FMRFamide-like immunoreactivity in the crayfish nervous system.

FMRFamide-like immunoreactivity (FLI) was detected in the nervous system of the crayfish Procambarus clarkii using an antiserum that recognizes extended RFamide peptides. Immunocytochemistry revealed FLI in neuronal somata, axons and varicose processes within the central nervous system. In the periphery, plexuses of immunoreactive varicosities were present in the pericardial organs (POs), in thoracic roots and on the hindgut. The hindgut plexus arose from 3-5 axons leaving the sixth abdominal ganglion (A6) via the intestinal nerve. The presence of FLI in these locations was confirmed by radioimmunoassay. In contrast, no FLI was detected in motor axons innervating exoskeletal muscles of the abdomen. The POs contained by far the largest amount of FLI of all tissues examined. The immunoreactive material was partially characterized by extraction and separation on two consecutive reversed-phase high performance liquid chromatography (RP-HPLC) columns. The largest amount of immunoreactivity on the second column co-eluted with a synthetic peptide, SDRNFLRFamide (F2), previously identified as one of two or more FMRFamide-related peptides contained in lobster POs. The immunoreactive fractions and peptide F2 elicited similar effects on isolated crayfish hearts; all increased the rate and amplitude of spontaneous cardiac contractions. As with the immunoreactivity, the highest level of bioactivity was contained in the fraction that co-eluted with F2. The results suggest that FMRFamide-related peptides act as neurohormones in crayfish and are likely to play roles in controlling circulation and defecation.

Lead, cadmium, and aluminum accumulation in the red swamp crayfish Procambarus clarkii G. collected from roadside drainage ditches in Louisiana.

The concentration of Pb, Cd, and Al in tissues of crayfish Procambarus clarkii were evaluated from several wetland sites located adjacent to roadways and were compared to crayfish harvested from a commercial site free from roadside influences. Abdominal muscle, hepatopancreas, alimentary tract, exoskeleton and blood were analyzed for metal content. Results indicated that levels of contamination obtained in almost all tissues of crayfish from roadside ditches contained significantly higher amounts of metals than those of the commercially harvested control crayfish (p = less than or equal to .05-.001). Detection limits of Pb, Cd, and Al ranged from 0.04 microgram Pb/g to 16.15 micrograms Pb/g, .001 microgram Cd/g to .13 microgram Cd/g, and 1.22 micrograms Al/g to 981 micrograms Al/g, respectively. Concentrations of Pb, Cd, and Al were highest in the hepatopancreas and alimentary tract. High levels of these elements were also detected in the exoskeleton. In contrast, muscle tissue was the least affected tissue. Several significant correlations among concentrations of metals were found when comparing a variety of tissues in Procambarus clarkii.

Anti-GABA antibodies label a subpopulation of chemical synapses which modulate an electrical synapse in crayfish.

Antibodies raised against gamma-aminobutyric acid (GABA) were used to stain sections from the crayfish abdominal nervous system, and the sections were examined under the electron microscope using a protein-A/gold conjugate secondary label. Sections were taken through the third ganglionic root, and through the interganglionic connective at the base of the third root posterior to the ganglia. The third root contains two very large motor axons, a non-GABAergic excitor (Motor Giant; MoG), and a GABAergic inhibitor (Flexor Inhibitor; FI). Only one of the two large axons stained positively for GABA, confirming that the antibody has high specificity for GABAergic neurones. The MoG is driven by powerful electrical synapses from the giant fibres, but also receives inhibitory chemical synaptic input which can gate the excitatory input. There is no physiological evidence for any other form of chemical input. However, at the ultrastructural level, the MoG is postsynaptic to three types of chemical profiles; SE-type containing round agranular vesicles, SI-type containing pleomorphic vesicles, and SM-type containing a mixture of round agranular and dense-cored vesicles. There is a highly differentiated staining pattern of these three synaptic types. Only the SI-type profiles stain positively with the GABA antibody, while the SE- and SM-type do not show significant staining. This suggests that the MoG can under some circumstances receive chemical input other than GABAergic inhibitory input. These other types of input have yet to be physiologically identified.

Effect of environs and seasonality on metal residues in tissues of wild and pond-raised crayfish in southern Louisiana.

Two commercially important species of Louisiana crayfish, Procambarus clarkii (Girard) and P. acutus acutus (Girard), from the Atchafalaya River Basin, from open ponds and from the sediment and water of these environs were sampled three times during two consecutive fishing (production) seasons. The abdominal muscle and hepatopancreatic tissue were analyzed separately. Lead, mercury, and cadmium, if present, were in concentrations below the detection limit. In the hepatopancreatic tissue, barium was present in concentrations below 8 mg/kg, copper 11-15 mg/kg, and iron below 640 mg/kg. Abdominal muscle samples had less than 3 mg/kg of most metals. Locations with the highest levels of metal residues in sediment were not necessarily locations where crayfish had the highest levels in their tissues.

Spectral properties of porphyropsin from an invertebrate.

Winter crayfish (Procambarus clarkii) contain both retinal and 3-dehydroretinal, as first described by Suzuki, Makino-Tasaka and Eguchi (1984). Using the detergent L-1695 we have extracted visual pigments from the rhabdoms of crayfish and have characterized spectrally both rhodopsin (P1) and porphyropsin (P2). Both P1 and P2 are converted by light to relatively stable meta-pigments (M1 and M2). We here show a method for estimating the absorbance spectra of all four pigment species. The spectra of P533(1) and M510(1) agree with previous microspectrophotometric measurements on isolated rhabdoms. P567(2) and M537(2) represent the first 3-dehydroretinal-based visual pigment system to be characterized from an arthropod.

An outbreak of type E botulism among common loons (Gavia immer) in Michigan's upper peninsula.

An epizootic of type E botulism (Clostridium botulinum) occurred among common loons (Gavia immer) along the Lake Michigan shore of Michigan's Upper Peninsula (USA) during October and November 1983. An estimated 592 dead loons washed ashore along the Garden Peninsula. Type E botulinal toxin was demonstrated in blood samples and stomach contents of dead loons, and in samples of three species of dead fish found on the Lake Michigan shore. We suspect that loons acquired botulism by ingesting sick or dead fish containing type E toxin.

Isolation and purification of a cell adhesion factor from crayfish blood cells.

Isolated granular haemocytes (blood cells) from the crayfish Pacifastacus leniusculus attached and spread in vitro on coverslips coated with a lysate of crayfish haemocytes. No cell adhesion activity was detected in crayfish plasma. The cell adhesion activity was only present in haemocyte lysates in which the prophenoloxidase (proPO) activating system (Söderhäll and Smith, 1986a, b) had been activated; either by lipopolysaccharide (LPS), the beta-1,3-glucan laminarin, or by preparing the lysate in 5 mM Ca2+. Both lysates of granular or of semigranular haemocytes could mediate adhesion. After A23187-induced exocytosis of the granular cells, cell adhesion activity could be generated in the secreted material if it was incubated with laminarin. The factor responsible for cell adhesion was isolated from an active haemocyte lysate and purified by ammonium sulfate precipitation, cation exchange chromatography and Con A-Sepharose; it had a molecular mass of approximately 76 kD on an SDS-polyacrylamide gel. An antibody to this 76-kD band inhibited cell adhesion. Ca2+ was necessary in the medium for the cells to adhere to the adhesion factor. With cyanide or azide, the cells attached but failed to spread. It is suggested that in vivo the cell adhesion factor is stored in the secretory granules of the semigranular and the granular cells in a putative inactive pro-form, which can be released during exocytosis and, in the presence of beta-1,3-glucans or LPS, be activated outside the cells to mediate cell attachment and spreading, processes of essential importance in arthropod host defense.

Identification of troponin-I of crayfish myofibrils.

Crayfish (Procambarus clarkii) myofibrils contain two basic proteins of molecular weights of 25,000 and 23,000. Both of the two proteins inhibit actomyosin ATPase as the vertebrate troponin-I does. These results differ from the previous one that troponin-I of crayfish (Astacus leptodactylus) showed a single band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE).

A neurosecretory hyperglycemic hormone from the sinus gland of the Mexican crayfish Procambarus bouvieri (Ortmann)--II. Structural comparison of two isoforms of the hormone.

1. The hyperglycemic activity of a crude extract of Procambarus bouvieri (Ortmann) sinus glands was resolved into two UV-absorbing peaks by means of a single step of reverse-phase high performance liquid chromatography (RP-HPLC) on a mu-Bondapak-Phenyl column. These peaks have been designated Crustacean Hyperglycemic Hormones CHH-B and CHH-C in the order of elution. 2. The ratio CHH-B/CHH-C was approximately 3:1, both in area under the curve and in protein content. 3. A structural comparison of the two isoforms of the CHH showed a substantial homology manifested in molecular weight (6000-6200), pI (4.79), number of residues (52-53), number of cysteines (4), number of acid residues, including their amides (12), number of basic residues (8), missing amino acids (methionine, histidine and tryptophane), amino end (blocked) and carboxyl end (isoleucine). 4. The only clear difference between the two isoforms of the CHH is their degree of hydrophobicity which might be due to minor differences in the number of neutral hydrophobic residues and/or postranslational modifications of the type amidation/deamidation of acid residues which cannot be detected in acid hydrolysates.

Biochemical analyses of the crustacean hyperglycemic hormone of the crayfish Astacus leptodactylus.

A biochemical analysis was made of the crustacean hyperglycemic hormone (CHH) of the crayfish Astacus leptodactylus, as present in the CHH-producing perikarya, in the axonal tract and in the sinus gland, respectively. Hyperglycemic material was analyzed by polyacrylamide gel electrophoresis (PAGE and SDS-PAGE) and high-pressure liquid chromatography (HPLC) in combination with a dotting immunobinding assay (DIA) and a bioassay for hyperglycemic activity. After electrophoretic analyses, the predominant biologically as well as immunologically detectable product present in all parts of the cell has an apparent molecular radius of approximately 7000 Da. In the perikarya extract, a second factor with lower electrophoretic mobility was found, which may represent the prohormone or precursor of CHH. The analyses by means of HPLC showed two predominant immunopositive peaks with an elution time of 28-29 and 52-54 min, respectively. For both HPLC peaks, electrophoretic analyses indicate a molecular weight of 7000 Da.

[Effect of temperature on the concentration of Ca2+, Mg2+, Na+ and K+ in the hemolymph and urine of Procambarus clarkii, Girard]. / Influencia de la temperatura sobre la concentración de Ca2+, Mg2+, Na+ y K+ en la hemolinfa y en la orina de Procambarus clarkii, Girard.

The concentration variability of Ca, Mg, Na, K ions in the haemolymph and urine has been analyzed in Procambarus clarkii during interecdysis instar, the animals having been kept under several temperature conditions (10, 15, 20, 25, 30 degrees C) during two different periods of time (48 h and 7 days). The environmental temperature did not affect the concentration of sodium and calcium in the haemolymph. Nevertheless the above parameter had an effect on the concentration of potassium and magnesium in the haemolymph as well as on the concentration of the four cations considered in the urine of Procambarus clarkii. No significant differences have been found in relation to the time of exposure.

Axonal microtubules of crayfish and spiny lobster nerve cords are decorated with a heat-stable protein of high molecular weight.

Axons of crayfish and spiny lobster ventral nerve cords contain large numbers of microtubules that are decorated with fine filaments. These microtubules can be stabilized in permeabilized axons using buffers that contain either polyethylene glycol or glycerol/dimethyl sulphoxide. In the former, the stabilized microtubules retain their filaments and their normal spacing; in the latter, the filaments are stripped off and the bare microtubules collapse onto one another. This observation has been used as the basis for a method of identifying some of the proteins that make up the filaments. Axons are first permeabilized and stabilized in either buffer and then treated with a microtubule-depolymerizing buffer. The axons treated first with polyethylene glycol release tubulin and significant quantities of microtubule-associated proteins (MAPs), while the axons pre-treated with glycerol release tubulin and only traces of associated proteins. One of the proteins released in largest quantity along with tubulin from polyethylene glycol-treated axons is a high molecular weight, heat-stable MAP that co-electrophoreses with MAP-2 from mammalian brain. This same protein co-purifies with tubulin that is obtained from crayfish nerve cords by two cycles of polymerization and depolymerization. It is concluded that this protein is a component of the filaments that decorate the axonal microtubules of the crayfish and spiny lobster.

3-Dehydroretinal (vitamin A2 aldehyde) in crayfish eye.

11-Cis-3-dehydroretinal was found in the eye of crayfish, Procambarus clarkii. The 11-cis-3-dehydroretinal was isomerized to all-trans isomer by light-illumination, as was also 11-cis-retinal. Irradiation with deep-red light (lambda greater than 680 nm) selectively isomerized the 11-cis-3-dehydroretinal. The 3-dehydroretinal was not extracted with petroleum ether from the tissue after freeze-drying. These facts suggest that the 11-cis-3-dehydroretinal is the chromophore of crayfish visual pigment. The 3-dehydroretinal content varied with season, high level in winter and low in summer. The crayfish may have a vitamin A1-A2 visual pigment system similar to those of freshwater fishes and amphibians.

Isolation and purification of a neurodepressing hormone from the eyestalk of Procambarus bouvieri (Ortmann).

1. A neurodepressing hormone has been isolated and purified to homogeneity from aqueous extracts of 2000 eyestalks of the Mexican crayfish Procambarus bouvieri (Ortmann). 2. Purification was achieved by gel filtration on Sephadex G-25 and G-15, and preparative paper electrophoresis at four pH valueimately 1200 molecular weight and composed of neutral amino acids. 5. No N-terminal group could be found. From its electrophoretic behavior it is concluded that the C-terminal group is also blocked.