Eyestalk Extract Research Articles

DUAL CONTROL OF THE LEUCOPHORES IN THE PRAWN,PALAEMONETES VULGARIS, BY PIGMENT-DISPERSING AND PIGMENT-CONCENTRATING SUBSTANCES

1. The prawn, Palaemonetes vulgaris, produces substances that cause dispersion and concentration of its white chromatophoric pigment.2. Injection of extracts prepared from one or more eyestalks triturated directly in isosmotic sea water produced concentration of the white pigment alone, dispersion alone, or concentration followed by dispersion. In contrast, similarly prepared fresh extracts of the supraesophageal ganglia with the circumesophageal connectives attached always produced white pigment dispersion alone.3. Chromatography of ethanol extracts of eyestalks and as well as supraesophageal ganglia with the circumesophageal connectives attached on the gel Sephadex LH-20 always yielded both the white pigment-dispersing substance and the -concentrating substance. The former preceded the latter off the gel column.4. The responses obtained with extracts prepared directly in isosmotic sea water were interpreted as having been due to the relative quantities of the white pigment-dispersing and -concentrating substances present in the organs in addition to an antagonism between these chromatophorotropins.

Influence of eyestalk extract on trehalos, fructose and glucose in the hemolymph of Oronectes limosus

Quantitative changes of glucose, fructose and trehalose in the hemolymph ofOrconectes limosus were measured after injection of eyestalk extracts. Especially in February and March high levels of trehalose were found after hormone injection.

Hormonal Effects on Calcium Metabolism in Crustacea

Cyclic shifts of calcium in the exoskeleton and soft tissues, as they are related to the intermolt cycle in crayfish, are reviewed. Regulatory factors, derived from the eyestalk, influence levels of exoskeletal calcium; eyestalk extracts prepared from animals in premolt decrease shell calcium, while reciprocally extracts from animals in intermolt increase it when these hormonal sources are injected into animals in the premolt stage (D-D4) . In addition, premolt eyestalk extract results in an increase in gastrolith calcium. In the exchange of calcium between the animal and its environment there is evidence for differentialdeposition of recently available calcium in the exoskeleton. Further, intermolt and early premolt animals maintained in Ca45-labelled water for 15 days concentrate it 4 and 3—fold in the exoskeleton and stomach, respectively. However, removal of a molt-inhibiting factor through ablation of eyestalks results in a 20 and 40—fold increase in incorporation inthese same sites relative to environmental concentrations. Treatment with mammalian parathyroid extract mobilizes both exoskeletal and gastric calciumand leads to a rise in blood calcium. However, there is little or no effect on levels of exoskeletal citric acid. Further, citric acid is higher in the crayfish carapace during stage C, the period of mineralization, than in stage D, the period of demineralization. There are both similarities and differences between the effects of crustacean and mammalianregulating factors with respect to the direction and extent of mineralization. Biochemical studies should elucidate the mechanisms regulated by these hormones.

Alterations in lipid metabolism associated with premolt activity in a land crab and fresh-water crayfish

1. 1. The initiation of premolt activity results in an increased lipid content in the hepatopancreas of both Orconectes virilis (fresh-water cryafish) and Gecarcinus lateralis (land crab) and an increase in the lipid content of the abdominal muscle of O. virilis. 2. 2. The increased lipid content of the hepatopancreas reflects and increased incorporation of lipid precursors into both the neutral lipid and phospholipid fractions of this tissue during premolt. 3. 3. Although the ratio of neutral lipid to phospholipid changes following the induction of premolt activity, the fatty acid compositions of these two fractions are remarkably similar in intermolt and premolt animals. 4. 4. The increase in the rate of lipid synthesis which results from eyestalk extirpation is partially negated by the administration of a saline eyestalk extract.

Neuroendocrine regulation of salt and water balance in the crayfish Procambarus clarkii

1. 1. The injection of brain extracts into eyestalk-ligated, nephroporeplugged crayfish prevents to some extent the rapid increase in weight found in eyestalk-ligated, nephropore-plugged animals. 2. 2. The injection of brain or eyestalk extract into eyestalk-ligated crayfish prevents the decrease in chloride concentration of the blood found in eyestalkless animals. 3. 3. The blood osmotic and sodium concentrations of eyestalk-ligated animals increase more rapidly than normal animals when placed in a hyperosmotic medium. 4. 4. There is a greater flow of urine in eyestalk-ligated animals as compared to normal animals. 5. 5. It is suggested that a brain-eyestalk neuroendocrine system is involved in the regulation of salt and water balance in crayfish.

A comparative study of leucophore-activating substances from the eyestalks of two crustaceans, Palaemonetes vulgaris and Uca pugilator.

1. The relationships between the response of the leucophores in Uca pugilator and the concentration of extracts of eyestalks from Uca pugilator and Palaemonetes vulgaris were determined. For the first time evidence is provided to show that substances capable of evoking pigment dispersion and pigment concentration in the leucophores of Uca are present in the eyestalks of Palaemonetes. Contrary to the situation recorded previously for melanin-dispersing activity, extracts prepared directly in saline and the ethanol-soluble fractions of the eyestalks from Uca evoked nearly identical white pigment-dispersing activity at all dilutions tested.2. White pigment-dispersing and -concentrating substances can both be demonstrated to be present in the ethanol-soluble fraction of the eyestalks of Uca as well as in the material directly extractable in water in spite of the fact that when these extracts are assayed immediately after preparation they evoke only white pigment dispersion because the white pigment-dispersing...

The effects of temperature on the responses of chromatophores in the crab, Ocypode macrocera

1. In the crab, Ocypode macrocera, the effects of temperature on chromatic adaptation and responses of chromatophores to chromatophorotropins were determined. 2. The pigments in the black and red chromatophores of intact Ocypode concentrated at high temperatures. The pigments in the black and white chromatophores dispersed at low temperatures. At temperatures between 22 and 28° C the crabs were able to achieve maximal chromatic adaptation to a given background. 3. The temperatures, 15, 22 and 28° C had no direct effect on the chromatophores of eyestalkless Ocypode macrocera. However, when identical doses of an eyestalk extract were injected into eyestalkless crabs maintained at the three different temperatures the responses of the chromatophores varied with the temperatures at which the assay animals had been maintained. 4. The integrated response of the black and white chromatophores to the injected eyestalk extract was inversely related to temperature. The rate of response was, however, directly related to temperature. 5. The amplitude of red pigment-concentrating response decreased at low temperatures, but the duration of response increased. However, the integrated response of the red pigment did not vary significantly with changes in temperature.

The influence of size on the response of melanophores in the fiddler crab, Uca pugilator, to eyestalk extracts

1. The influence of size on the response of melanophores in Uca pugilator from Alligator Point, Florida and West Falmouth, Massachusetts was determined. 2. For the first time evidence is presented to indicate that the melanin-dispersing response evoked in eyestalkless crabs after injection of eyestalk extract increases with increase in the size of the crabs used in the assay. 3. The blood of small crabs has a higher titer of melanin-concentrating substance than does the blood of large crabs. Consequently smaller crabs would be more readily able to antagonize injected melanin-dispersing hormone, resulting in a smaller response. In addition, it is possible that the sensitivity of the chromatophores may increase with increase in size of the crab.

Molt cycle regulation of nucleotide pyrophosphatase in the hepatopancreas of the blue crab, Callinectes sapidus rathbun

1. 1. Three substrate specific nucleotide pyrophosphatases are reported in 12,000 g supernatants obtained from the hepatopancreas of the blue crab. 2. 2. The activity of UDPG, UDPGA and UDPNAG nucleotide pyrophosphatase is related to the stage of the molt cycle of the blue crab. UDPGA nucleotide pyrophosphatase rates are relatively low in all stages of the molt cycle except D 1–2. 3. 3. Crude eyestalk extract preparations from D 3–4 crabs cause a decrease in UDPG nucleotide pyrophosphatase activity and an increase in UDPGA nucleotide pyrophosphatase activity in hepatopancreas enzyme preparations from B-stage crabs. 4. 4. A discussion of the possible relationship of these three enzymes to molt cycle activity is presented.

Rate of production and release of chromatophorotropins in crustaceans

It is now well accepted that the crustacean chromatophores are largely controlled by the condition of light and by hormonal effectors, the chromatophorotropins, which are the product of neurosecretory systems. Distribution of these “hormones” can be measured by bioassay methods in the freshwater prawn Palaemon paucidens. The red pigment-dispersing hormone (RPDH) is mainly localized in ganglionic tissues such as the supraesophageal ganglion or the “brain” (BR) and thoracic ganglion (ThG), whereas the tritocerebral commissure (TrC) is the source of the most potent red pigment-concentrating hormone (RPCH). Other nervous tissues, including the eyestalk (ES) and circumesophageal connectives (CC), contain both these hormones, but in smaller amounts. Two questions arise from these results: Is an RPDH present in the BR chemically identical with corresponding substances contained in other tissues? And, is there a relationship between the two, physiologically antagonistic hormones which coexist within a tissue? The prawns, when placed on a specially devised “neutral” background, manifest a diurnal variation in chromatophoral behavior under natural light: they are darker by day than at night. Chromatophorotropins contained in several nervous tissue-extracts from such animals were shown to have a definite rhythm of potency. The BR contained more RPDH during daytime than at night time, whereas the ES contained more RPDH during night than by day. On the other hand, amounts of the RPCH present in the TrC changed little during the 24-hour period. These results seem to show (1) that the BR-RPDH, after synthesis, is released promptly into circulation to bring about dispersion of red pigment, and (2) that the TrC, containing the RPCH at about the same level throughout the 24-hour period, does not play a principal role in diurnal variation of bodily color in Palaemon. Bioassay of particular tissue extracts from the prawns that had previously received other tissue extracts gives interesting features of the relationship between hormones of different origins. Thus, a significant decrease in potency was noticed of the BR-RPDH in animals that had received injection of the ES extracts, suggesting that the ES contains a substance which liberates the tissue-bound RPDH of the BR. On the contrary, the potency of the ES-RPCH increased greatly after the animals received the TrC extracts, suggesting that the TrC-RPCH is transported to the ES before its release into circulation. The nauplius eye is suggested as the site of another hormonal factor that might be involved in physiology of chromatophorotropins. The evidence to support this concept is discussed.

Effects of Eyestalk Removal On Rhythmic Locomotor Activity in Carcinus

ABSTRACT Male Carcinus collected in summer and given several days to recover from eyestalk ablation showed a continuously high level of activity. Injection of eyestalk extract inhibited activity. Unlike control animals from which retinae were removed, experimental animals did not show a rhythm after chilling. Chilling the eyestalks alone elicited a rhythm similar to that appearing when the whole animal was chilled. The results are consistent with the view that locomotor rhythmicity is mediated via an inhibitory hormonal mechanism in the neurosecretory cell groups of the eyestalk. There is no direct evidence that the neuroendocrine mechanism alone constitutes an autonomous clock, however, and it is in any case probably co-ordinated by one or more other rhythmic systems within the crab.

Endocrine control of glycogen synthesis in crabs

1. 1. Eyestalk removal in crabs Hemigrapsus nudus and Cancer magister (intermolt stage) is followed by an increase in the activity of UDPG-Glycogen transglucosylase system (UDPG-GT) in muscle tissue. 2. 2. Injection of eyestalk extract into eyestalkless animals decreases the enzyme activity nearly to normal levels. 3. 3. Net synthesis of glycogen in muscle and hepatopancreas, from either glucose or maltose, is not increased by eyestalk removal or depressed by the eyestalk extract injection. 4. 4. The turnover of glucose in glycogen in muscle and hepatopancreas is increased, as indicated by increased specific activity of the glycogen following injection of C 14 glucose or C 14 maltose, by eyestalk removal. 5. 5. Eyestalk extracts have been fractionated by chromatography on Sephadex and the inhibitory factor localized in a definite fraction. 6. 6. The increased turnover is treturned to normal by the injection of crude eyestalk extract or of the purified fraction containing the transglucosylase inhibitor. 7. 7. Eyestalk removal is followed by a marked decrease in total phosphorylase and an increase in the percentage of “inactive” phosphorylase “ b”, but these changes are not reversed by injection of purified fraction of eyestalk extracts. 8. 8. The purified fraction does not increase blood glucose when injected, but does increase the maltose concentration in the blood. 9. 9. These results support the hypothesis that the inhibitor of UDPG-GT acts as a hormone in the control of glycogen synthesis and suggests the possibility that other factors may also be involved in the control of carbohydrate metabolism.

Neuroendocrine repression of ribonuclease in the prawn, palaemonetes vulgaris

Crustacean eyestalk extract repressed the activity of hepatopancreas ribonuclease in the prawn, Palaemonetes vulgaris.

Ovary-inhibiting effect of extract of crab eyestalk on female American cockroaches

Administration of an eyestalk extract in Ringer solution, of the crab Paratelphusa hydrodromous (Herbst) suppresses ovarian function in adult females of the cockroach Periplaneta americana (L). The effective principle, which may be the ovaryinhibiting hormone (OIH) contained in the eyestalk, or some substance mimicking OIH functionally, results in an inhibition of ovarian growth accompanied by an accumulation of stainable material in the brain-corpus cardiacum-corpus allatum neuroendocrine complex. The “OIH” may thus act indirectly on the ovary. Polyacrylamide-dise electrophoresis of the plasma of treated animals reveals an accumulation of materials with protein and carbohydrate properties in a fraction which is normally utilized by the ovary and thus seems to represent the sex-limited blood protein.

Solubility and stability properties of the melanin-dispersing substances from the eyestalks of the fiddler crab,Uca pugilator

1. Extracts of fresh, freeze-dried, and heat-dried eyestalks prepared directly in physiological saline evoked nearly identical melanin-dispersing responses. The highly active ethanol-soluble fraction extractable from fresh eyestalks ofUca was to a great extent unextractable after the eyestalks were freeze-dried, heat-dried, or pretreated with a variety of organic solvents, whereas the melanin-dispersing substance in the ethanol-insoluble fraction of the eyestalk was resistant to these treatments. The melanin-dispersing substance obtained by direct extraction of the eyestalk in water is more resistant to treatment with organic solvents than the substance present in the ethanol-soluble fraction. 2. The melanin-dispersing activities of the eyestalk material soluble in various solvents showed no overall relationship with the dielectric constants of the solvents used. However, the melanin-dispersing activity was related to the dielectric constants of the normal alcohols used in the following decreasing order: methanol>ethanol>propanol>butanol>amyl alcohol. 3. Ether and isopropyl ether destroyed most of the melanin-dispersing material present in the ethanol-soluble and water-soluble fractions of the eyestalk, possibly due to excessive oxidation. 4. The melanin-dispersing material in the ethanol-soluble fraction of the eyestalk is greatly destroyed by heat. In contrast, the melanin-dispersing material in the water-soluble fraction of the eyestalk is resistant to heat. 5. The possibility that two melanin-dispersing substances are present in the eyestalks ofUca is considered and discussed.

Characterization of the melanin-dispersing hormone fraction of the eyestalks of Uca pugilator by electrophoresis

Extracts prepared from the eyestalks of Uca pugilator were subjected to highvoltage vertical starch-gel electrophoresis to study the behavior of the melanindispersing fraction of the eyestalk extract in an electrical field. Separations were made in a pH range from 2.05 to 8.15 with McIlvaine buffer and at pH 8.9 with barbital buffer. During electrophoresis the apparatus was placed at about 3°C and an aluminum plate closely applied to the Saran-wrapped gel mold acted as a heat sink to dissipate the heat created in the gel. Over the range of hydrogen ion concentrations employed the biologically active fraction migrated as a single component. The isoelectric point appeared to be about pH 5.15. The hormonal activity migrated as much as 10 cm and responded in a manner predictable for an ampholyte to changes in voltage and pH. The assay procedure resolved the width of the active band within 1 or 2 cm of gel.

Temperature Dependence of Ionizing Radiation Effect on Dry Preparations of Two Melanophore-Stimulating Hormones

Dry preparations of α-MSH and of fiddler crab eyestalk extracts were irradiated with fast electrons. The irradiations were carried out in a vacuum and at absolute temperatures from 90°K to 420°K. The direct action of the radiation on melanin-dispersing activity in the chromatophores of appropriate assay animals can be interpreted according to target theory and also in terms of Arrhenius activation energies for yield in relation to temperature. At temperatures around 300°K the radiation sensitivity of α-MSH corresponds by target theory to a molecular mass of 1100 gm/mole. For the crab hormone the radiation-sensitive mass is about 3750 gm/mole from irradiations at that temperature. Results at various temperatures for α-MSH indicate only roughly an activation energy between 350 and 600 cal/mole. For the crab hormone two activation energies are evident, one at 200 cal/mole and the other at around 3300 cal/mole. The patterns of activation energies are reasonably distinct from those evident in such work with en...

Relationship between the response of melanophores in the fiddler crab, Uca pugilator, and the concentration of eyestalk extract

1. Eyestalks from the fiddler crab, Uca pugilator, were extracted directly in either water or absolute ethyl alcohol. Dosage-response curves for these extracts were then determined. 2. The melanin-dispersing response produced by the alcohol soluble material increased sharply with increasing concentration of the extract between 1 and 80 eyestalks/ml. In contrast, the dosage-response curves for the extracts prepared directly in water did not rise at all as sharply as did that of the alcohol extract. 3. A water extract seems to contain a substance which at the higher concentrations antagonized the melanin-dispersing hormone. Elimination of this antagonist by the technique of gel filtration resulted in an increased melanin-dispersing activity. 4. The possible action of absolute ethyl alcohol which results in a preparation of melanin-dispersing hormone from the eyestalk that is much more active at higher concentrations than a similar extract prepared in water is discussed.

Direct stimulation of respiratory enzyme activity by crustacean eyestalk extract

Crustacean eyestalk extract stimulated the activity of cytochrome oxidasem succinic dehydrogenase and malic dehydrogenase of oyster mantle homogenate systems.

Separation and purification of crustacean eyestalk hormones.

proteinaceous in nature, with several of them apparently polypeptides of relatively low molecular weight. Some of the reported pigmentary-effector hormones are recognizable as distinct molecular species on the basis of chromatographic behavior and molecular size. The diabetogenic hormone is a much larger molecule and is a protein. The properties of the molt-inhibiting hormone of the eyestalk, although still too little known for specific classification, indicate that it belongs to one of these two categories. Tests of functional specificity and structural studies, which in most cases depend on further chemical purification, seem attainable in the near future. The subsequent comparative studies that may become feasible will be of physiological and biochemical interest. Modern crustacean may be considered to have started with the discovery (Perkins, 1928; Koller, 1928) that integumentary chromatophores are influ? enced by a blood-borne substance originat? ing in the eyestalk. These studies in physio? logical color change were considerably ex? tended by Brown (1935) who presented experimental evidence for the existence of a number of physiologically-different chromatophorotropins. Another pigmentaryeffector hormone was demonstrated with the discovery (Kleinholz, 1936) that photomechanical movements of retinal pigments in crustaceans are regulated by eyestalk extracts. The diversity of physiological processes in crustaceans that has since been reported under endocrine control has been reviewed recently by Barrington (1964), Charinaux-Cotton and Kleinholz (1964), and Fingerman (1965). The best-known physiological effects of eyestalk hormones are shown diagrammatically in Figure 1. One consequence of this hormonal abundance, reminiscent of the variety of con? trolling hormones found in the anterior pituitary gland of vertebrates, is the ques? tion of specificity of physiological effect. That is, does one eyestalk hormone have one specific role only, and hence does the multiplicity of reported endocrine effects imply a multiplicity of different hormones, or, are a few substances produced in the neurosecretory system of the eyestalk, each of which may have more than one physio? logical action? This very question was being argued some thirty years ago for the eyestalk chromatophorotropins. The prob? lem was later further complicated by the discovery of other neurosecretory struc? tures, outside the eyestalk, extracts of which also showed chromatophorotropic activity. Barrington's (1964) comment It should be remembered, however, that the indi? vidual substances (the color change hor? mones) have not yet been characterized, either structurally or functionally, with the rigorous precision that we encounter in many fields of vertebrate endocrinology is even more pertinent with the larger num? ber of endocrine effects now known.