • • •• •• • 9 Tissue Preparation • • • •• ••••• •• •••• •• • 11 Separation and Purification •• •• • •• • • •• •• • • • 14 Properties of the Active Material · • • • •• • • •• • • • 17 Other Activity-Containing Substances •• • •• ••• •• • 18 Effector Tissues •• • •• •• · •• ••• · •• •• • 19 Results •• • ••• • •• ••••• • ••• •• •• • • •• 22 Discussion • • · • • • • • • •• •• •••••• • • · •• •• • 48 Literature Cited •• • • • • • • · • • • • ••• • •••• • • 65 List of Tables
Table Page
1 Water and sodium influx into crabs injected with minimally treated ventral ganglion (VG) and brain (Br) extract •••• 24
2 Water and sodium influx into crabs injected with boiled, minimally treated VG extract •••••••••••••• • • 25 3 Water and sodium influx into crabs injected with muscle tissue extract ••••••••••••••••••••• • • 26 4 Water and sodium influx into crayfish injected with minimally treated VG and Br extract •••••••••• •• 26
5 Water and sodium influx into crayfish injected with minimally treated Br extract from 50% and 100% sea water adapted crabs ••••••••••••••••••••• •• 27
6 Water and sodium influx into crayfish injected with fully processed VG and Br extract •••••••••••••• •• 28
7 Water and sodium influx into crayfish injected with collected VG fractions from Sephadex G-50 ••••••• •• 29
8 Water and sodium influx into crayfish injected with Br fractions collected from Sephadex G-50 ••••••••••• 30
9 Water and sodium influx into crayfish injected with active VG and Br fractions from Sephadex G-75 ••••••••••• 32
10 Water and sodium influx into crayfish injected with VG fractions collected from Sephadex G-25 ••••••••••• 34
11 Water and sodium influx into crayfish injected with Fraction II of VG eluted through Sephadex G-IO. ~ ••••• 37
12 Water and sodium influx into crayfish injected with the active fraction from VG and Br eluted through GM Sephadex • 38
13 Water and sodium influx into T. crenata injected with ~Sephadex the active VG fraction from elution ••• • • • 38 14 Water and sodium influx into crayfish injected with the acetone soluble VG and Br fraction. •••••• • • • • 41 15 Water and sodium influx into crayfish injected with oxytocin and vasopressin •••••••••••••• •• • • 42 ~i
Table Page
16 Water and sodium influx into crayfish injected with 5-hydroxytryptamine, norepinephrine, acetylcholine, and glutamic acid ••••••••••••••••••••• 42
17 Water and sodium influx into crayfish injected with pericardia1 organ extract ••••••••••••••••• 43
18 Water and sodium influx into crayfish injected with ecdysterone •••••••••••••••••••••••• 43
19 Influx of tritiated water into crabs after gill chamber perfusion ••••••••••••••••••••• 44 List of Illustrations
Figure Page
1 Flow diagram of tissue preparation •••••••••••• 13 2 Diagram of separation procedure • • • • · • •••• • •• • 15 3 Tritiated water saturation times into crabs. • • · • • · • 23 4 Chromatograms: ventral ganglion (VG) extract through Sephadex G-50 • • •• • • •• • • • ••• · • ••• •• • • 28 5 Chromatogram: brain (Br) extract through Sephadex G-50 • • 30 6 Chromatogram: VG and Br extracts through Sephadex G-75 • • 31 7 Chromatogram: VG active fraction through Sephadex G-25 • • 33 8 Chromatogram: VG extract through Sephadex G-25 • •• • 33 9 Chromatogram: Br extract through Sephadex G-25 • · • • • • 35 10 Chromatogram.: VG active fraction through Sephadex G-15 • • 35 11 Chromatogram: VG active fraction through Sephadex G-10 · • 36 12 Chromatogram: active Br fraction from G-10 eluted through CM Sephadex • • •• • • •• · • • • • • •• • • • · 37 13 Molecular weight estimation of the water soluble active material ••••••••••••••••••••••• • • 40 14 Tritiated water flux from the lumen of the isolated crayfish intestine into the bathing medium •• •• • •• • 45 15 Tritiated water flux from the bathing medium into the lumen of the isolated crayfish intestine •••• •• ••• 47 16 Diagram. of proposed secretions and effector tissues in the crab •••••••••••••••••••• •• • • 64 Introduction
Marine animals living in brackish water respond to salinity
fluctuations in two ways. They adjust to the changes, thus maintaining
their body fluid osmoconcentration as it was prior to environmental
changes, or they conform, having no control over their internal blood
concentration, the blood changing isotonically with changing environ mental salinity. The animal which is able to adjust its internal body
fluid against an external change is referred to as an osmoregulator while the animal whose body fluid concentration changes directly pro portional with the changing medium is··termed an osmoconformer (Krogh,
1939; Prosser and Brown, 1961).
Euryhaline crustaceans are generally osmoregulators and must have
specific physiological means to maintain their internal osmoconcentra
tion. These physiological means must be such that they can maintain
the animal in a hypo-osmotic or hyperosmotic state to the environment; they may even have to be such as to allow the animal to adjust from a hypo-osmotic regulator to a hyperosmOtic regulator. Osmoregulators rely on the influx or efflux of water and/or elimination from, or accumulation of ions in, the body fluid to maintain their internal osmoconcentration (Robertson, 1953; Lockwood, 1962 and 1964; Potts and
Parry, 1964). In some animals mobilization or a concentration of amino acids aid in this (Schoffeniels and Gilles, 1970). More than
likely it is a combination of the three methods that enable the animal to maintain its body fluid composition when osmotically stressed. The water and ionic movements may take place through the gills (Bryan, 1960; 2
Bielawski, 1964 and 1971; Copeland, 1968; Bergmiler and Bielawski,
1970; Smith and Linton, 1971), gut (Croghan, 1958; Dall, 1967 and
1970), kidney (Riegel and Lockwood, 1961; Dehnel and Stone, 1964;
Kirschner, 1967; Rudy, 1967; Kamemoto and Ono, 1968; Pritchard and
Kerley, 1970), and possibly the integument, while the mobilization of amino acids usually occurs between the cells and extracellular fluids.
Thus, an os~regulator can maintain its original internal osmotic concentration through a variety of processes.
During the intermolt period of euryhaline crustaceans the environmental water moves into the animal at a rather fixed rate and contributes to the maintenance of the animal's steady state. This influx o~ water into the animal can vary under two normal conditions:
1) a salinity change in the environment can cause either an increase or decrease in water movement with the osmotic gradient which must be controlled to maintain its internal osmoconcentration and 2) the influx of water into the animal during ecdysis. Therefore, within the animal, a mechanism must be present to control water movement in the above two conditions and during the steady state.
Of the two conditions water influx into the animal during ecdysis has been the most thoroughly investigated. The mechanism that deals with ecdysis in crustaceans is a combination of both neural and endo crine changes acting in the X-organ-sinus gland complex, generally located in the eyestalk, and the Y-organ. Neurosecretory products, specifically the molt-inhibiting hormone, from the X-organ-sinus gland complex inhibits ecdysis while the Y-organ, by secreting the molting hormone or ecdysone, initiates the process. This integrated mechanism 3 has been carefully worked out and reviewed by Passano (1953), Carlisle and Knowles (1959), Echalier (1959), Passano and Jyssum (1963), Gabe
(1966), and Lockwood (1968). When ecdysis occurs, the animal takes in a much larger amount of water than when in the steady state; this causes it to expand, displacing the hydroderm (epidermis) and resulting in an increase in size.
Passano (1953) suggested that water balance in general, including water balance during the animal's steady state, may be controlled through the X-organ-sinus gland complex mainly by the molt-inhibiting hormone. However, Guyselman (1953), after observing a diurnal and seasonal rhythm in water content of crustaceans, and Carlisle (1955) suggested that although this complex probably contained and produced a neurosecretory hormone which controlled general water balance, it must be separate and distinct from the molt-inhibiting hormone. This would imply that there is a different mechanism which controls general water balance. From the investigations of Bliss et al. (1966) and
Bliss (1968) using pericardial sac size as an index of water influx, there appeared to be two hormones which affected water balance. They suggested that the hormones worked antagonistically during ecdysis and also during the crab's steady state. A third hormone, diuretic in action and probably liberated by the central nervous system, was also postulated.
Scudamore (1947) found that by removing eyestalks from crayfish the proecdysis water balance is upset with an increase of water content in the animal. Similar results were shown by Bliss et al. (1966) in which eyestalkless crabs increased their carapace size, yet with the 4
implantation of central nervous system tissue, these eyestalkless animals showed much less increase in size. They thus concluded that
there was a water release factor (diuretic) working during ecdysis which was produced in the central nervous system. Kamemoto et al.
(1966) found that eyestalk litigation caused crayfish to increase in weight and decrease blood osmoconcentration beyond that of normal crayfish. Also, eyestalkless Metopograpsus messor (Forskal), a
semiterrestrial grapsid crab, tended to conform their internal osmo concentration to that of the external medium. Thus, if an eyestalkless crab was put in a hypertonic medium, it increased its blood osmocon centration; the opposite happened when placed in a dilute medium--the blood osmotic concentration moved toward isotonicity. The normal crabs, being excellent osmoregulators, kept their blood osmoconcentra tion stable over a wide external salinity change. Injections of ventral ganglion homogenate or implantations of ventral ganglion tissue into normal crabs caused the internal osmoconcentration to change toward that of the medium. This was similar to the response of the eyestalkless crabs. Kato (1968) demonstrated significant weight increases in nephropore-plugged, eyestalk-ligated M. messor while Kato and Kamemoto (1969) found that injection of eyestalk extract into eye stalkless crabs in dilute media prevented the decrease in blood osmoconcentration as seen in the uninjected eyestalkless control animals. Kamemoto and Ono (1969) also found that the injection of brain homogenate into nephropore-plugged, eyestalkless crayfish pro duced a reducation of weight gain when compared to the weight gain in
the non-injected, eyestalkless controls. The investigators proposed 5 6
crayfish. Ramamurthi and Scheer (1967) showed that prawn cephalo
thorax contained a substance that decreased sodium efflux in shore
crabs and presumed this substance to be neurosecretory.
Neurosecretory material has been isolated from the spider crab pericardia1 organ (Cooke, 1966; Berlind and Cooke, 1970), and this
substance has been shown to increase the crab's heartbeat. Kleinholz
(1966 and 1970) and Kleinholz et ale (1967) observed and isolated
several neurosecretory hormones from crustacean eyestalks which were involved with pigmentation, carbohydrate metabolism, and ecdysis.
The role and the isolation of neurosecretory substances involved with pigmentation has also been investigated by Fingerman (1966), Bartell et ale (1971), and Hallahan and Powell (1971).
As stated above, the sites of water uptake could be the gills, gut, or integument. On the other hand, the increase in water content and weight or the decrease in osmoconcentration of the blood could ',. also be due to the retention of water by the animal. The retention of water could be caused by 1) a decrease in urine production (De
Leersnyder, 1967; Ono and Kamemoto, 1969) and/or 2) a decrease in the outward permeability of gills, gut, or integument. Whatever the method, either by increased influx or by retention, the central nervous
system appears to contain an active substance which alters water movements.
An investigation of the possible role of the central nervous
system in controlling water influx in decapod crustaceans seemed in order. In particular neurohormones or hormone-like substances from
the ventral ganglion and brain and their specific effects on water 7
influx was studied. The isolation and purification of these sub
stances as well as their biological influence on the animal was
discussed. The effects on water influx by other biological substances and, to some degree, sodium influx, was also investigated. Material and Methods
Central nervous system tissue, specifically the ventral ganglion
and brain, was obtained from the brackish water, euryhaline crab
Thalamita crenata Latreille which inhabits a variety of areas on the
southern coast of Oahu, Hawaii. The main collecting sites were Paiko
Lagoon, the Ala Wai Canal, and Fort Kamehameha Reef. The semi
terrestrial crab Metopograpsus messor (Forskal), used in the tritiated water saturation experiments, was collected at Paiko Lagoon. The
crabs, 1.:. crenata, were brought to the laboratory and held in running
sea water holding tanks where they were induced to autotomize their
chelae by the use of a small shock from a six-volt battery. Specimens
of both sexes in the intermolt stage weighing over 4 g were used.
Within a day or two the ventral ganglion and brain were removed from
the crabs and prepared for immediate experimental use or frozen,
lyophilized, and stored at _20oC until further processing or experi mentation. Some of the central nervous system tissue was stored as
long as six months without destroying the activity.
In the majority of experiments crayfish were used as the bioassay
animal. The fresh water crayfish, Procambarus clarkii (Girard), was
obtained from watercress farms on the southern coast of Oahu, Hawaii,
in the vicinity of Pearl City. They were sorted and held according
to weight and sex in running tap water and were fed weekly with liver
or crabmeat. Only males were used in bioassays. 9
Bioassay
The bioassay method was generally the same for both crayfish and crabs in the intermolt stage (Drach and Tchernigovtzeff, 1967) although the adaptation procedure differed prior to the experiment. Intermolt crayfish with matched weights (± 1 g) were transferred from running tap water to 0.1 M NaCl 48 hours before use in the bioassay. This was done in order to stabilize the internal ionic concentration of the animals. The crabs were transferred from running sea water (about
360 /00 salinity) to 50% sea water and held for two days. In some experiments the crabs were kept in 100% or 125% sea water for the two days.
The bioassay can be summarized as follows: at the end of the two day holding period, the animals were injected with ventral ganglion or brain extract and returned to the holding medium for two hours. They were then placed in an isotopic solution of an equivalent dilution for
30 minutes and 50 ~l of haemolymph were removed and counted for iso topic activity.
Injection of the central nervous system material into the bioassay crayfish was through the ventral side of the second abdominal segment while injections into the crabs were through the arthrodial membrane between the fourth and fifth walking legs. Prior to the injections, the central nervous system material was brought up to the standard
50 ~l/animal injection volume with deionized water (DIW) while 50 ~l of DIW was injected into each of the control animals. After injection of either the ventral ganglion, brain extract, or DIW, all animals were replaced in the adaptation medium for another two hours before 10 transfer to the isotopic medium. The medium containing the isotopes was exactly the same (salinity, temperature, pH, etc.) as the adapta tion medium except that it had been aerated for one hour prior to the bioassay.
Tritium labeled water and sodium-22 were the two isotopes commonly used. They were obtained in the form of tritiated water (T20) and sodium-22-chloride (New England Nuclear or Amersham), and the concen tration in the medium made so that it contained 1.0 mCi and 0.04 mCi per liter, respectively. The experimental and control animals were kept in separate 20 em diameter finger bowls during the bioassay.
Four to seven animals were placed in one bowl, each containing about
700 ml of isotopic medium.
The assay animals were left in the labeled solution for exactly
30 minutes. They were removed, rinsed in non-isotopic solution, and damp-drie~. A SO pI haemolymph sample was removed via the arthrodial membrane of the fourth walking leg. Crab blood was drawn from the opposite side of the injection site. Each SO pI sample was mixed directly into a high quality polyethylene scintillation vial containing a toluene:2,S-diphenyloxazole (PPO):1,4-di(2-(S-phenyloxazolyl)) benzene (POPOP) scintillation solvent (100:4:0.S::v:w:w) in which a solubilizer (Beckman BioSolv BBS-3) was added to the solvent in a
1:10 (v:v) ratio. Prior to the addition of the sample, 0.1 ml of lS% ascorbic acid was added to each vial. This enhanced the oxidation process of the haemolymph by the solubilizer. The resulting sample solvent mixture was clear, with no coagulation, precipitation, or coloring occurring, and there was a minimum of quenching. The samples 11 were stored in darkness for two hours and then counted at ambient temperature in a Beckman LS-lOO Liquid Scintillation System.
In some of the preliminary experiments the 50 pI samples of haemolymph were treated by a freeze-drying method (Rudy, 1967) in order to remove the tritiated water from the rest of the blood components; this water was collected from a freezing trap and added directly to scintillation vials containing a dioxane-base solvent (Bray, 1960) and counted. This method offered no advantages over the above method and its use was discontinued.
Significant differences between control and experimental groups were determined by the Student's T Test. The sample standard devia tion was also calculated.
Tissue Preparation
Two major methods were used for preparing ventral ganglion and brain tissue extract. These allowed for a minimally treated extract or a fully processed preparation that could eventually be utilized in column chromatography.
The minimally treated extract was prepared by simply removing the ventral ganglion or brain tissue and homogenizing it in a 2 ml
Tenbroeck glass tissue grinder using a minimum amount of DIW. The homogenate was heated in a boiling water bath for 10 minutes, centri fuged at 12,800 x g, and the supernatant injected in 50 pI amounts.
Two or three ventral ganglion or brain equivalents were injected per animal. 12
In order to process this tissue for column chromatography 40 to
60 ventral ganglia or brains were used. This tissue was quickly removed from the crab, frozen, lyophilized, and stored at _20oC. In the preliminary experiments the frozen tissue was acetone-dried for
24 hours with at least four changes of acetone. This acetone-dried material was then glass homogenized in 0.1 N HCl, heated in a boiling water bath for 10 minutes, and centrifuged for 10 minutes at 2,000 x g.
The supernatant was collected and the residue washed and centrifuged once more. The pooled supernatant was neutralized to pH 7.2 with
0.2 N NaOH and centrifuged at 12,800 x g for 30 minutes, the residue being discarded and the supernatant frozen, lyophilized, and stored at
_20oC. However, because a buffered extract did not appear to be necessary to maintain the activity found in the central nervous system tissue, this procedure was replaced with the following one (Figure 1): tissue that was to be used with column chromatography was glass homoge nized in acetone and centrifuged for IS minutes at 1,200 x g and the supernatant collected; the residue was acetone-washed and centrifuged three additional times, dried in a vacuum or by a nitrogen stream, and stored. The pooled acetone supernatant was then put under a dry nitrogen stream (less than 1 psi) until the acetone had completely evaporated thus leaving a residue. This residue was then solubilized in chloroform-methanol (1:2) and centrifuged at 40,000 x g at -SoC for two hours, the supernatant being discarded, and the residue dried with a nitrogen stream and stored. This residue was referred to as the acetone soluble fraction. eNS Tissue IFreeze, lyophilize Tissue + Acetone Homogenize with acetone; centrifuge 1,200 x 9; 3 washes
Residue Acetone soluble Re-homogenize in O/Wi bOl'lj centrifuge /3,000 x g; ~N2 stream combine 3 washes
Residue Supernatant Residue (discord) Chloroform: metOH(/:?) Lyophilize centrifuge 40,000 x g
Sephadex G- 50
I Residue Supernatant Sephadex G- 25 Wash wl'lh OIWj (discord) I lyophilize Sephadex G-15 I Sephodex G-IO I eM Sephodex
Figure 1. Flow diagram of the tissue preparation procedure t-' for Thalamita crenata central nervous tissue. \..oJ 14
The dried residue from the acetone-wash was glass homogenized in
DIW, boiled for 10 minutes, sonicated for 10 seconds, and centrifuged
at 13,000 x g for 30 minutes. This supernatant and the supernatant
from three additional washes were collected and pooled, the residue
.~~ing discarded after the last wash. The pooled supernatant containing
a water soluble active fraction was frozen, lyophilized, and stored at
_20 0 C.
Separation and Purification
Sephadex G gels (Pharmacia Fine Chemicals) were employed for the
fractionation and separation of the water soluble active substance.
The main gels utilized were G-50 (fine), G-25 (fine), G-15, and G-10.
A variety of columns were used but in general, the respective column
sizes for the above gels were 1.5 x 30, 1.5 x 30, 0.9 x 30, and
1.1 x 62 (ID cm x L cm). All but the last were Pharmacia K type
columns, the last being laboratory manufactured. Except for tris-HCl
buffer of pH 8.0 used in a few preliminary experiments, the eluant
for all columns using Sephadex G gels was DIW, and the sample volume
applied to the column was 0.5 ml. The flow rate varied according to
the gel, length of column, and sample; all columns were of the
descending type and gravity fed (Figure 2). The eluant passed through
an ultraviolet (280 mu) spectrophotometer (LKB 8300A Uvicord II), and
the resulting chromatogram was recorded ona chopper-bar recorder
(LKB 6520) and plotted as percentage transmittance (%T). Fifty drop
(2.5 ml) samples were collected on an automatic fraction collector
(LKB 7000 UltroRac). The columns, UV spectrophotometer, and fraction 15
D.C. r-"" Recorder II ,-- /B~fW\ .-.....--...
Seporation \ Column I. -.... ~
Control U.V. "- Unit Spectrophotometer
Stream Analyzer
l Drop Counter I ...... I I I) '-::===:::::..
Fraction Collector "',~\~, ~ ~ "0'\:'\.. "',."-.... ,, ~ ....,.
Figure 2. Diagram of the separation procedure. 16 collector were kept at a constant temperature of 5.0oC within a Uni
Therm (Puffer Hubbard) double-door refrigerator. The fractions were collected, pooled, frozen, lyophilized, and stored at -ZOoC. Occa sionally, ultrafiltration was used to separate the large macromolecules from the supernatant; a magnetically stirred ultrafiltration cell with
Diaf10 PM-lO (Amicon) membrane filters under 40 psi of nitrogen appeared to be satisfactory for this. However, the majority of the separations was done with Sephadex G gels.
Ion exchange chromatography, utilizing CM Sephadex A-Z5 cellulose
(Pharmacia Fine Chemicals). and two different buffer systems, was employed to further resolve the active material after maximum resolu tion of Sephadex G-IO. A gradient mixture apparatus was made to produce continuous ion concentration gradients, thus all fractions from the ion exchange columns were eluted relative to a particular ion concentration. This same apparatus was also used to produce a con tinuous pH gradient. CM Sephadex with a 0.01 M KHZP04 starting buffer of pH 6.6 eluted through a 0.9 x 30 cm column with the upper ion concentration 0.1 or 0.5 M KHZP04 appeared to resolve the active material from other substances present from the Sephadex G-IO elution.
The other buffer system used with CM Sephadex was 0.01 to 0.1 M
NH HC0 of pH 7.4. This buffer had the advantage of eliminating the 4 3 desalting procedure necessary when using the potassium buffer.
Desalting was usually carried out by eluting the collected fraction with DIW through Sephadex G-10 or through an uncharged DEAE Sephadex column, thus trapping the salts and allowing the weakly positive charged active material to pass freely through the column. The 17 isoelectric point was roughly calculated by eluting the activity containing fraction through CM Sephadex with a pH gradient of 5.9 to
8.0 in a KH P0 buffer of 0.1 M ion concentration. 2 4 Thin layer electrophoresis was used to check the homogeneity and purity of the active fraction. ITLC-SA gels (Gelman) containing silicic acid impregnated in glass microfibers produced adequate results when used with a tris-barbital buffer of pH 8.8 and 0.05 molar concen tration. The biologically active material was spotted on damp-dry gels in about 5 ~l amounts (containing less than lOb pg of active material) and electrophoresed for 25 minutes at 250 volts at less than
4 amperes per electrophoretic strip. The electrophoretograms were air dried overnight and sprayed with ninhydrin. After marking the ninhydrin-positive spots, the electrophoretograms were chlorinated in a chlorine gas chamber and treated according to the halogenation method of Stahl (1969) in order to determine any additional peptide areas.
These purity tests were carefully compared to the UV chromatograms to ascertain the purity.
Properties of the Active Material
To determine the protein nature of the water soluble active material eluted through Sephadex G and CM Sephadex, enzyme hydrolysis of the active fraction was attempted. Pancreatin (Merck) was used as the proteolytic enzyme because of its multienzyme composition. The activity of the enzyme mixture was verified by observing its action on soluble egg albumin and determining substrate breakdown by two dimensional thin layer chromatography (lTLC-SA, Gelman); the enzymes 18 were highly active. The active fraction from the ventral ganglion eluted off of Sephadex G-lO was added to a 5% enzyme-KH P0 buffer Z 4 solution adjusted to pH 7.6 with NaOH and diluted 1:4 with DIW. This mixture was divided into two aliquots, one of which was heated in a boiling water bath for 10 minutes to denature the enzymes. Both aliquots were then incubated and gently agitated in a 380 C water bath for eight hours. They were then boiled, centrifuged at lZ,800 x g for 30 minutes, and the supernatant passed through a PM-lO Diaflo membrane. The filtrate was collected, frozen, lyophilized, and bio assayed for activity. A small portion (10 pI) of the bioassay material of both the control and experimental aliquots was subjected to IILe chromatography.
The molecular weight was estimated by Diaflo membranes as well as by the elution volume from Sephadex G gels compared to elution volumes of compounds of known molecular weights. Glutathione (MW 147),
L-1eucyl-L-tyrosine (MW 295), vitamin BIZ (MW 1,357), and bacitracin
(MW 1,411) were eluted through Sephadex G-10 and the elution volumes plotted on a curve as a function of molecular weight. The active material's elution volume was added to the curve, and its molecular weight was estimated from its placement on the curve.
Other Activity-Containing Substances
Experiments were carried out to observe what effects, if any, other substances may have on water or sodium influx. The neuro transmitter substances found in crustacean central nervous system tissue or those that are involved in water balance in other animals 19 were used. Since norepinephrine;' 5-hydroxytryptamine, acetylcholine, and glutamic acid are common neurotransmittors in crustaceans, 50 or
500 ~g (Nutritional Biochemicals Corp.) were brought up to injection volume with DIW or dilute salt solution and assayed for influx activity.
Similar experiments were done using oxytocin (Pitocin) and vasopressin
(Pitressin, both from Parke, Davis and Co.), and these octapeptides were injected in 50 ~U amounts. The effect of ecdysterone, a molt inducing hormone found in arthropods, was also observed. Natural ecdysterone (Schwarz/Mann) was brought up to injection volume by pure mineral oil or DIW and injected into crayfish at concentrations of
300 J,lg/animal.
The water or sodium influx activity that may be contained in the crab pericardial organ, which is neurosecretory in nature, was inves tigated. The pericardial organs were removed from ~ crenata, frozen, and lyophilized. After glass homogenation in DIW, the extract was injected into crayfish and the tritiated water and sodium-22 influx determined by the normal bioassay.
Effector Tissues
Because in the normal bioassay animals were submerged in the isotopic medium, it was difficult to determine the specific effector tissues involved in water and ion fluxes. As mentioned earlier, water and ion movements may take place through the gills and gut, hence, experiments were designed to determine the influx of tritiated water through these tissues. 20
The gill chambers of 50% sea water adapted crabs, ~ crenata, were perfused at the rate of 100 ml/hour with the same isotopic medium used in the normal bioassay. The isotopic medium entered the posterior region of the gill chamber and was circulated anteriorly over the gills. None of the isotopic medium entered the mouth which was plugged with p. v. tubing. The gill chambers are reasonably water-tight com partments, thus only the gills and walls of the gill chamber were subjected to the medium. The active material was"injected (five ventral ganglion equivalents/animal) into the perfused crab, and a blood sample was taken after 30 minutes and analyzed for activity.
Isolated crayfish intestines were set up by removing the intes tine and attaching one end of it to a peristaltic pump through which
Ringer or one-third Ringer solution could be perfused; the efferent end was attached to a waste collector. The isolated intestine was then immersed in an aerated bathing chamber filled with Ringer or one third Ringer solution. Tritiated water flux from the lumen to haemolymph or from the haemolymphto lumen was observed after the addition of the active material to the bathing medium (haemolymphside).
The concentration gradient was created by diluting van Harreveld's crayfish Ringer solution by two-thirds with tritiated water (1 ~Ci/ml) against a full-strength Ringer solution. Depending on which side of the intestine the dilute Ringer's was on, the direction of the gra dient could be into or out of the lumen. The effect of the water soluble (from Sephadex G-lO) and ace~one soluble fractions were inves tigated relative to the increase or decrease of tritiated water flux as a function of time. These fractions were added to the bathing 21 medium 50 minutes after the beginning of perfusion, and the perfusion rate was 20 ml/hour. Ten microliter samples were collected from the bathing medium or the perfused effluent and counted for activity. Results
In the past one method of detennining the relative influx of
water between control animals and eyestalk ligated animals utilized
crabs and crayfish with plugged nephropores (Kato, 1968; Kamemoto and
Ono, 1969); these animals could not eliminate urine, thus gaining in
weight, and the weight difference was used as a measure of the relative
difference of water influx. It was thought that if the animals could
be assayed in a more natural condition, that is, with their nonnal
excretory passage open, the results would be more meaningful since the
animals would be under less stress, and turgor pressure would be
eliminated.
Therefore, the tritiated water-sodium-22 isotope method was
developed. The length of time necessary to leave the assay animals in
the isotopic medium was dependent on the length of time it took before
the animal became saturated with the isotope. To detennine the optimum
length of time in which the greatest influx of tritiated water occurred
as a function of time, a saturation curve was obtained using the crab
Metopograpsus messor (Forskal) that had been adapted to 5~1o sea water
for two days (Figure 3). The animals were about 4~1o tritiated water
saturated within the first 30 minutes. From similar experiments with
crayfish in 0.1 M NaCl and Thalamita crenata Latreille in 50% sea water,
it was found that complete saturation occurred within six hours, which
was similar to ~ messor, and that optimum influx of tritiated water
took place within the first 30 minutes. Hence, 30 minutes was used as
the bioassay time for these animals. That tritiated water, as a water 23
100%
..:z: o
O%'---'-~__,- --r "'" 0.5 1.0 3.0 6.0 Time (hrsl
Figure 3. Tritiated water saturation tUnes due to its influx into normal Metopograpsus messor adapted to 50% sea water. influx indicator, may behave differently from normal water when passing through biological membranes because of its nuclear properties was not of concern. Takashina et a1. (1962) demonstrated with dog pericardium that transfer differences between water, deuterium, and tritiated water were insignificant.
The preliminary experUnents with central nervous system tissue were designed to show any change in water influx that may be elicited by unknown factors in the ventral ganglion or brain of ~ crenata. The crabs, ~ crenata, were placed in 50% sea water for two days prior to the bioassay in order to stabilize the animals and produce an osmotic gradient needed for the bioassay. The fresh central nervous system 24 tissue was minimally treated, but not boiled, and injected into the experimental animals (Table 1).
Table 1. Influx of tritiated water and sodium-22 into Tha1amita crenata injected with DIW (controls) and minimally treated ventral ganglion (VG) or brain (Br) extract. Two VG or Br equivalents! animal. The crabs were adapted to 50% sea water for two days.
Experiment Treatment n cpm 3H
1 Control 3 17,053 ± 735 Br 3 4,283 ± 830** 2 Control 3 2,605 ± 270 VG 3 1,955 ± 170**
3 Control 3 10,837 ± 615 637 ± 248 Br 4 8,213 ± 370** 502 + 208 4 Control 4 16,565 ± 1,785 383 + 253 VG 4 6,868 ± 3,485** 195 ±41
** p is less than 0.01
This fresh, minimally treated extract from the ventral ganglion and brain consistently decreased the influx of tritiated water into
~ crenata. Although the brain is about one-third the size of the ventral ganglion, it is difficult to conclude from these data which tissue contained the highest water influx decreasing activity. It may appear that the brain contained more activity per unit weight.
After heating the minimally treated extract to 100oC, the results obtained again showed a significant decrease in water influx (Table 2).
Therefore, heating this material to 1000 C appeared to have no effect on the active factor responsible for the decrease in tritiated water influx; this material was heat-stable. 25
Table 2. Influx of tritiated water and sodium-22 into Thalamita crenata injected with DIW (controls) and boiled, minimal treated ventral ganglion (VG) extract. Two ventral ganglion equivalents injected per animal. The crabs were adapted to 50% sea water for two days.
3 Experiment Treatment n cpm H cpm 2~a
1 Control 5 4,113 ± 248 225 + 55 VG 5 2,291 ± 735** 178 ±47 2 Control 4 13,243 ± 641 811 + 161 VG 4 9,769 ± 1,672** 670 ±218 3 Control 5 14,099 ± 1,133 509 + 158 VG 5 9,952 ± 1,157** 521 ±91
**p is less than 0.01
Since this active material could, in fact, have been a substance
common to all tissues, a control experiment was done to determine if
the water influx activity was contained solely in the central nervous
system tissue. Boiled muscle tissue extract from the walking legs of
~ crenata, prepared like minimally treated ventral ganglion or brain,
was injected into the crabs (Table 3). Since the water influx rate of
the experimental animals did not differ significantly from that of the
controls, it was concluded that the substance eliciting a decrease in
water influx into the animals was present in and discrete to the central
nervous system tissue.
Fresh water crayfish were examined for their suitability as the
bioassay animal because of the apparent ionic and influx variation
within ~ crenata, the difficulty in determin~ng true intermolt indi-
vidua1s, and the scarcity of these crabs. Crab ventral ganglion and
brain tissue was minimally treated, heated to 100oC, and assayed in the
~~~~------=-======--~""""""LL14J2;1;Y~~I':l'!"'!~~ 26
Table 3. Tritiated water and sodium-22 influx into 50% sea water adapted Thalamita crenata after injection of boiled muscle extract. DIW injected into the controls.
3 22 Experiment Treatment n cpm H cpm Na
1 Control 5 13,093 + 1,478 408 + 56 Muscle 5 13,305 +2',289 370 ±71 2 Control 5 14,442 ± 468 377 ± 51 Muscle 5 14,587 ± 453 361 ± 53
crayfish (Table 4). The factor contained in the central nervous system tissue when bioassayed on ~ crenata appeared to be active in the cray- fish since there was a significant decrease of water influx into the animals. These data were the basis for using crayfish as the major bioassay animal in future experiments. Periodic assays on T. crenata were made to verify the activity in the crabs.
Table 4. Influx of tritiated water and sodium-22 into the crayfish Procambarus clarkii adapted to 0.1 M NaCl for two days. Two ventral ganglion (VG) or brain (Br) equivalents injected per animal.
3 Experiment Treatment n cpm H cpm 2~a
1 Control 5 3,469 ± 215 33 ± 6 VG 5 2,746 ± 247** 35 + 7 Br 5 2,603 ± 109** 33 ±3 2 Control 5 4,042 ± 199 36 + 4 VG 6 3,132 ± 153** 33:+ 4
** P is less than 0.01
An experiment was conducted assaying the effect of Cancer magister
Dana brain extract on crayfish. The mean tritiated water influx cpm 27
of 3,015 (± 177) was recorded for the control while that of the injected crayfish was 801 (± 221); four animals were used in each group. These
data were similar to those obtained from ~ crenata with regard to the
significant decrease in the influx of tritiated water but not of
sodium-22.
In order to detennine if the salinity of the external environment
had any effect on the availability of this water influx decreasing
factor, brain extract from 50% and 100% sea water adapted ~ crenata was compared for activity (Table 5). No difference in tritiated water
influx was caused by the extracts from these two groups of animals.
Table 5. Relative influx of tritiate~ water and sodium-22 into 0.1 M NaCl adapted crayfish injected with brain extract from Thalamita crenata adapted to 50% and 100% sea water. n= 5.
3 Treatment cpm. H cpm. 2~a
Control 3,604 ± 244 34 ± 5
Brain from 50% SW 1,151 ± 157** 32 + 2 Brain from 100% SW 1,234 ± 286** 32 + 4
** P is less than 0.01
In preparing the ventral ganglion and brain tissue for column
chromatography the material was homogenized in acetone, and water
soluble and acetone soluble portions were collected. The water soluble
fraction was assayed for activity and the results show that the crayfish
injected with the water soluble extract, prepared for column chroma-
tography, did indeed decrease the water influx (Table 6). Therefore,
the water soluble portion of the central nervous system tissue 28
Table 6. Influx of tritiated water and sodium-22 into crayfish adapted to 0.1 M NaCl and injected with Thalamita crenata ventral ganglion and brain water-soluble fractions after acetone homogenation.
3 22 Treatment n cpm H cpm Na
Control 5 4,136 ± 119 47 + 10 VG 5 3,123 ± 515** 50 ±11 Control 5 4,399 ± 452 47 + 9 Br 5 3,593 ± 119* 49 ±11
* P is less than 0.05 ** p is less than 0.01 preparation was eluted through Sephadex G-50 (Figure 4).
~ I+-f----''---+lI....!!-.I+-ji-.::.:UI_-tl,o-----:.IV"'"""-1r----+1 E o CD N
I •:>t
5 \0 15 20 25 Fraction Number
Figure 4. Chromatogram showing the separation of Thalamita crenata ventral ganglion extract on Sephadex G-50. Eluant: DIW; Column: 1.5 x 30 em; Sample: 50 drops at 12 ml/hr. 29
Fractions I, II, III, and IV were collected separately from the appropriate sample tubes, frozen, lyophilized, and bioassayed for water and sodium influx using crayfish (Table 7). These results were repre- sentative of several similar bioassays made with ventral ganglion fractions eluted through Sephadex G-50. These data indicated that only Fraction III contained activity which decreased the influx of water into the crayfish. The other fractions do not differ signifi- cantly from the control, nor was there any apparent effect on sodium-22 influx by any of the four fractions.
Table 7. Relative tritiated water and sodium-22 influx activity from Thalamita crenata ventral ganglion fractions eluted on Sephadex G-50 and assayed in 0.1 M NaCl adapted crayfish. n= 5.
3 Treatment cpm H cpm 2~a
Control 3,212 ± 116 44 ± 8 Fractions I & II 3,343 ± 386 53 + 10 Fraction III 2,678 + 175** 47 + 11 Fraction IV 3,281 +201 46 ± 2
** P is less than 0.01
The water soluble fraction of the brain, when eluted on Sephadex
G-50, produced a chromatogram similar to the ventral ganglion except that the third peak was consistently smaller (Figure 5). By bioassay it was determined that water influx was decreased in animals injected with pooled Fractions III and IV (Table 8).
Subsequent experiments showed that the activity was specifically in Fraction III. The active material from the brain, contained in 30
~ o CD C\I.. I- *
5 10 15 20 Fraction Number
Figure 5. Chromatogram showing the separation of Tha1amita crenata brain extract on Sephadex G-50. Eluant: DIW; Column: 1.5 x 30 em; Sample: 50 drops at 12 m1/hr.
Table 8. Relative tritiated water and sodium-22 influx activity from Thalamita crenata brain fractions eluted on Sephadex G-50 and assayed in 0.1 M NaC1 adapted crayfish. n= 5.
3 Treatment cpm H cpm 2~a
Control 3,774 ± 287 45 ± 8 Fractions I & II 3,594 + 146 37 + 7 Fractions III & IV 1,217 ±743** 31 +7* Fraction V 3,682 ± 211 41 ±3
* P is less than 0.05 ** P is less than 0.01 31
Fraction III, was eluted into the same sample tube number as was the active material from the ventral ganglion (Figure 4). The chromato- graphic elution pattern of the peaks containing the active material from the ventral ganglion and brain extracts was the same for elutions using the same gel, column, and flow rate. Thus it appeared that the active material from these tissues was very similar.
In order to further resolve and separate the active material from other compounds closely eluted with it, these activity-containing fractions were chromatographed on Sephadex G-75 (Figure 6). The eluted fractions, namely the fractions corresponding to the large peaks, were bioassayed for activity (Table 9).
'0 I~ 10 Fraction Number Fl"Qction Nymber
Figure 6. Chromatograms showing the separation of the activity containing fractions (III from Sephadex G-50) of Tha1amitacrenata brain (left) and ventral ganglion (right) on Sephadex G-75. Eluant: DIW; Column: 0.9 x 60 cm; Sample: 50 drops at 9 m1/hr. 32
Table 9. Relative activities of the ventral ganglion and brain fractions of Thalamita crenata after the active fractions from G-50 (fraction III) have been eluted on Sephadex G-75. n= 5.
3 Treatment cpm H cpm 2~a
Control 3,446 ± 449 36 + 5
VG 569 + 267** 32 + 8 Br 1,007 ±188** 28 + 2
** P is less than 0.01
It was apparent that the resolution and separation of the frac- tions containing the active material from Fraction III off of Sephadex
G-50 (Figures 4 and 5) was not enhanced by eluting them through
Sephadex G-75. Therefore, these activity-containing fractions,
Fraction III from Sephadex G-50, were eluted through a less porous gel, Sephadex G-25 (Figure 7). The elution through this gel afforded better resolution of closely eluted substances. The large peak repre- sented by Fraction II (Figure 7) appeared to correspond with the large peaks eluted through Sephadex G-50 (Fraction III, Figures 4 and 5).
Since this activity-containing fraction was better resolved, ventral ganglion extract was eluted through Sephadex G-25 as the primary separation step, eliminating Sephadex G-50 from the purification process (Figure 8). Fractions I, II, III, IV, and V eluted from this gel were assayed for activity, and, as with the fractions eluted from
Sephadex G-50, the activity was found in the second peak or Fraction
II (Table 10). 33
/+-(-----'-----... +---,1--...::---- ++-----:.1:.:..11---7
I 5 10 Sample Number
Figure 7. Chromatogram showing additional resolution in the separation of Tha1amita crenata ventral ganglion Fraction III from Sephadex G-50 eluted on Sephadex G-25. Eluant: nIW; Column: 0.9 x 60 cm; Sample: 50 drops at 3 m1/hr.
I IV ,I, V I' 'II E" 0 5 10 15 20 Sample Number Figure 8. Chromatogram showing the separation of Tha1amita crenata ventral ganglion extract on Sephadex G-25. Eluant: nIW; Column: 1.5 x 30 cm; Sample: 50 drops at 2 ml/hr. 34 Table 10. Relative tritiated water and sodium-22 influx of Thalamita crenata ventral ganglion fractions after separation on Sephadex G-25 and bioassayed on 0.1 M NaC1 adapted crayfish., n = 5. 3 22 Treatment cpm H cpm Na Control 2,623 ± 176 35 + 4 Fraction I 2,784 + 243 38 + 2 Fraction II 1,709 =+ 281** 33 =+ 5 Fraction III 2,932 =+ 322 40 =+ 6 Fractions IV & V 2,775 ±260 38 =+ 5 ** P is less than 0.01 The similarity between the chromatograms of the separation of ventral ganglion on Sephadex G-50 (Figure 4) and on Sephadex G-25 (Figure 8) was not only apparent in the elution patterns but in the assay results in which the activity was present in the second peak (Tables 7 and 10). The elution pattern from brain extract separated on Sephadex G-25 (Figure 9) also showed similarity with the ventral ganglion separation on the same gel; the activity was again found in fractions collected from the second peak. The brain and ventral ganglion fractions that contained activity after being eluted through Sephadex G-25 were again chromatographed on Sephadex G-15 (Figure 10); additional resolution occurred. At this purification step there was no difference between the elution profiles of ventral ganglion and brain activity-containing fractions. Fraction II, the large peak from Sephadex G-15, was rechromato- graphed on Sephadex G-10 (Figure 11), and the fraction represented by 35 ::l E 1 I ....--ji---+--'-----...... --1-----:;,..1 ---~III------tl ..1.!..V__ o ++-+---1\ (-( I(-(__ -7l! '"N.- I- <;e 5 10 15 Sample Number Figure 9. Chromatogram showing the separation of Thalamita crenata brain extract through Sephadex G-25. Eluant: DIW; Column: 1.5 x 30 cm; Sample: 50 drops at 2 ml/hr. III ) If In If ~ 0 CD C\I I- "e0 3 5 7 Sample Number Figure 10. Chromatogram showing addition resolution of Thalamita crenata ventral ganglion Fraction II from Sephadex G-25 when eluted on Sephadex G-15. Eluant: DIW; Column: 0.9 x 30 cm; Sample: 50,drops at 4 ml/hr. 36 :::l E 0 (l) C\I II I- ~ ~ 5 10 15 Sample Number Figure 11. Chromatogram showing resolution of Thalamita crenata ventral ganglion Fraction .11 from Sephadex G-15 when eluted on Sephadex G-lO. Eluant: DIY; Column: 1.1 x 62 cm; Sample: 50 drops at 10 ml/hr. the large peak, Fraction II, was bioassayed for tritiated water and sodium-22 influx activity. The data indicate that Fraction II con- tained activity, and that this activity had been maintained through at least three chromatographic elutions (Table 11). To further resolve the activity-containing fraction that was eluted from Sephadex G-10 (Fraction II, Figure 11) from other sub- stances eluted close to it, ion exchange chromatography with CM Sephadex and a continuous ionic strength gradient was used (Figure 12). Two different buffer systems were used (potassium phosphate and ammo- nium bicarbonate), and, from these, two similar chromatograms were 37 Table 11. Relative influx of tritiated water and sodium-22 into 0.1 M NaCl adapted crayfish after injection of Thalamita crenata ventral ganglion Fraction II after separation on Sephadex G-lO. n= 6. 3 Treatment cpm H cpm 2~a Control 3,429 ± 257 37 ± 3 Fraction II 2,641 ± 141** 42 ± 5 ** P is less than 0.01 0.3 ::J 0.2 .&: E '60 c 0 CD :! C\I.- 0.001 5 10 15 20 20 Fraction Number Figure 12. Chromatogram showing separation of Thalamita crenata brain Fraction II from Sephadex G-lO when eluted on CM Sephadex. Eluant: 0.01 to 0.5 M KH2P04' pH 6.6; Column: 0.9 x 30 em; Sample: 50 drops at 8 ml/hr. obtained. Both buffer systems fully resolved the active material from other substances, something that Sephadex G-lO could not accomplish. The resulting bioassay demonstrated that the activity was present in 38 the large peak (Table 12). Table 12. Relative influx of tritiated water and sodium-22 into 0.1 M NaCl adapted crayfish after injection of Thalamita crenata ventral ganglion (VG) and brain (Br) fractions eluted on CM Sephadex ion exchange column. n= 5. 3 Treatment cpm H cpm 2~a Control 3,1l5 ± 284 34 ± 7 VG 2,364 ± 163** 36 ± 5 Br 2,645 ± 171** 38 ± 7 ** P is less than 0.01 Confirmation that the factor from ~ crenata central nervous sys- tern was, in fact, active in the crab was obtained from bioassaying the activity-containing fraction from CM Sephadex elution in the crab itself (Table 13). It was observed that the fraction was active in the crab and to a greater degree than it was in the crayfish. Table 13. Relative influx of tritiated water and sodium-22 into 50% sea water adapted Thalamita crenata after injection of T. crenata ventral ganglion (VG) fraction eluted on CM Sephadex. n= 5. 3 Treatment cpm H cpm 2~a Control 10,763 ± 248 571 ± 108 VG 3,259 ± 1222** 467 ± 208 ** P is less than 0.01 A purity check of this fraction using thin layer electrophoresis produced only one ninhydrin-positive spot on the electrophoretogram, 39 and the halogenation reaction took place only at this spot. This was suggestive that the eluted activity-containing fraction from ion exchange chromatography was reasonably pure and corroborated the single peak on the chromatogram (Figure 12). A problem was encountered desalting the active fraction eluted from the ~ Sephadex ion exchange column. Since a control desalting elution was successful separating the active fraction from NaCl on Sephadex G-10, this gel was used to desalt the fractions eluted with KHzP04 buffer. However, this salt was eluted very close to the active frac tion, and complete resolution was impossible on the first elution. Hence, several DIW elutions were needed when using Sephadex G-10 to desalt the ion exchange fractions. Nor could ultrafiltration through UM-05 membranes be used successfully, for although the active substance did not pass through the membrane with the filtrate containing the salts, it could not be recovered from the membrane. This difficulty was inherent in the membrane's binding properties with this particular unknown substance. Desalting was also accomplished by eluting the active fraction through an uncharged ion exchange gel of the same ionic charge as the active substance. Since thin layer electrophoresis indicated that this material was weakly positive, DEAE Sephadex was used as the gel. The active material was rejected by the gel while the salts were held in the gel when eluted with DIW. It was found, however, that a column could be used only once when desalting by this method. There was no noticeable effect of enzyme hydrolysis on the active material. The influx of tritiated water and sodium-22 was similar 40 after injection of the denatured and "live" enzyme aliquots; both decreased the influx of tritiated water into crayfish but had no effect on sodium movement. E1ectrophoretograms of these a1iquots indicated that no hydrolysis occurred, for the ninhydrin-positive spots were the same in number and Rf'S. Estimation of the active material's molecular weight from its elution volume as compared to the elution volumes of compounds of known molecular weight is shown in Figure 13. The molecular weight of this substance appears to be between 600 and 700. sol A Ve Imll DE 20l...;-;::------::"!::::---:r=:::--=:::------100 500 700 1000 Moleculor Weigh! Figure 13. Molecular weight estimation of the water soluble active material (X) when eluted with compounds of known molecular weight (A-E) on Sephadex G-10. A, NaCl (MW 58); B, glutathione (MW 147); C, L-1eucy1-L-tyrosine (MW 295); D, vitamine B12 (MW 1,357); E, bacitracin (MW 1,411). 41 During the preparation of the central nervous system for column chromatography, an acetone soluble fraction was obtained from acetone homogenation, the preliminary step in the tissue processing procedure. The acetone soluble fraction was subjected to bioassay to determine if it contained water influx activity (Table 14). It was apparent that this fraction contained an active factor opposite of that elicited by the water soluble fraction. Table 14. Relative influx of tritiated water and sodium-22 into 0.1 M NaCl adapted crayfish after injection of the acetone soluble fraction of Thalamita crenata ventral ganglion and brain. n= 7. 3 Treatment cpm H cpm 2~a Control 3,488 + 313 46 ± 5 Ventral ganglion 4,153 +415** 39 ± 13* Control 2,655 + 219 32 + 3 Brain 3,234 ±101** 38 ± 4 * P is less than 0.05 ** P is less than 0.01 Bioassays designed to detect changes in tritiated water or sodium- 22 influx into crayfish produced by substances other than those from the ventral ganglion or brain were carried out using vertebrate octapeptides, crustacean neurotransnittors, pericardial organ extract, and ecdysterone. Oxytocin and vasopressin, vertebrate octapeptide hormones from the neurohypophysis, were ineffective in changing water or sodium influx into crayfish (Table 15). Similarly, the common crustacean neurotransnitter substances 5- hydroxytryptamine, acetylcholine, norepinephrine, and glutamic acid 42 Table 15. Influx of tritiated water and sodium-22 into 0.1 M NaCl adapted crayfish after injection of vasopressin or oxytocin in 50 ~U/animal amounts. n= 5. 3 Treatment cpm H cpm Z;a Control 3,993 ± 371 41 ± 7 Vasopressin 3,787 ± 440 35 ± 6 Oxytocin 3,786 ± 327 34 ± 3 did not appear to effect changes in water influx. However, there were consistent results showing significant increases in sodium-22 influx when 5-hydroxytryptamine was injected (Table 16). Table 16. Influx of tritiated water and sodium-22 into 0.1 M NaCl adapted crayfish after injection of 5-hydroxytryptamine, acetylcholine, norepi nephrine, or glutamic acid in 500 ~g/animal amounts. n= 5. 3 Treatment cpm H cpm 2~a Control 2,247 ± 100 37 ± 4 5-hydroxytryptamine 2,695 + 584 52 ± 3** Acetylcholine 2,372 ±469 39 +11 Norepinephrine 2,368 ± 410 41 +14 Glutamic acid 2,541 ± 351 33 ±5 ** P is less than 0.01 The crab pericardial organ extract also appeared to cause an increase in sodium-22 influx; the water movement was not affected (Table 17). Three experiments conducted to test the effect of ecdysterone on water and sodium influx suggested that this substance might be involved 43 Tabl~_17. Relative influx of tritiated water and sodium-22 into 0.1 M NaCI adapted crayfish injected with Thalamita crenata pericardiaI organ extract in amounts not exceeding 2 PO equivalents/animal. n=6. 3 Treatment cpm H cpm 2~a Control 2,429 ± 216 28 ± 2 PO extract 2,439 ± 289 56 ± 10** ** P is less than 0.01 in water fluxes. Although only one experUnent showed significant results (ecdysterone injected with mineral oil rather than with DIW), all demonstrated increases in water influx (Table 18). Table 18. Relative influx of tritiated water and sodium-22 into 0.1 M NaCl adapted crayfish injected with 300 J.1g of ecdysterone. n= 5. 3 Treatment cpm H cpm 2~a Control 2,470 ± 128 34 ± 4 Ecdysterone*** 2,798 ± 156** 35 ± 4 Control 2,256 ± 312 30 ± 4 Ecdysterone 2,526 ± 306 33 ± 2 Control 4,089 ± 302 39 + 2 Ecdysterone 4,610 ± 836 46 ±8 ** P is less than 0.01 *** Ecdysterone injected with mineral oil The perfusion of tritiated water and sodium-22 through the gill chambers of ~ crenata resulted in similar influx patterns as when the crab was completely submerged in the nonnal bioassay medium. After 44 injection of the water soluble fraction there was a decrease in tri- tiated water influx into the animal, whereas an increase occurred after injection of the acetone soluble fraction. There was no difference in sodium-22 influx (Table 19). Table 19. Influx of tritiated water into Thalamita crenata after 50% sea water-3H20 was perfused into the gill chambers. Control and experimental animals were paired for sex and weight and five ventral ganglion equivalents/animal fractions were injected. Fraction Control Experimental Injected ~% ~~ Water soluble 4304 2636 4206 3111 4681 2387 Acetone soluble 4645 5266 4663 5350 4146 5053 When one-third Ringer-tritiated water solution was perfused through the isolated crayfish intestine, there was a linear flux of tritiated water into the bathing medium as a function of time. No change was observed in the flux of tritiated water into the bathing medium after the addition of the water soluble fraction, thus the flux rate was similar to the control intestine (Figure 14). There was an increase of tritiated water flux into the bathing medium after the acetone soluble fraction had been added. After reversing the osmotic gradient by having one-third Ringer- tritiated water solution as the bathing medium and sampling the perfused Ringer solution from the intestine, no differences were 45 40 Acetone Soluble 20 o ~::::::::::::===------...------,.- 40 Water Soluble 0/0 of 20 Perfused Medium 0 40 Control 20 50 100 150 Time (minutes) Figure 14. The flux of tritiated water from the lumen of the crayfish intestine into the bathing medium. One third Ringer-tritiated water solution perfused 20 ml/hr through the intestine; full-strength Ringer's bathing medium. Acetone and water soluble fractions added to the bathing medium after 50 min (arrow); 10 pI bathing medium samples taken every 10 min. 46 observed between the control effluent and the effluent from intestines bathed in the medium to which water and acetone soluble fractions had been added (Figure 15). 2.9F Control ~ 2.3 --- 5'0 Ibo I~ Time (minutes) Figure 15. The flux of tritiated water from the bathing medium into the lumen of the crayfish intestine. Full-strength Ringer's perfused 20 ml/hr through the intestine; one-third Ringer-tritiated water solution bathing medium. Acetone and water soluble fractions added to the bathing medium after 50 min (arrow); 10 ~l perfusion medium samples taken every 10 min. :El Discussion Water influx in decapod crustaceans has been Unplicated by various observations. The most common observation has been the change in weight in the anUnal after the occlusion of the nephropores, thus pre venting the release of urine (Bryan, 1960; Kamemoto et al., 1966; Kato, 1968; Kato and Kamemoto, 1969). Urine production and flow has also been used as an indicator of total water intake (Kamemoto and Ono, 1968). The deteDninations of water influx in the current study are based on a modification of a method used by Rudy (1967). NODnal anUnals were subjected to an isotopic bathing medium with the influx of tri tiated water as the indicator of water influx rather than the changes in weight or osmotic concentration of the blood. This method has several advantages, the most Unportant being that the measurement of tritiated water influx is direct. The bioassay is completed within a short period of tUne, and any change in the rate of influx of tritiated water can easily be recorded. The level of isotope is low so that even long exposure to the isotopic medium is not damaging to the anUnal; the amount of handling is also reduced. In general, the anUnals are under a minUnum of stress. Crayfish were used as the bioassay animal in most of the experi ments since they responded to the central nervous system extracts and fractions being assayed. The magnitude of their response, however, was not as great as in the crab, Thalamita crenata Latreille. The use of crayfish afforded an anUnal that was relatively easy to obtain, maintain, and stage according to molt cycle. 49 The separation and isolation of crustacean neurosecretory sub stances is not unique. K1einho1z (1970) summarized his work with eyesta1k neurohormones; his column chromatography separations utilized Sephadex G gels and DEAE and CM Sephadex ion exchangers. With a variety of gels and buffer systems he was able to fractionate at least five separate hormones from prawn eyesta1ks. In an earlier work K1einho1z et a1. (1967) isolated a hyperglycemic hormone from prawn eyesta1ks by using Sephadex G-100. Bartell et a1. (1971), investiga ting pigment hormones, isolated an active substance with Bio-Ge1s and Sephadex gels as did Hallahan and Powell (1971). Belamarich and Terwilliger (1966) and Berlind and Cooke (1970) using Sephadex G-25 separated an active substance from crab pericardial organs which increased the rate of heart beat, and Dall (1971) separated honu~rine from crustacean blood with Sephadex G-25. With the exception of the cardio-excitor hormone (Belamarich and Terwilliger, 1966) and homarine (Da1l, 1971) all of the crustacean hormones isolated were peptides or proteins with molecular weights over 1,000. Rao (1965), doing preli minary isolation of crustacean mo1t-irihibiting hormone, suggested that this hormone is a peptide. The water soluble active material effecting decreases in water influx also appears to be a peptide. It is ninhydrin positive, absorbs 280 mu light wavelength, and reacts with halogenation reagents; heating to 1000C does not destroy its activity nor do the proteolytic and other enzymes contained in pancreatin Merck. Because of this and its ultrafiltration characteristics and chromatographic elution patterns, this active material is thought to be a small molecule. 50 Chromatograms from column chromatography of the water soluble fractions show that separation of the active substance causing a decrease in water influx can be obtained with Sephadex gels. Because of the low molecular weight of the active substance, fractionation through progressively less porous gels is necessary, but even Sephadex G-10, the least porous gel, is unable to completely resolve substances closely eluted to the active material. Chromatograms of ventral ganglion and brain extract separated on Sephadex G-25 and G-15 Show that the active material is eluted in the middle of the fractionation range. From the elution profile of the Sephadex G-10 fractionation (Figure 11), it is observed that the active material is eluted close to the void volume; but other substances are eluted before it, and complete separation is not possible on the columns used. It is apparent from the chromatograms that the activity-containing fractions from the ventral ganglion and brain are eluted at the same sample tube number when fractionated on Sephadex gels G-50 through G-10. There are, however, differences in the total elution profile between the ventral ganglion and the brain, but these differences disappear from chromatograms of Sephadex G-l5 and G-10 elutions. By discarding the undesired portion of the collected fractions and rechromatographing only the activity-containing fractions, the chromatograms of ventral ganglion and brain elutions become similar due to the elimination of undesired substances. Semi-pure active material is obtained by rechromatographing through this series of less porous gels. Determination of the purity of the eluted fractions by thin layer electrophoresis is very suitable and also gives information about the 51 electrical properties of the active substance. Although polyacrylamide gel electrophoresis is commonly used for protein and polypeptide analy sis, this method proved unsatisfactory, for the active substance, although somewhat resolved within the gel, appeared to be washed out of the gel during destaining procedures. As mentioned above, the active substance is ninhydrin positive and reactive with halogenation reagents, thus, it is a simple method to determine purity from the number of spots on the electrophoretogram using small aliquots of the bioassay material. Since the electrophoretograms and chromatograms from the activity-containing fraction show several spots and over lappings, it is not pure at the end of the Sephadex G-10 step. eM Sephadex, a cationic ion exchanger, is successful in resolving the activity-containing fraction. A continuous ionic strength gra dient buffer is eluted through the column, and the active material is separated from the other substances. This is confirmed by thin layer electrophoresis done on all fractions bioassayed for activity. From its elution pattern from the ion exchange chromatography and by the electrophoretic behavior, the active substance appears to have a weak positive charge with an isoelectric point between 6.0 and 7.0. Since CM Sephadex is a weakly acidic exchanger, the results may have been more exact by using a strongly acidic exchanger such as SF Sephadex. The choice of gel, buffer, and ionic strength of the buffer is deter mined by the electrical properties and size of the active material. The molecular weight of the active substance can be estimated from its elution characteristics off of different gels as well as by the comparison of elution peaks with compounds of known molecular 52 weights. The active material is eluted within the fractionation range of Sephadex G-10 which is capable of separating peptides of up to a molecular weight of about 700, its exclusion limit. When plotted against their molecular weights the elution volumes of known molecular weight compounds produce a curve that suggests that the molecular weight of the active substance is between 600 and 700. This is in line with the inability of this material to be passed through UM-05 Diaflo ultrafiltration membranes with the upper limit of 500 molecular weight peptides. However, the upper and lower limits of the Sephadex sieving and fractionation range and of Diaflo membranes are somewhat dependent on the subtle interactions by the compound being chromatographed and filtered; a molecule's shape and charge can alter the elution and fil tration patterns, thus the molecular weight estimation. The vertebrate neurohypophyseal hormones oxytocin and vasopressin had no effect on water or sodium influx in crayfish. It thus appears that vertebrate-like octapeptides do not play a role in the water balance of crustaceans. With the exception of 5-hydroxytryptamine, the common crustacean neurotransmitter substances also effect no change in water or sodium movements. When treated with 5-hydroxytryptamine, there is a consistently significant increase in the rate of sodium-22 influx. It is possible that this substance can produce humoral or neural changes at the sites associated with sodium transport. 5-hydroxytryptamine is known to mimic crab pericardial organ cardio excitor activity (Cooke, 1966), and Rao and Fingerman (1970) discuss this biogenic amine's role in stimulating the release of a red pigment dispersing substance from the eyestalk of the fiddler crab. This 53 increase of sodium-22 influx is similar to that described by Kamemoto and Tullis (1972) for crayfish injected with brain extract from fresh water crustaceans. It seems reasonable to assume that 5-hydroxy- tryptamine either acts directly at the target organs as a humoral stimulus or indirectly as a neurotransmittor stimulating the release of an effector substance. Related to this are the results from the pericardial organ experi- ments. Although no change in tritiated water influx is observed after injection of pericardial organ extract, the crayfish show an increase in sodium-22 influx. This, then, would relate the pericardial organ to mineral regulation in crustaceans, if not directly, at least by inference. Brain tissue from fresh water crustaceans, 5-hydroxytryptamine, and crab pericardial organ extract all function alike in increasing sodium-22 influx. Since there is no consistent passive movements of water that accompanies this influx, it is suggestive that sodium move- ment is under separate and different control as compared with that of water, and that this control is regulated by products from the central nervous system or the pericardial organ. ~ crenata, being a brackish water crab, a good osmoregulator, and one subjected to changing salinities, was chosen as the crab from which the central nervous system would be investigated for its water influx activity. Results from preliminary experiments with minimally treated ventral ganglion and brain extracts do not confirm what was originally predicted: Kamemoto et ale (1966) found that Metopograpsus messor 54 (Forskal) ventral ganglion produced a decrease in blood osmotic con centration when administered to crabs held in hypotonic media. They suggested that the ventral ganglion contains a factor which increases water permeability in these crabs. Therefore, it was thought that ventral ganglion extract from~ crenata would increase the water permeability resulting in an increased tritiated water influx into the crab. On the contrary, when ventral ganglion or brain extract is injected into the animal in two or three tissue equivalents per animal, there is a significant decrease in water influx. This suggests that a factor is present which produces the opposite effect as was predicted. The original prediction is rather meaningless since it was based on results from experiments not designed to measure water influx directly and used a different animal. The activity of this water influx decreasing factor is maintained after acetone homogenation and boiling and is water soluble; it is also stable through column chromatography and can be isolated as discussed earlier. Brain extracts from other marine decapods also produce a decrease in water influx when injected into crayfish. Extracts from Cancer magister Dana and the prawn Spirontocaris marmoratus (Olivier) show significant decreases while there are non-significant decreases ~th ~ messor brain extract (Kamemoto and Tullis, 1972). Results from experiments by these inves tigators indicate that brain extracts from fresh water decapods contain no water influx decreasing factor, although a decrease in water influx would be' '~xpected in them because of hypotonic stress. There is also a factor present in the ventral ganglion and brain 55 which is acetone soluble that produces an opposite effect, that is, when this fraction is injected into the bioassay animal, it causes an increased influx of tritiated water. Thus, there is a factor in the central nervous system of ~ crenata which has the same effect as that suggested by Kamemoto et al. (1966) for M. messor ventral ganglion. The experimental results strongly suggest that there are two factors present which are antagonistic and, through their opposing effects, control the over all steady state water balance. Bliss et al. (1966) and Mantel (1968) have previously suggested the presence of two sub stances which regulate water movements in Gecarcinus lateralis (Fremdnville). Rao et al. (1967) and Fingerman et al. (1971) also obtained results indicating that chromatophorotropins from the eye stalks of the fiddler crab, Uca pugilator (Bose), produce opposing effects on the chromatophores and that these chromatophorotropins have different solubility characteristics. The water soluble and acetone soluble fractions, having opposite effects on water flux, may have different target organs or effector tissues. It has been shown that both factors act on the gills of the crab by changing the influx of tritiated water into the animal (Table 19). In these experiments the tritiated water was perfused over the gills without the isotope coming in contact with the mouth; the only way the tritiated water could enter the crab was through the gills or across the body wall of the gill chamber which is unlikely. The order of magnitude of the tritiated water influx was equal to that found in whole animal assays. It is concluded that the gills of the crab are the major source of water entry into the animal during the period of 56 the assay and that the two central nervous system factors may play important roles in the regulation of this water entry process. The crayfish responds to the ligation of eyestalks by increasing water movement into the hemocoel (Suwanrumpha, unpublished) suggesting an influence of hormones upon the process. When the two factors from the crab central nervous system were tested on isolated crayfish intes tines, only the acetone soluble fraction was effective in producing permeability changes (Figure 14). When the osmotic gradient is in the direction of the bathing medium (into the haemolymph from the lumen), the acetone soluble fraction produces an increase in flux rate; the water soluble fraction has no effect. The gut, as with the gills and kidney, has been suspected of being involved with water as well as salt absorption in aquatic and marine crustaceans, and this acetone soluble factor which increases the flux of water into the bathing medium supports this idea. It is observed that neither of the influx factors affect the flux rate of tritiated water when the osmotic gra dient is reversed, that is, the movement of tritiated water from the bathing medium into the intestine (Figure 15). Without elaborating, this excludes these factors from consideration for any role they may have in the elimination of water through the crayfish intestine. It is of interest that Mantel (1968) observed tritiated water movements in both directions in the foregut of ~ lateralis. In her experiments tritiated water movement from the haemolymph to lumen increased with the addition of ventral ganglion extract to the haemolymph side. In the above experiments intestines and foreguts from T. crenata were not used because of size and fragility. 57 Why the action of the acetone soluble water influx increasing factor is not observed in animals injected with minimally treated ventral ganglion and brain extract is not clear. It may be that its stored amount in the central nervous system is very low compared to the decreasing factor; the steady state production and titer of the water influx decreasing factor may be normally high as compared to the increasing factor. The central nervous system tissue from animals maintained in dilute sea water for one week prior to experimentation elicits the same influx decreasing effect and at the same magnitude as does the tissue extract taken from animals kept in normal sea water. One might expect that animals in dilute sea water would deplete to below normal this water influx decreasing factor when continually stressed, hence, producing no decrease. These results show that at least a steady state amount of this material is present within the central nervous system of these animals. The administration of ecdysterone into crayfish may produce increased water influx but has no effect on sodium movement. In this respect a similarity exists with the acetone soluble fractions which cause increased water influx to occur. However, data from these bio assays are not exclusively significant and only the suggestion of influx activity can be considered. There are conflicting reports as to the dosage of ecdysterone needed to stimulate ecdysis (Warner et al., 1969 versus Krishnakumaran and Schneiderman, 1969), but these investi gators and others (Faux et al., 1969; Jegla and Costlow, 1970; Blanchet and Charniaux-Cotton, 1971) agree that ecdysterone and other insect and plant polyhydroxy steroids initiate ecdysis in crustaceans. The steady 58 state water and sodium fluxes are upset at the beginning of ecdysis with more water entering the animal in preparation for the molt. Per haps the reason that the results were not significant was that the ecdysterone effect produces long term changes, changes that cannot be clearly seen in the relatively short bioassay period. The crustacean molting hormone, secreted from the Y-organ, is thought to be synonymous, and even homologous, with thE!' ecdysones produced in the insect thoracic gland (reviewed by Highnam and Hill, 1969). This would eliminate the crustacean central nervous system from being a source for the ecdy sterones, and it is improbable that the acetone soluble factor is an ecdysone. These studies strongly suggest that at least part of the steady state water balance of ~ crenata is regulated by the acetone and water soluble factors contained in the central nervous system. It is diffi cult, however, to incorporate the present findings into an overall scheme that interacts meaningfully with the results from previous investigators. The main problem encountered in attempting to incor porate all of the information is that there are few "common denominators" shared by the experiments. For example, in the present study, a direct measurement of water influx is used to determine the effect of various central nervous system components on water influx in ~ crenata, whereas Kato and Kamemoto (1969) show the effect of eyestalk ligation in ~ messor on blood and urine osmotic concentrations. Both studies attempt to define neuroendocrine control of water balance in crustaceans, yet they have little methodology in common. At the same time, information from each study can help "piece" together the neuroendocrine control 59 mechanisms of crustacean water balance. As mentioned earlier, changes in water influx in euryha1ine decapod crustaceans can occur if tissue permeability is modified and/ or the urine flow increases or decreases. If an increase of water influx occurs while the urine flow remains static, then the animals will gain water provided it is in dilute medium. This can result in a lower blood osmotic concentration and an increase in weight if the internal salt content is not upset. Carlisle (1955) reported that eyesta1k1ess Carcinus maenas (Pennant) show greater size increases at molt than do the normal molt ing crabs. Injections of eyesta1k extract into the eyesta1k1ess molting crabs resulted in reducing the size increase. He correlated the relative size differences with the amount of water the crabs took in. Bliss et a1. (1966) found that the carapace size of ~ 1atera1is increased after ligating the eyestalks of crabs during ecdysis, the increase in size being due to water retention. The water that is retained by these eyesta1k1ess crabs can be released by injections of the central nervous contents of the eyesta1k or thoracic ganglia. They proposed that the substance responsible for the release of water is a diuretic hormone while the material producing water retention is antidiuretic, and that retention of water is partially due to an increase in water influx. Kamemoto et a1. (1966) found that the blood osmotic concentration was lower in eyesta1k~ligated M. messor in dilute sea water than in normal crabs with eyesta1ks. The injection of ventral ganglion extract also produced a lowering of the blood osmotic concentration. Kato and 60 Kamemoto (1969) observed the lowering of osmotic concentration of the blood, as well as that of the urine, in eyesta1k-1igated M. messor held in 25% sea water. In nephropore-p1ugged crabs held in dilute sea water the eyestalk-ligated animals gained more weight than the normal control crabs. The investigators interpreted the lowering of osmotic concentration and the weight increase as increased water movement into the animals. Injections of eyesta1k extract into eyestalk-1igated crabs maintain the blood osmotic concentration as that of normal animals. De Leersnyder (1967) also observed an increase in urine flow in eyesta1k-1igated Eriocheir sinensis H. Milne-Edwards which she attributed to increased water influx. The ligation of the eyesta1ks of T. crenata resulted in a lowering of blood osmotic concentration, as noted by the decreased chloride concentration (Kamemoto and Tullis, 1972). Kamemoto and Ono (1969) found that the blood osmotic concen tration is lowered and urine flow increased in eyesta1k1ess crayfish. These results suggest that the influx of water was increased after removal of the eyesta1ks. It is concluded that by isolating the eyesta1k neuroendocrine system from the animal, a component that helps maintain steady state water balance in the crab is removed. When this component is present, the influx of water is stable, and when it is absent, water influx increased. When this eyestalk component is replaced into the eye stalkless crab, by injecting eyestalk extract, the influx of water decreases towards that of normal animals. The acetone soluble factor from T. crenata central nervous system described in the present studies produces an increased water influx 61 into crabs and crayfish, mainly through the gills (dashed lines, Figure l6}, with little accompanying sodium influx. Because of the apparent ensuing dilution of the haemolymph, a lowering of the blood osmotic concentration may occur providing there is no modification of internal salt content. The lowered blood osmotic concentrations and the increases in weight and size, as observed by the previous investi gators, may have occurred this way. The increase in water influx is probably due to the increase in tissue permeability to water. If, however, the animal actively maintains its original salt-water ratio after the injection of the acetone soluble factor, then no change of blood osmotic concentration would be observed. Therefore, increased water influx does not necessarily lead to lower blood osmotic concen trations. It appears, then, that both the eyestalk complex and the central nervous system contain substances which are involved in controlling water influx in crabs. The active material in the central nervous system may be under inhibitive neural or humoral control from the eye stalk. By removal of the eyestalks, the inhibition is released and increased influx of water occurs. At the same time, the eyestalk con tains an active factor which reduces water influx. This inhibitory control by the eyestalk over the ventral ganglion was proposed by Kamemoto et ale (1966). Injections of the water soluble factor from the central nervous system of ~ crenata produce decreases in water influx in the crab (mainly in the gill tissue, Figure 16). This decrease in water influx may be caused by a decrease of the permeability of gill tissue to 62 water. Since there app~ars to be no decrease in sodium-22 influx with that of water, the internal salt content could be actively maintained while the permeability is decreased. This could lead to an increase in blood osmotic concentration, possibly paralleling in action that caused by eyestalk replacement therapy in which the addition of eyestalk extract into eyestalkless crabs rendered the blood osmotic concentration normal (Kato and Kamemoto, 1969). In the steady state the action pro duced by the water soluble factor may counteract that of the acetone soluble factor. The digestive tract appears to respond to the central nervous system factors. Bliss (1968) suggests that the thoracic ganglion of G. lateralis contains a substance that increases water flux from the haemolymph-into the foregut of normal intermolt crabs and of eyestalk less animals after ecdysis. Mantel (1968) shows that water flux increases in the isolated foregut of the crab with the addition of extracts of the thoracic ganglia to the haemolymph side. The acetone soluble factor increases the water flux rate in the isolated crayfish intestine from the intestinal lumen into the haemolymph (Figure 16); this probably occurs with the crab intestine, also (dotted lines, Figure 16). Isolated crayfish intestines from destalked crayfish show increased flux rate in the same direction (Suwanrumpha, unpublished). There is no doubt that the digestive tract is capable of responding to central nervous system factors. These factors can increase the per meability of the gut to water, and their secretion from the central nervous system may be controlled via the eyestalk complex. 63 Experimental animals must be treated individually, and extrapola tions from one group of animals to another group must be considered carefully. As Bliss (1968) and Kamemoto and Tullis (1972) point out, terrestrial, semiterrestrial, euryhaline, and fresh water decapods do not, and need not, react similarly to experimental manipulations. For instance, brain extracts from the fresh water prawn Macrobrachium rosenbergii (De Man) and the marine prawn ~mannoratus produce dif ferent water and ionic influx responses when assayed in the crayfish ~ c1arkii (Kamemoto and Tullis, 1972). The extracts from the marine prawn produce a decrease in tritiated water influx (similar to the water soluble factor), while extracts from the fresh water prawn produce increased sodium influx. 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