An ultrastructural analysis of mature sperm from the crayfish Pacifastacus leniusculus,Dana

Sperm from the crayfish, Pacifastacus leniusculus, resemble other reptantian sperm in that they are composed of an acrosome, subacrosomal region, nucleus, membrane lamellar complex, and spikes which radiate from the nuclear compartment. The acrosome (PAS positive vesicle) can be subdivided into three regions: the apical cap, crystalline inner acrosomal material, and outer acrosomal material which is homogeneous except for a peripheral electron dense band. The nucleus contains uncondensed chromatin and bundles of microtubules which project into the spikes. The orientation of the microtubule bundles relative to the nuclear envelope near the base of the subacrosomal region suggests that the nuclear envelope may function in the organization of the spike microtubules.     

State-dependent responses of two motor systems in the crayfish,Pacifastacus leniusculus

The expression of both swimmeret and postural motor patterns in crayfish (Pacifastacus leniusculus) were affected by stimulation of a second root of a thoracic ganglion. The response of the swimmeret system depended on the state of the postural system. In most cases, the response of the swimmeret system outlasted the stimulus.Stimulation of a thoracic second root also elicited coordinated responses from the postural system, that outlasted the stimulus. In different preparations, either the flexor excitor motor neurones or the extensor excitor motor neurones were excited by this stimulation. In every case, excitation of one set of motor neurones was accompanied by inhibition of that group's functional antagonists.This stimulation seemed to coordinate the activity of both systems; when stimulation inhibited the flexor motor neurones, then the extensor motor neurones and the swimmeret system were excited. When stimulation excited the flexor motor neurones, then the extensor motor neurones and the swimmeret system were inhibited.Two classes of interneurones that responded to stimulation of a thoracic second root were encountered in the first abdominal ganglion. These interneurones could be the pathway that coordinates the response of the postural and swimmeret systems to stimulation of a thoracic second root.Abbreviations TSR thoracic second root - epsp excitatory post-synaptic potential - ipsp inhibitory post-synaptic potential - EJP excitatory jonctional potential - PS power-stroke - RS return-stroke - INT interneurone - N1 first segmental nerve - N2 second segmental nerve - N3 third segmental nerve - A1 abdominal ganglion 1     

Processing of an antibacterial peptide from hemocyanin of the freshwater crayfish Pacifastacus leniusculus

An antibacterial peptide with 16 amino acid residues was found in plasma of the freshwater crayfish, Pacifastacus leniusculus. This peptide, designated astacidin 1, was purified by cation-exchange column chromatography and reverse-phase high performance liquid chromatography. Astacidin 1 has a broad range of antibacterial activity, and it inhibits growth of both Gram-positive and Gram-negative bacteria. The primary sequence of astacidin 1 was FKVQNQHGQVVKIFHH-COOH. The molecular mass was 1945.2 Da, and no carbohydrate-linked amino acid residues could be found by mass spectrometry. A synthetic astacidin 1 resulted in similar activity as the authentic astacidin 1 against Gram-positive bacteria, whereas it had less or no activity against Gram-negative bacteria. Three amino-terminal-truncated synthetic peptides were made; they all showed low activity, suggesting that the amino-terminal part of astacidin 1 contributes to the antibacterial activity. The structure of astacidin 1 based on the CD results showed that it has a beta-sheet structure in citric acid buffer at pH 4, 6, and 8. Cloning of astacidin 1 shows that it is the carboxyl-terminal part of crayfish hemocyanin and that astacidin 1 is produced by a proteolytic cleavage from hemocyanin under acidic conditions. The processing and release of astacidin 1 from hemocyanin is enhanced when crayfish are injected with lipopolysaccharide or glucan.     

Purification and characterization of a beta-1,3-glucan binding protein from plasma of the crayfish Pacifastacus leniusculus

The plasma of the crayfish Pacifastacus leniusculus contains a protein which is able to bind to laminarin (a soluble beta-1,3-glucan) and which has been isolated by two independent methods, affinity precipitation with a beta-1,3-glucan or immunoaffinity chromatography. The purified beta-1,3-glucan binding protein was homogenous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is a monomeric glycoprotein with a molecular mass of approximately 100,000 Da and an isoelectric point of approximately 5.0. Amino acid analysis showed a very high similarity with the amino acid composition of beta-1,3-glucan binding proteins recently purified from two insects, the cockroach Blaberus craniifer and the silkworm Bombyx mori. The N-terminal amino acid sequence was determined to be: H2N-Asp-Ala-Gly-X-Ala-Ser-Leu-Val-Thr-Asn-Phe-Asn-Ser-Ala-Lys-Leu-X-X-Ly s--- Using monospecific rabbit polyclonal antibodies, the presence of this protein has also been shown within the blood cells. The purified beta-1,3-glucan binding protein did not show any peptidase or phenoloxidase activity but was able to enhance the activation of hemocyte-derived peptidase and prophenoloxidase only in the presence of the beta-1,3-glucan, laminarin, whereas mannan, dextran (alpha-glucan), or cellulose (beta-1,4-glucan) incubated with the beta-1,3-glucan binding protein had no effect on these enzyme activities. The beta-1,3-glucan binding protein could only be affinity-precipitated from crayfish plasma by the beta-1,3-glucans laminarin or curdlan (an insoluble beta-1,3-glucan), while mannan or dextran did not bind to the beta-1,3-glucan binding protein. No hemagglutinating activity of the purified beta-1,3-glucan binding protein could be detected.     

5-HT-like immunoreactivity in the central nervous system of the crayfish,Pacifastacus leniusculus

An immunocytochemical technique with the use of three different antibodies raised against serotonin was applied to localize the immunoreactive neurons in the central nervous system of the crayfish, Pacifastacus leniusculus. Immunoreactive neurons were found in three optic ganglia (medulla externa, interna and terminalis). They appeared in three layers of the medulla externa and interna. The medulla terminalis displayed three prominent groups of immunoreactive perikarya and mainly marginal immunoreactive fibres. Immunoreactive areas of the brain comprised the protocerebral bridge, central body, paracentral lobes and two loci in the anterior portion of the protocerebrum, i.e., the terminal areas for immunoreactive fibres from the optic centres. The olfactory lobes showed a specific immunoreactive pattern. In addition, diffusely and sparsely distributed immunoreactive fibres were found throughout the brain. The immunoreactive neurons are largely localized in the same areas of the central nervous system as the catecholaminergic neurons although some distinct differences occur.     

Movement and dispersal of the invasive signal crayfish Pacifastacus leniusculus in upland rivers

1. The American signal crayfish Pacifastacus leniusculus, an invasive species widely introduced throughout Europe, is a major threat to native European crayfish species and is causing increasing concern because of its wide impact on aquatic ecosystems. 2. Whilst various control and management methods have been proposed, very little is known about the factors influencing dispersal and movements of signal crayfish. 3. Sixty‐four adult signal crayfish (carapace length 31.9–63.8 mm) were radiotagged in upland rivers in northern England, during four periods. Tracking was carried out at two sites, a low‐density establishing population and a high‐density established population. Tracking was carried out at both sites concurrently during midsummer (June to August 2002), during late summer (August to September 2001) at the low‐density population site and during autumn to winter (October to February 2000/01) at the high‐density population site. 4. Maximum movement occurred during midsummer. Temperature appeared to be a major factor influencing the timing and extent of movements between tracking periods. 5. The frequency distribution of the maximum distance moved upstream and downstream by radiotagged crayfish showed an inverse power relationship. The median maximal upstream and downstream distances moved were 13.5 m (range 0–283 m) and 15 m (range 0–417 m), respectively. There was a significant difference between the distributions of upstream and downstream ranges, with greater distances moved downstream. 6. All downstream movements made by crayfish appeared to be active movements and not the result of passive movement during periods of high discharge. There was no apparent influence of size, sex or density on the amount of movement recorded. 7. The study provides important information on the spatial and temporal behaviour of introduced crayfish in upland lotic systems. In contrast to lowland rivers, our results suggest that flow or gradient may influence the invasive potential of signal crayfish in an upstream direction in upland rivers.     

Amino acid sequence of the tryptic peptide containing the alkylamine-reactive site from human alpha 2-macroglobulin. Identification of gamma-glutamylmethylamide

Human alpha 2-macroglobulin (alpha 2M) is inhibited by covalent reaction with alkylamines. The site of methylamine incorporation has been proposed to be an activated glutamyl residue (Swenson, R. P., and Howard, J. B. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4313-4316). A large, 56-amino acid residue glycopeptide derived from tryptic cleavage of [14C]methylamine-labeled alpha 2M was isolated. Based upon recovery of the specific radioactivity in the peptide, there appears to be only a single site of incorporation per Mr = 185,000 subunit. The complete amino acid sequence was deduced from Edman degradation and carboxypeptidase Y digestion of the tryptic peptide and of several small peptides derived from it. The structure of the radiolabeled amino acid was determined to be gamma-glutamylmethylamide by mass spectral analysis of the phenylthiohydantoin and N-benzoyl-O-methylester derivatives. The putative structure was confirmed by a comparison of the mass spectral and chromatographic properties of the authentic compound and the protein-derived amino acid residue. The 10 amino acid residues following the methylamine-reactive glutamyl residue were identical with the first 10 amino acid residues of the pyroglutaminase-deblocked, Mr = 65,000 fragment generated by heat denaturation of alpha 2M (Howard, J. B., Vermeulen, M., and Swenson, R. P. (1980) J. Biol. Chem. 255, 3820-3823).     

The behavioural responses of juvenile signal crayfish Pacifastacus leniusculus to stimuli from perch and eels

• 1 Experiments were designed to determine the relative importance of chemical and visual stimuli in eliciting predator avoidance behaviour in juvenile freshwater crayfish Pacifastacus leniusculus (Dana).
• 2 Crayfish placed in visual and/or chemical contact with one of two predators exhibited marked avoidance behaviour, spending less time walking and climbing and more time within shelters.
• 3 The combined effects of both visual and chemical stimuli increased crayfish shelter use and reduced walking and climbing activity to a greater degree than either stimulus when presented alone.
• 4 Crayfish exhibited avoidance behaviour in response to chemical stimuli during periods of light and darkness. Visual detection of predators elicited avoidance behaviour during the day.
• 5 It is suggested that the behavioural response of P. leniusculus to chemical stimuli reduces the likelihood of being detected by visual predators, and that chemical stimuli lower the response threshold for avoidance behaviour in crayfish reacting to visual stimuli. The adaptiviry of using chemical cues to detect predators is emphasized.

     

Characterization of a pattern recognition protein, a masquerade-like protein, in the freshwater crayfish Pacifastacus leniusculus

A multifunctional masquerade-like protein has been isolated, purified, and characterized from hemocytes of the freshwater crayfish, Pacifastacus leniusculus. It was isolated by its Escherichia coli binding property, and it binds to formaldehyde-treated Gram-negative bacteria as well as to yeast, Saccharomyces cerevisiae, whereas it does not bind to formaldehyde-fixed Gram-positive bacteria. The intact masquerade (mas)-like protein is present in crayfish hemocytes as a heterodimer composed of two subunits with molecular masses of 134 and 129 kDa. Under reducing conditions the molecular masses of the intact proteins are not changed. After binding to bacteria or yeast cell walls, the mas-like protein is processed by a proteolytic enzyme. The 134 kDa of the processed protein yields four subunits of 65, 47, 33, and 29 kDa, and the 129-kDa protein results in four subunits of 63, 47, 33, and 29 kDa in 10% SDS-PAGE under reducing conditions. The 33-kDa protein could be purified by immunoaffinity chromatography using an Ab to the C-terminal part of the mas-like protein. This subunit of the mas-like protein has cell adhesion activity, whereas the two intact proteins, 134 and 129 kDa, have binding activity to LPSs, glucans, Gram-negative bacteria, and yeast. E. coli coated with the mas-like protein were more rapidly cleared in crayfish than only E. coli, suggesting this protein is an opsonin. Therefore, the cell adhesion and opsonic activities of the mas-like protein suggest that it plays a role as an innate immune protein.     

Experimental infection of white spot syndrome virus in freshwater crayfish Pacifastacus leniusculus.

The signal freshwater crayfish Pacifastacus leniusculus was found to be susceptible to infection with white spot syndrome virus (WSSV). Histopathological observations of various tissues of virus-injected crayfish showed similar symptoms to those from WSSV-infected penaeid shrimp, but no appearance of white spots on the cuticle or reddish body colour were observed, although these are the prominent gross signs of white spot disease in shrimp. A gene probe for detecting WSSV was developed in order to detect the virus in affected cells and tissues using in situ hybridisation. Strong signals were observed in cells of virus-injected crayfish, but not in control-injected crayfish. The number of granular haemocytes in virus-injected crayfish was significantly higher than in sham-injected and non-injected crayfish from Days 5 to 8 (p < or = 0.05) and Days 3 to 8 (p < 0.01) post-injection, respectively. The proportion of granular haemocytes in virus-injected crayfish was also significantly higher than in sham-injected controls from Days 3 to 8 (p < 0.01). These results indicate that WSSV has a significant effect on the proportion of different haemocyte types in the freshwater crayfish.     

Amino acid sequence of crayfish troponin I

Troponin I is the actomyosin ATPase inhibitory subunit present in the thin filament regulatory complex. The complete amino acid sequence of crayfish tail muscle troponin I has been determined. The protein is composed of 201 amino acid residues and has a molecular weight of 23,547. The N terminus is blocked, likely by an acetyl group. Crayfish troponin I shows a rather low (20-25%) sequence identity with vertebrate troponin Is as compared to the 60-82% identity within the vertebrate phylum. Similar to vertebrate cardiac troponin I, crayfish troponin I contains a 30-residue-long N-terminal extension. In crayfish troponin I, this segment bears significant sequence homology with the heavy or light chains of particular myosins. The actin-binding domain of crayfish troponin I, which displays 57% sequence homology with vertebrate troponin Is, possesses 2 unusual trimethyllysine residues. The consensus sequence of this domain in five troponin Is is as follows: D-L-R-G-K-F-X-R*-P-X-L-R*-R*-V, where R+ stands for Arg/Lys, R* for Arg/trimethyllysine, and X for any amino acid residue. Troponin I possesses two Ca2+-dependent interactive sites for troponin C; one partly overlaps with the actin binding domain and is highly conserved, and the other, corresponding to the 30-residue-long segment following the N-terminal extension in vertebrate cardiac and crayfish troponin I, is poorly conserved in the different troponin Is. Troponin I also interacts with troponin T. The consensus sequence for the interacting site on troponin I is as follows: h-D- -X-D- -R+-Y-D-h-E-h, where h stands for a hydrophobic residue, D- for Asp/Glu, R+ for Arg/Lys, and X for any residue. The five troponin Is further possess one more 15-residue-long segment of high sequence identity near the C terminus. Its evolutionary conservation suggests that this domain is involved in protein-protein interaction.     

A cell adhesion protein from the crayfish Pacifastacus leniusculus, a serine proteinase homologue similar to Drosophila masquerade

A cDNA encoding a protein resembling masquerade, a serine proteinase homologue expressed during embryogenesis, larval, and pupal development in Drosophila melanogaster, was identified in hemocytes of the adult freshwater crayfish, Pacifastacus leniusculus. The crayfish protein is similar to Drosophila masquerade in the following aspects: (a) overall sequence of the serine proteinase domain, such as the position of three putative disulfide bridges, glycine in the place of the catalytic serine residue, and the presence of a substrate-lining pocket typical for trypsins; (b) the presence of several copies of a disulfide-knotted motif in the putative propeptide. This masquerade-like protein is cleaved into a 27-kDa fragment, which could be detected by immunoblot analysis using an affinity-purified antibody against a synthetic peptide in the C-terminal domain of the protein. The 27-kDa protein could be immunoaffinity-purified from hemocyte lysate supernatant and exhibited cell adhesion activity in vitro, indicating that the C-terminal domain of the crayfish masquerade-like protein mediates cell adhesion.     

Diurnal variations in physiology and behaviour of the noble crayfish Astacus astacus and the signal crayfish Pacifastacus leniusculus

Heart rate, locomotor activity, and oxygen consumption were recorded simultaneously and continuously in seven individuals of the noble crayfish Astacus astacus (Linneus 1758) and seven individuals of the signal crayfish Pacifastacus leniusculus (Dana 1852). The recordings were made in the laboratory over 7 days at 15°C under a 12 : 12 h dark : light regime. Circadian rhythms in heart rate, locomotor activity and oxygen consumption were found both in A. astacus and P. leniusculus. Increased heart rate, locomotor activity, and oxygen consumption levels during night time in both A. astacus and P. leniusculus illustrated expression of nocturnal behaviour. No differences in oxygen consumption levels were observed between A. astacus and P. leniusculus. Also, no significant difference between heart rate levels or heart rate variances was found in A. astacus and P. leniusculus at night. During day, however, heart rate levels, heart rate variances and locomotor activity were higher in P. leniusculus than in A. astacus. The higher activity level in P. leniusculus than in A. astacus during daytime indicates that P. leniusculus is less strictly nocturnal than is A. astacus.     

Bacteria-Induced Dscam Isoforms of the Crustacean, Pacifastacus leniusculus

The Down syndrome cell adhesion molecule, also known as Dscam, is a member of the immunoglobulin super family. Dscam plays an essential function in neuronal wiring and appears to be involved in innate immune reactions in insects. The deduced amino acid sequence of Dscam in the crustacean Pacifastacus leniusculus (PlDscam), encodes 9(Ig)-4(FNIII)-(Ig)-2(FNIII)-TM and it has variable regions in the N-terminal half of Ig2 and Ig3 and the complete Ig7 and in the transmembrane domain. The cytoplasmic tail can generate multiple isoforms. PlDscam can generate more than 22,000 different unique isoforms. Bacteria and LPS injection enhanced the expression of PlDscam, but no response in expression occurred after a white spot syndrome virus (WSSV) infection or injection with peptidoglycans. Furthermore, PlDscam silencing did not have any effect on the replication of the WSSV. Bacterial specific isoforms of PlDscam were shown to have a specific binding property to each tested bacteria, E. coli or S. aureus. The bacteria specific isoforms of PlDscam were shown to be associated with bacterial clearance and phagocytosis in crayfish.     

Tyrosine hydroxylase activity in the central nervous system of the crayfish,Pacifastacus leniusculus (Crustacea,Decapoda)

Summary Tyrosine hydroxylase, responsible for the formation ofl-dopa froml-tyrosine, has been identified in the central nervous system of the crayfish,Pacifastacus leniusculus (Crustacea, Decapoda). It requires pterine as cofactor and is inhibited by a number of known tyrosine hydroxylase inhibitors; iron-chelators, tyrosine analogues and also by the catecholamines, dopamine and noradrenaline. Iron enhances the activity of the enzyme. It differs from the vertebrate tyrosine hydroxylase in having a more alkaline pH optimum and a higher affinity for the pterine cofactor. Kinetic studies were performed andK _m andV _max values are presented. Dopa formed was identified and quantitatively measured by high pressure liquid chromatography (HPLC) and electrochemical detection.     

Amino acid sequence of a unique protease from the crayfish Astacus fluviatilis

The amino acid sequence of a protease from the crayfish Astacus fluviatilis has been determined from overlapping sets of peptides derived largely by cleavage at Met, Lys, or Arg residues. The protein comprises 200 amino acid residues in a single polypeptide chain, corresponding to a molecular mass of 22,614 daltons. Two disulfide bonds link Cys-42 to Cys-198 and Cys-64 to Cys-84. The sequence of this invertebrate protease appears to be unique since it has no homologous relationship to any of the known protein sequences.     

Ammonia uptake and its effects on ionoregulation in the freshwater crayfish Pacifastacus leniusculus (Dana)

Exposure of adult crayfish Pacifastacus leniusculus to Artificial Freshwater (AFW) media containing 1.5 m and 0.15 mmol x l(-1) total ammonia [Tamm; 0.1 x acute lethal concentration (24 h LC50) and 0.01 x 24 h LC50] and adjusted to pH 6.5, pH 8.2 and pH 10.5 resulted in significant increases in haemolymph ammonia over a 24-h period. Ammonia accumulated most rapidly at pH 10.5. These media were chosen to expose animals to a range of different un-ionised ammonia (UIA) [NH3] and ionised ammonia [NH4+] concentrations. From comparisons of measured transepithelial potential differences (PDte) with calculated Nernst potentials (PDNH4+) for the known haemolymph-to-medium gradients of [NH4+], it was deduced that, in pH 8.2 and pH 6.5 AFW, NH4+ was not in thermodynamic equilibrium across the integument (presumably gill epithelium). In pH 10.5 AFW with 1.5 mmol x l(-1) Tamm (predominantly NH3), the accumulation of ammonia in the haemolymph was in the NH4+ form due to haemolymph pH regulation by the crayfish in this alkaline external medium. Measured net fluxes of ammonia (Jamm(net)) were inwardly directed and maximal when [NH3] was the main component externally, but were also significant at pH 8.2 with high [NH4+] ([NH4+]:[NH3] approximately 20:1). Haemolymph Na+ depletion was significant and, over the 24-h exposure period, most rapid in high [NH3] medium but [Cl-] was unaffected. However, paradoxically, sodium uptake (measured JNa(in) on immediate transfer to high Tamm medium) was not significantly inhibited when [NH3] was the predominant ammonia species. In 1.5 mmol x l(-1) Tamm (mainly [NH4+), VNa(in) (the active component of JNa(in)) was significantly inhibited, particularly at low external [Na+]. This inhibition could not be demonstrated as one of competition at an Na+/NH4+ apical gill exchange site. The resultant net efflux of sodium from the animal showed that the ability of the animals to balance sodium losses at low external [Na+] was severely affected. Longer exposure to pH 10.5 AFW with high [NH3] (12 h) resulted in significantly increased JNa(out), while not significantly affecting JNa(in). Analysis of urinary Na+ losses showed that, while urinary flow rate and water reabsorption was most likely unaffected by ammonia exposure, final urine [Na+] was significantly elevated. The resulting urinary Na+ loss accounted for 63% of the increased JNa(out) in high [NH3] medium.     

The first report on the occurrence of twins in a freshwater crayfish,Pacifastacus leniusculus (Decapoda,Astacoidea)

Twin stage 1 juveniles from the signal crayfish, Pacifastacus leniusculus, were observed in this study. No such observations have been found in the literature. The twins consist of two stage 1 juveniles that are fused at the head. Each juvenile has a separate abdomen and thorax and has the appropriate number of appendages and a telson, all characteristic of a normal stage 1 juvenile.     

Amino acid sequence homology between the C3 chain of rat prostatic steroid binding protein and human alpha 2-macroglobulin

Using 32P-labeled DNA complementary to mouse submaxillary gland renin mRNA, we probed mRNA gel blots from mouse testis and kidney tissues. Poly(A)-RNA from testis contained a hybridizable RNA species which was blotted onto nitrocellulose paper. The molecular size of testicular renin mRNA (approximately 1600 nucleotides in length) was not significantly different from tht of kidney renin mRNA. Densitometric scan revealed that the content of renin mRNA in mouse testis was approximately 5-fold lower than that in mouse kidney. These results support the proposal that mouse testicular cells synthesize renin.