WLLIS, Richard Eugene, 1936- NEDROENOOCRINE CONfROL OF WATER BALANCE IN TIlE DECAPOD CRUSTACEAN ~ 'THALAM!TA' CRENATA. University of Hawaii, Ph.D., 1972 Zoology f-------.----------------------------- -- ----- -- ! \ 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