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Two types of neurosecretory fibers, designated as Type 5 and Type 6 axons, in the sinus gland of the freshwater prawn, Palaemon, establish contact with other neurosecretory axons by means of synaptic junctions. This finding strongly supports the view that release of some neurohormones from the eyestalk may be regulated by neurosecretory neurons through synaptic transmission.
In the medulla terminalis ganglionic X-organ (MTGX) of the eyestalk of the freshwater prawn, Palaemon paucidens, six peptidergic neurosecretory cell types (A-, B-, C-, D-, E-, and F-cells) are distinguishable on the basis of the different morphology of their elementary granules and rough endoplasmic reticulum (rER). All of these cell types seem to correspond to Type-IIIa cells or dispersing Type-IV cells, that have previously been differentiated at the light microscopic level (Hisano, 1974), as judged from the dimensions of their cell bodies and nuclei. Two other peptidergic neurosecretory cell types that are apparently comparable to the Type-II and Type-IIIb cells (Hisano, 1974), respectively, are recognized in parts of the optic ganglia other than MTGX, and these are now designated as G- and H-cells, respectively. All the remaining cell types, designated as Type-I, cluster-forming Type-IV, Type-V and Type-VI cells in our previous light microscopic study, have small cored-vesicles in their cytoplasm. It remains undecided whether these, possibly aminergic, neurons are neurosecretory or not.
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In crustaceans, all the steps in the assimilation of food take place in the hepatopancreas. To facilitate the study of this organ, a method for the dissociation of cell types was developed. The hepatopancreas of the prawn Palaemon serratus was mechanically dissociated and the cells separated by Percoll density-gradient centrifugation. The E and R cells had similar densities of around 1.05 g/ml. The F cells were separated into two distinct fractions with densities of 1.075 and 1.082 g/ml. The B cells sedimented at a density of 1.12 g/ml. The ratio between the two populations of F cells was found to vary during the intermolt cycle while B cells disappeared after the molt. When the density gradient fractions were incubated with 3H-leucine, incorporation was highest in the F cell fractions. Measurements of alpha-amylase activity, indicated that the two populations of F cells may be derived from the same cell type.
The pattern of copper utilization during the embryogenesis of the freshwater prawn, Palaemon lamarrei has been described. Throughout the incubation period, lasting for 14 to 16 days, the egg of P. lamarrei is permeable to salts and against a concentration gradient, it absorbs 11 mug of salt from the freshwater medium. Out of this total salt uptake, as much as 0.143 mug is due to the absorption of copper. Intake of copper, as a function of incubation time, exhibit a more or less similar trend to that of total salt intake and this can be attributed to the increased synthesis of haemocyanin.
Antiserum against Man beta 1-4Glc beta 1-1Ceramide (MIOse2Cer), a mannolipid isolated from spermatozoa of the fresh-water bivalve, Hyriopsis schlegellii, has been elicited in rabbits by repeated injection of a mixture of hapten-bovine serum albumin (1:1, mg/ml) with Freund's adjuvant. The specificity of the affinity-purified antibody (immunoglobulin G type) obtained from the serum was examined, using other glycosphingolipids and glyco-proteins structurally related to MIOse2Cer, by means of ELISA and TLC-immunostaining. The purified antibody was highly specific to MIOse2Cer and lacked reactivity with other glycolipids and glycoproteins including glucosylceramide, lactosylceramide, dimannosylglucosylceramide (MIOse3Cer), glucosaminylmannosylglucosylceramide (ArOse3Cer), thyroglobulin and alpha 1-acid glycoprotein. The antibody was found to bind, although less efficiently, to certain other compounds containing the group Man beta 1-4Glc and/or Man beta 1-4GlcNAc at their termini, such as MIOse2-sphingosine and Man beta 1-4GlcNAc beta 1-p-aminobenzoic acid ethylester derivatives. The present antibody was applied to the detection of the natural hapten in crustacean glycolipids. The purified antibody reacted with a neutral glycosphingolipid present in the two kinds of crustacean, Euphausia superba (antarctic krill) and Macrobrachium nipponense (fresh-water shrimp) as shown by TLC-immunostaining. The crustacean glycolipid antigen was isolated and characterized to be the Man beta 1-4Glc-Cer. This is the first report on the presence of a mannose-containing glycosphingolipid in the crustacean.
Surface changes induced by sea water were analyzed in the ovulated oocyte of the prawn Palaemon serratus. They depended on the presence of external Mg2+ but not on external Ca2+ alone. Increasing external Mg2+ from 0 mM to 30 mM stimulated first a progressive disappearance of preexisting microvilli, which was over within 30 min of incubation. This is correlated with membrane removal via internalization of coated vesicles, ascertained by observations of endocytosis of an extracellular fluid-phase marker and by measurement of a diminution in membrane capacitance (Cm). Thirty-five minutes after sea water contact, the prawn oocyte underwent a cortical reaction independent of fertilization. It consists in a heavy exocytosis of ring-shaped elements, leading to the deposition of a thick capsule, and requiring a threshold Mg2+ concentration of greater than or equal to 10 mM and at least a 3-min incubation with Mg2+. Concurrently, the values of the membrane capacitance (Cm) and conductance (Gm) increased about 2 and 10 times their initial values, respectively. The calcium ionophore ionomycin, added to Mg(2+)-free artificial sea water, stimulated the cortical reaction with requirement of external Ca2+. Other divalent cations (Mn2+, Zn2+, Co2+, Ni2+, Cd2+) instead of Mg2+, induced the cortical reaction, but Ba2+, Sr2+, and La3+ did not. When eggs are fertilized, the cortical reaction takes place in two steps, the first being a discrete exocytosis of a foamy material and the second always involving ring-shaped elements.
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Ecdysteroid levels during the embryogenesis of the giant freshwater prawn, Macrobrachium rosenbergii, were determined by radioimmunoassay and high-performance liquid chromatography. Ecdysteroids consisting of significant amounts of 20-hydroxyecdysone and high-polarity products (HPP) and lesser amounts of ecdysone and low-polarity products (LPP) were detected in mature ovaries and newly laid eggs. All ecdysteroid groups decreased gradually during the nauplius phase. With the formation of the compound eye and the appearance of the carapace and other body-like structures, marking morphogenesis to the zoeal stage, embryos showed the beginning of a continuous and dramatic increase in ecdysteroid concentrations sustained until larval hatchout. Ecdysteroid levels at hatchout were above 20-fold greater than ecdysteroid levels in newly laid eggs. More specifically, HPP and 20-hydroxyecdysone increased concomitantly, with a decrease in 20-hydroxyecdysone only at the end of the embryogenic period, while ecdysone and LPP levels remained low or undetectable. It may be postulated that the presence of ecdysteroids in ovaries and eggs represents a reserve of maternal ecdysteroids which are necessary at the commencement of embryonic development; with the differentiation of embryonic tissue capable of ecdysteroid synthesis, ecdysteroids increase rapidly to play a role in later embryonic development.
Mature males of the freshwater prawn, Macrobrachium rosenbergii (de Man), may change from one to another morphotype, according to a set sequence. Small males may develop into orange-claw males and orange-claw males into dominant blue-claw males. Each of the three morphotypes demonstrates distinctive reproductive behavior and secondary sexual characteristics. The role of the androgenic gland in this morphotypic transformation was examined experimentally by bilateral androgenic gland ablation (andrectomy) of small males and orange-claw males. For andrectomy initiated in the small male morphotype, transformation to the next morphotype was permitted (orange-claw), but subsequent transformation to the blue-claw morphotype was blocked. Andrectomy of orange-claw males did not prevent transformation into the blue-claw. Andrectomy on both small and orange-claw males caused disappearance of the genital papillae and atrophy of the sperm ducts and testes. The growth rates of the andrectomized small and orange-claw males were significantly lower than those of the unoperated and sham-operated controls. We conclude that androgenic gland factors control not only the differentiation of male secondary sexual characteristics but also morphotypic differentiation. Bioassays based on the results of this study will be instrumental in the characterization of such a factor(s).
Putative neuroendocrine mediation of osmotic and ionic responses to acute exposure to high salinity medium was investigated in the freshwater shrimp Macrobrachium olfersii (Wiegmann). Homogenates of supra-esophageal or thoracic ganglia, prepared from shrimps exposed to seawater of 21% S for 6 hr, were injected into the abdominal musculature of shrimps previously exposed to freshwater and subsequently exposed to either freshwater or seawater (21% S). Osmotic, sodium, chloride, potassium, magnesium, and calcium concentrations were determined in hemolymph samples removed by intracardiac puncture at time = 0, 1, 3, or 6 hr after homogenate application. Control shrimps were injected with filtered seawater, isosmotic to the hemolymph, and treated similarly. In control shrimps, the osmotic, Na+, Cl-, K+, Mg2+, and Ca2+ concentrations in the hemolymph increased (P less than or equal to 0.05) after 1-hr exposure to seawater. In shrimps injected with homogenates of supra-esophageal ganglion and exposed to seawater, osmotic and ionic concentrations in the hemolymph did not vary with exposure time; in injected shrimps exposed to freshwater, Na+, Cl-, K+, and Mg2+ concentrations decreased (P less than or equal to 0.05) with time. In shrimps injected with homogenates of thoracic ganglion and exposed to seawater, hemolymph osmotic, K+, and Mg2+ concentrations increased (P less than or equal to 0.05); Na+, Cl-, and Ca2+ concentrations remained unchanged. In injected shrimps exposed to freshwater, hemolymph osmotic concentration alone increased (P less than or equal to 0.05) after 1 hr, all other ionic concentrations remaining unchanged. These data suggest that neurofactors apparently located within the ganglia of the central nervous system of M. olfersii may alter the apparent ionic permeabilities of this shrimp, depending on the salinity characteristics of the external medium. The data support the notion that invasion of the freshwater biotope by estuarine crustaceans has necessitated the evolution of specific physiological mechanisms capable of compensating for the osmotic dilution and ion loss typically encountered by such organisms.
The presence of putative neurofactors within the central nervous system, i.e., the eyestalks (ES), ventral nerve cord (VNC), and supra-esophageal (SEG) and thoracic ganglia (TG), which are involved in osmotic and ionic regulation, was investigated in the euryhaline, freshwater shrimp, Macrobrachium olfersii. Homogenates were prepared from shrimps exposed for 6 hr to a high salinity medium (HSM, 21/1000 S) and were injected into shrimps subsequently maintained for 1, 3, or 6 hr in freshwater (FW, 0/1000 S) or HSM. Osmolality and sodium, chloride, and calcium concentrations were determined in single hemolymph samples removed at each time interval. Heart rates and wet weights were measured before and after experimental treatments. Exposure to HSM increased [Na+] and [Cl-] and heart rate. Injection of ES homogenate increased osmolality, [Na+] and [Cl-], and wet weight in shrimps maintained in FW; VNC homogenate also increased hemolymph [Cl-] in shrimps maintained in FW after injection, but reduced heart rate in shrimps subsequently exposed to HSM. Injection of TG homogenate reduced heart rate to a lesser extent in shrimps maintained in FW. Hemolymph [Ca2+] was not altered by homogenate injection. The exposure period of 6 hr to HSM appears to result in the accumulation of factors within the central nervous system that regulate the osmotic and ionic concentrations of the hemolymph, in addition to exerting antidiuretic and cardio-depressor actions. The coordinated action of these factors is intimately involved in the hyporegulatory processes that permit the survival of M. olfersii in media of elevated salinity.
Membrane preparation from the optic lobe of the giant freshwater prawn, Macrobrachium rosenbergii de Man, was examined for the presence of specific L-(3H) glutamate binding. The optic lobes were isolated from live animals. The tissue was homogenized and the membrane fraction isolated by differential centrifugation. The membrane suspension was incubated with 10-1,000 nM of L-(3H) glutamate at 37 degrees C for 60 min. Nonspecific binding was determined by incubating the mixture with 100 microM L-glutamate. L-(3H) glutamate specifically bound to the membrane fraction with a dissociation equilibrium constant (Kd) of 205 nM and maximum number of binding sites (Bmax) of 2.04 n mol/mg protein. By using LIGAND computerized program, the saturation isotherm binding pattern indicates a single type of binding. To determine the type of glutamate receptors, competitive inhibition and IC50 of several glutamate agonists and antagonists were determined. The study reveals a metabotropic type of binding site.
Giant freshwater prawns, Macrobrachium rosenbergii de Man, were reared under three different lighting conditions: continuous darkness (DD), 12 hr of light and 12 hr of darkness (LD 12:12) and continuous light (LL). After one month, the prawns were sacrificed and optic lobes isolated from the eyestalks were determined for N-acetyltransferase (NAT) activities and melatonin concentrations. Gonads were weighed and examined under light microscopy. The optic lobes from LL prawns contained significantly higher activities of NAT than those from LD 12:12 prawns. The melatonin concentrations and size and histological features of the gonads from the three groups of prawns did not differ. The results indicate that continuous light increases NAT activities in the optic lobe of M. rosenbergii but has no drastic effect on gonadal growth.
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Organophosphorus compounds and carbamates are pesticides which inhibit acetylcholinesterase activity. The toxicity of phosalone and carbaryl was studied for 29 days in the adult common prawn (Palaemon serratus). Induction thresholds for inhibitory effects were determined and the toxicity of these two pesticides was investigated. For monitoring purposes, several methods of assessing the effects of these two pesticides in the marine environment are considered and discussed.
1. Chemoreceptor cells in the first pereiopods (legs) of the freshwater prawn, Macrobrachium rosenbergii, were investigated using single-unit, extracellular electrophysiological recording techniques on an isolated, perfused leg preparation. 2. The cells were responsive to aqueous extracts of food (shrimp, mullet, trout chow), a salt mixture (artificial sea-water), amino acids (L-arginine HCl, taurine), a quaternary ammonium compound (betaine HCl) and ammonium chloride. 3. The response specificity of individual cells ranged from narrow to broad, but on average was broad, being more similar to chemoreceptor cells of freshwater crayfish than of marine spiny or clawed lobsters. 4. Responses were generally excitatory. However, some responses were inhibitory, the first such demonstration in aquatic crustaceans. 5. These electrophysiological results highly correlate with results of feeding behavioral assays carried out on M. rosenbergii.