Ultrastructural changes of the microvillar cytoskeleton in the photoreceptor of the crayfish Orconectes limosus related to different adaptation conditions

In the retina of crayfish microvilli of seven of the eight photoreceptor cells build highly organized structures, the rhabdoms. Cytoskeletal elements inside the microvilli were investigated in conventional and slightly extracted electron microscopical preparations. In conventional preparations the ultrastructure of these cytoskeletal elements depended on the adaptational state of the animal. They appeared as central filament-like structures inside each microvillus when dark-adapted retinae were prepared and fixed at night in the absence of calcium. Changes of these conditions (light, daytime, or calcium concentration) impaired the detectability of these central filaments; in light-adapted eyes prepared at midday they were rarely seen. Nevertheless, single microvillar filaments were present in light-adapted retinae after mild cell permeabilization with the saponin beta-escin. They appeared as a regular structure in each microvillus, often attached to the membrane. Their fine structure was consistent with the ultrastructure of single actin filaments as indicated by fast-Fourier-analysis and further supported by the presence of anti-actin immunoreactivity in electron microscopical and immunocytochemical preparations. These results indicate that microvillar filaments are not necessarily destroyed by light as previously described; we suggest that their appearance inside the microvillus might be altered by the properties of associated, maybe sidearm-like proteins.     

Spiny-cheek crayfish Orconectes limosus carry a novel genotype of the crayfish plague pathogen Aphanomyces astaci

The oomycete Aphanomyces astaci causes mass mortalities of European crayfish. Different species of North American crayfish, original hosts of this parasite, seem to carry different strains of A. astaci. So far, four distinct genotype groups have been recognised using Random Amplification of Polymorphic DNA (RAPD-PCR). We succeeded in isolating A. astaci from the spiny-cheek crayfish Orconectes limosus, a widespread invader in Europe, and confirmed that this species carries a novel A. astaci genotype. Improving knowledge on the diversity of this parasite may facilitate identification of genotypes in mass mortalities of European crayfish, thus tracing the sources of infection.     

The diet of the spiny-cheek crayfish Orconectes limosus in the Czech Republic

The composition of the diet of the invasive spiny-cheek crayfish Orconectes limosus was studied using qualitative and quantitative analyses of stomach contents. A total of 368 specimens collected in 2003–2005 and 2008 in Czech localities were examined, predominantly from the Labe (Elbe) and Vltava River basins. Food components were compared for three size classes of crayfish and both sexes. The following conclusions were reached: (1) the spiny-cheek crayfish is an omnivorous species consuming plants, animals and detritus; (2) quantitatively, the main food component of O. limosus is detritus, while the plant component was second; (3) O. limosus may swallow whole food particles up to 4 mm in size, and the bodies of small animals may sometimes be found undamaged in their stomachs.     

Orcokinin: a novel myotropic peptide from the nervous system of the crayfish, Orconectes limosus.

A myotropic peptide, named orcokinin, was isolated from approximately 1200 abdominal nerve cords of the crayfish, Orconectes limosus. Its amino acid sequence was determined as follows: Asn-Phe-Asp-Glu-Ile-Asp-Arg-Ser-Gly-Phe-Gly-Phe-Asn. This structure was confirmed by synthesis. There is no sequence similarity to any known neuropeptide. Orcokinin exhibits high potency on the crayfish hindgut, enhancing both frequency and amplitude of spontaneous contractions. The threshold of biological activity in vitro was determined to be approximately 5 x 10(-11) M.     

Characterization of a molt-inhibiting hormone (MIH) of the crayfish, Orconectes limosus, by cDNA cloning and mass spectrometric analysis

The structure of the precursor of a molt-inhibiting hormone (MIH) of the American crayfish, Orconectes limosus was determined by cloning of a cDNA based on RNA from the neurosecretory perikarya of the X-organ in the eyestalk ganglia. The open reading frame includes the complete precursor sequence, consisting of a signal peptide of 29, and the MIH sequence of 77 amino acids. In addition, the mature peptide was isolated by HPLC from the neurohemal sinus gland and analyzed by ESI-MS and MALDI-TOF-MS peptide mapping. This showed that the mature peptide (Mass 8664.29 Da) consists of only 75 amino acids, having Ala75-NH2 as C-terminus. Thus, C-terminal Arg77 of the precursor is removed during processing, and Gly76 serves as an amide donor. Sequence comparison confirms this peptide as a novel member of the large family, which includes crustacean hyperglycaemic hormone (CHH), MIH and gonad (vitellogenesis)-inhibiting hormone (GIH/VIH). The lack of a CPRP (CHH-precursor related peptide) in the hormone precursor, the size and specific sequence characteristics show that Orl MIH belongs to the MIH/GIH(VIH) subgroup of this larger family. Comparison with the MIH of Procambarus clarkii, the only other MIH that has thus far been identified in freshwater crayfish, shows extremely high sequence conservation. Both MIHs differ in only one amino acid residue ( approximately 99% identity), whereas the sequence identity to several other known MIHs is between 40 and 46%.     

Cross-species amplification of microsatellite markers in the invasive spiny-cheek crayfish (Orconectes limosus): Assessment and application

The North American spiny-cheek crayfish,Orconectes limosus (Cambaridae), endangered in its native range, is a widespread invasive species in European waters and conservationally important carrier of crayfish plague. However, its population structure is poorly known, and no informative genetic markers for the species are available. We tested cross-species transfer of microsatellite loci to spiny-cheek crayfish from 5 other crayfish species. Variability of 10 successfully amplifying loci derived from 4 species was then tested in 60 individuals ofO. limosus originating from 3 natural populations: the river Danube at Bogyiszló in Hungary, a pond in Stary Klíčov, and the brook Černovicky, both in the Czech Republic. The allele number within the populations ranged from 4 to 10 alleles per locus, while heterozygosity levels varied from 0.650 to 0.900 forHo and from 0.660 to 0.890 forHe. No linkage disequilibrium and no null alleles were detected. The selected markers are useful for assessing population structure, intraspecific variation, and paternity studies in spiny-cheek crayfish.     

Amino acid sequence of crustacean hyperglycemic hormone (CHH) from the crayfish, Orconectes limosus: emergence of a novel neuropeptide family.

The primary structure of the major form of CHH from sinus glands of the crayfish, Orconectes limosus, was determined by manual Edman microsequencing. It is a 72-residue peptide with a calculated Mr of 8400 Da. In the number of residues, it is identical to the CHH of Carcinus maenas and very similar to MIH (moult inhibiting hormone) of Homarus americanus. All three peptides have pGlu as N-terminus in common, and Val-NH2 is the C-terminal residue in Orconectes and Carcinus CHH. Six Cys residues occupy identical position in the three peptides. There is a 61% sequence identity with Carcinus CHH, and an 81% identity with Homarus MIH.     

High activity of NADP-dependent malic enzyme in mitochondria from abdomen muscle of the crayfish Orconectes limosus.

1. Mitochondria isolated from abdomen muscle of crayfish Orconectes limosus exhibit malic enzyme activity in the presence of L-malate, NADP and Mn2+ ions after addition of Triton X-100. Under optimal conditions about 230 nmole of reduced NADP and an equivalent amount of pyruvate are produced per min per mg of mitochondrial protein. 2. The pH optimum for decarboxylation of L-malate is about 7.5. 3. The apparent Km for L-malate, NADP and Mn2+ ions was found to be 0.66, 0.012, and 0.0025 mM, respectively. 4. The requirement for Mn2+ can be replaced by Mg2+, Co2+ and Ni2+ ions; however, higher concentrations of these ions than Mn2+ are required for a full stimulation of malic enzyme activity. 5. Oxaloacetate and pyruvate inhibited the enzyme activity in a competitive manner with apparent Ki values of 0.05 mM and 5.4 mM, respectively.     

Description of intracerebral ocelli in two species of North American crayfish: Orconectes limosus (Cambaridae) and Pacifastacus leniusculus (Astacidae)

Abstract. Among malacostracan crustaceans, intracerebral ocelli were first discovered in Isopoda, but they have been more recently reported from a crayfish ( Cherax destructor ) and a sandhopper ( Talitrus saltator ). This electron microscopic study increases the number of crayfish taxa in which intracerebral ocelli are now known to occur by two: Astacidae and Cambaridae. These photoreceptors are always integrated into the anteromedio-dorsal part of the brain and are not visible externally. Each ocellus is made up of 4–5 photoreceptor cells and is characterized by the presence of a fused rhabdom. The occurrence of different kinds of lysosomes in the cytoplasm is indicative of metabolic activity and perhaps membrane turnover. One typical feature of crayfish ocelli is their extraordinary variability in number. This trait is exemplified by individuals of Pacifastacus leniusculus , where as many as 14 ocelli were identified in a single brain. The arrangement of the ocelli is often not symmetrical with regard to the brain's midline and the ocelli always lack dioptric structures. Thus, it is difficult to see how they are involved in image formation. However, further research is needed to determine the precise role of these hidden receptors.     

Introductory study of an oocyte membrane protein that specifically binds vitellogenin in the crayfish Orconectes limosus

Summary

After solubilization of membranes from vitellogenic oocytes of the crayfish Orconectes limosus, a component with an apparent molecular weight between 28 and 30 kDa that specifically binds vitellogenin, could be identified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and by electroblotting. Because this component was pronase sensitive, we assume that we are dealing with a protein. A polyclonal rabbit antiserum was produced against this component and its tissue specificity was verified.     

Antioxidant defence enzyme activities in hepatopancreas, gills and muscle of Spiny cheek crayfish (Orconectes limosus) from the River Danube

The aim of our study was to determine the activity of antioxidant defence (AD) enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR) and the phase II biotransformation enzyme glutathione-S-transferase (GST) in the hepatopancreas, the gills and muscle of Spiny cheek crayfish (Orconectes limosus) from the River Danube and to compare tissue specificities of investigated enzymes. Our results indicated that both specific and total SOD activities in the hepatopancreas were lower compared to the gills and muscle. Total SOD activity in the gills was lower with respect to that in muscle. CAT and GSH-Px (both specific and total) activities were higher in the hepatopancreas compared to those in the gills and muscle. In the gills the specific and total GR activities were higher than in the hepatopancreas and muscle. The specific and total GST activities were higher in the hepatopancreas compared with the gills and muscle. Our study represents the first comprehensive report of AD enzymes in tissues of O. limosus caught in the River Danube. The noted tissue distributions of the investigated AD enzyme activities most likely reflected different metabolic activities and different responses to environmental conditions in the examined tissues.     

Structure, distribution, and biological activity of novel members of the allatostatin family in the crayfish Orconectes limosus.

In the central and peripheral nervous system of the crayfish, Orconectes limosus, neuropeptides immunoreactive to an antiserum against allatostatin I (= Dipstatin 7) of the cockroach Diploptera punctata have been detected by immunocytochemistry and a sensitive enzyme immunoassay. Abundant immunoreactivity occurs throughout the central nervous system in distinct interneurons and neurosecretory cells. The latter have terminals in well-known neurohemal organs, such as the sinus gland, the pericardial organs, and the perineural sheath of the ventral nerve cord. Nervous tissue extracts were separated by reverse-phase high-performance liquid chromatography and fractions were monitored in the enzyme immunoassay. Three of several immunopositive fractions have been purified and identified by mass spectroscopy and microsequencing as AGPYAFGL-NH2, SAGPYAFGL-NH2, and PRVYGFGL-NH2. The first peptide is identical to carcinustatin 8 previously identified in the crab Carcinus maenas. The others are novel and are designated orcostatin I and orcostatin II, respectively. All three peptides exert dramatic inhibitory effects on contractions of the crayfish hindgut. Carcinustatin 8 also inhibits induced contractions of the cockroach hindgut. Furthermore, this peptide reduces the cycle frequency of the pyloric rhythms generated by the stomatogastric nervous system of two decapod species in vitro. These crayfish allatostatin-like peptides are the first native crustacean peptides with demonstrated inhibitory actions on hindgut muscles and the pyloric rhythm of the stomatogastric ganglion.     

Dynamics of biosynthesis and release of crustacean hyperglycemic hormone isoforms in the X-organ-sinus gland complex of the crayfish Orconectes limosus.

The crustacean hyperglycemic hormone (CHH) is the major neuropeptide produced by the X-organ-sinus gland neurosecretory system of the crayfish, Orconectes limosus. This hormone is synthesized by two different cell types, as two isomers (CHH and D-Phe3-CHH) which display different activities The aim of this report is to analyze and compare the synthetic and secretory activities of these specialized cells. In vitro pulse-chase incubations and time-course experiments were conducted on isolated X-organ-sinus gland (XO-SG) complexes, followed by analysis of the labeled peptides. The different steps of the post-translational processing of the CHH precursor, including proteolytic cleavage of the propeptide, C-terminal amidation and N-terminal pyroglutamylation were characterized and the kinetics of CHHs maturation were estimated in the different parts of the neuroendocrine complex. Furthermore, synthesis of CHHs in XO-SG complexes and release in incubation media were investigated using combined HPLC/immunoassay. Under basal conditions, i.e. without stimulation, similar dynamics for both isomers were found and results indicate that newly synthesized CHHs are preferentially released.