Two novel ficolin-like proteins act as pattern recognition receptors for invading pathogens in the freshwater crayfish Pacifastacus leniusculus

To isolate pathogen-associated molecular patterns (PAMPs)-binding molecules, the bacterium, Staphylococcus aureus was used as an affinity matrix to find bacteria-binding proteins in the plasma of the freshwater crayfish, Pacifastacus leniusculus. Two new bacteria-binding ficolin-like proteins (FLPs) were identified by 2-DE and MS analysis. The FLPs have a fibrinogen-related domain (FReD) in their C-terminal and a repeat region in their N-terminal regions with putative structural similarities to the collagen-like domain of vertebrate ficolins and mannose binding lectins (MBLs). Phylogenetic analysis shows that the newly isolated crayfish FLP1 and FLP2 cluster separately from other FReD-containing proteins. A tissue distribution study showed that the mRNA expression of FLP occurred mainly in the hematopoietic tissue (Hpt) and in the hepatopancreas. Recombinant FLPs exhibited agglutination activity of Gram-negative bacteria Escherichia coli and Aeromonas hydrophila in the presence of Ca(2+) . The FLPs could bind to A. hydrophila, E. coli as well as S. aureus as judged by bacteria adsorption. Moreover, the FLPs may help crayfish to clear Gram-negative bacteria, but not Gram-positive bacteria which had been injected into the hemolymph. When Gram-negative bacteria coated with FLPs were incubated with Hpt cells, a lower death rate of the cells was found compared with control treatment. Our results suggest that FLPs function as pattern recognition receptors in the immune response of crayfish.     

Properties of the prophenoloxidase activating enzyme of the freshwater crayfish, Pacifastacus leniusculus.

The prophenoloxidase activating enzyme (ppA), a serine proteinase catalyzing the conversion of prophenoloxidase to an active phenoloxidase, has a molecular mass of about 36 kDa in its active form. This protein was cloned from a blood cell cDNA library and its corresponding cDNA of 1736 base pairs encodes a zymogenic protein (proppA) of 468 amino acids. An antibody raised against a synthetic peptide derived from a region of the cDNA sequence could efficiently inhibit the beta-1,3-glucan triggered activation of prophenoloxidase in vitro. The C-terminal half of the proppA is composed of a typical serine proteinase domain, with a sequence similar to other invertebrate and vertebrate serine proteinases. The N-terminal half contains a cationic glycine-rich domain, a cationic proline-rich domain and a clip-domain, in which the disulfide-bonding pattern is likely to be identical to those of the horseshoe crab big defensin and mammalian beta-defensins. Antibodies made against both the C- and the N-terminal halves recognize two proppAs under reducing conditions. However, under nonreducing conditions only the anti-C antibody recognized the two proppAs, which suggests that a conformational change takes place upon reduction that allows the anti-N to react with the N-terminal half of proppA. The recombinant clip-domain in crayfish proppA was overexpressed in Escherichia coli and the resulting peptide exhibited antibacterial activity against Gram-positive bacterial strains such as Micrococcus luteus Ml11 and Bacillus megaterium Bm11 with 50% growth inhibitory concentrations of 1.43 microM and 17.9 microM, respectively. These results suggest that the clip-domains in proppAs may function as antibacterial peptides.     

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.     

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.     

Color variation in signal crayfish Pacifastacus leniusculus

External coloration in animals depends on the interaction of several different factors including the genetics and epigenetics processes that underlie the color expression,the mechanisms of color perception,and the general mechanisms controlling color evolution and function.Among all,camouflages from predators and conspicuousness are of particular interest because pose animal to choose between opposite adjustment in coloration.The external coloration of crustaceans is mainly due to the accumulation of carotenoids in the exoskeleton and the epidermal layer,and the trade-off between camouflage and communication had led to a variety of responses,involving signal partitioning,spectral sensibility,changing coloration,or signaling behavior.Here,we used digital images to explore intrapopulation variability of the external coloration of Pacifastacus leniusculus among body regions within an individual and between sexes.We found that 1)ventral coloration of claws are more saturated and brilliant than upper parts,2)males express a more saturated and brightness coloration than females,especially on the lower portion of claws,3)color intensity and brightness increases with size differently in different body regions,and 4)brightness is more variable in males than in females.All the above patterns support the hypothesis that color in this species could be the result of a compromise between camouflage from predators and conspicuousness for communication.The results of this study suggest that carotenoid might have something to do with intraspecific communication and perform more complex functions than that of a simple pigment.     

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.     

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.     

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     

A lipopolysaccharide- and beta-1,3-glucan-binding protein from hemocytes of the freshwater crayfish Pacifastacus leniusculus. Purification, characterization, and cDNA cloning

A lipopolysaccharide- and beta-1,3-glucan-binding protein (LGBP) was isolated and characterized from blood cells (hemocytes) of the freshwater crayfish Pacifastacus leniusculus. The LGBP was purified by chromatography on Blue-Sepharose and phenyl-Sepharose, followed by Sephacryl S-200. The LGBP has a molecular mass of 36 kDa and 40 kDa on 10% SDS-polyacrylamide gel electrophoresis under reducing and nonreducing conditions, respectively. The calculated mass of LGBP is 39,492 Da, which corresponds to the native size of LGBP; the estimated pI of the mature LGBP is 5.80. LGBP has binding activity to lipopolysaccharides as well as to beta-1,3-glucans such as laminarin and curdlan, but peptidoglycan could not bind to LGBP. Cloning and sequencing of LGBP showed significant homology with several putative Gram-negative bacteria-binding proteins and beta-1, 3-glucanases. Interestingly, LGBP also has a structure and functions similar to those of the coelomic cytolytic factor-1, a lipopolysaccharide- and glucan-binding protein from the earthworm Eisenia foetida. To evaluate the involvement of LGBP in the prophenoloxidase (proPO) activating system, a polyclonal antibody against LGBP was made and used for the inhibition of phenoloxidase (PO) activity triggered by the beta-1,3-glucan laminarin in the hemocyte lysate of crayfish. The PO activity was blocked completely by the anti-LGBP antibody. Moreover, the PO activity could be recovered by the addition of purified LGBP. These results suggest that the 36-kDa LGBP plays a role in the activation of the proPO activating system in crayfish and thus seems to play an important role in the innate immune system of crayfish.     

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.     

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.     

Over-invasion in a freshwater ecosystem: newly introduced virile crayfish (Orconectes virilis) outcompete established invasive signal crayfish (Pacifastacus leniusculus)

Biological invasions are a key threat to freshwater biodiversity, and identifying determinants of invasion success is a global conservation priority. The establishment of introduced species is predicted to be hindered by pre-existing, functionally similar invasive species. Over a five-year period we, however, find that in the River Lee (UK), recently introduced non-native virile crayfish (Orconectes virilis) increased in range and abundance, despite the presence of established alien signal crayfish (Pacifastacus leniusculus). In regions of sympatry, virile crayfish had a detrimental effect on signal crayfish abundance but not vice versa. Competition experiments revealed that virile crayfish were more aggressive than signal crayfish and outcompeted them for shelter. Together, these results provide early evidence for the potential over-invasion of signal crayfish by competitively dominant virile crayfish. Based on our results and the limited distribution of virile crayfish in Europe, we recommend that efforts to contain them within the Lee catchment be implemented immediately.     

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.     

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.     

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.     

Fluoride bioaccumulation in the signal crayfish Pacifastacus leniusculus (Dana) as suitable bioindicator of fluoride pollution in freshwater ecosystems

Fluoride bioaccumulation in the signal crayfish Pacifastacus leniusculus (Dana) was examined through field and laboratory studies, considering in the latter the effects of several biotic and abiotic factors: water chloride content, developmental stage, sex, and tissue. The potential use of P. leniusculus as bioindicator of fluoride pollution in freshwater ecosystems was also assessed by testing the capability of juvenile crayfish to release fluoride during depuration periods. After discussing the obtained results, we concluded that: (1) fluoride pollution by industrial effluents may significantly increase the fluoride content in signal crayfish inhabiting polluted freshwater ecosystems; (2) although high chloride levels in the aquatic medium may significantly reduce the fluoride content in signal crayfish exposed to fluoride pollution conditions, fluoride bioaccumulation can still occur in significant amounts; (3) sex does not seem to be an important biotic factor affecting fluoride bioaccumulation in signal crayfish; (4) in contrast, the type of tissue is an important biotic factor affecting fluoride bioaccumulation in signal crayfish, with the exoskeleton accumulating more fluoride than the muscle, in absolute terms, but with the muscle accumulating more fluoride than the exoskeleton, in relative terms (in relation to pre-effluent/control values); (5) the developmental stage seems to be another important biotic factor affecting fluoride bioaccumulation in signal crayfish, with juveniles being able to accumulate fluoride more rapidly than adults under fluoride pollution conditions; (6) although, during depuration periods, signal crayfish may significantly release fluoride, they can still retain significant amounts of the fluoride previously bioaccumulated during exposure periods. We can overall conclude that fluoride bioaccumulation in signal crayfish may be used as suitable bioindicator of fluoride pollution in those freshwater ecosystems where it is already present. However, this notable capability to accumulate and retain fluoride poses a potential risk to human health when signal crayfish from fluoride polluted areas are consumed.