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.     

Aggression and competition for shelter between a native and an introduced crayfish in Europe

1. The introduced North American crayfish, Pacifastacus leniusculus Dana, is expanding its range in Europe and locally often replaces two native crayfish species, Astacus astacus L. and Austropotamobius pallipes Lereboullet. Pacifastacus leniusculus is also expected to invade the habitat of a third native crayfish, the endangered Austropotamobius torrentium Schrank. Interspecific aggressive interactions and competition for shelter were experimentally studied in the laboratory to assess the potential impact of P. leniusculus on A. torrentium .
2. Neither species was inherently dominant in aggressive interactions, but dominance was strongly size-dependent, favouring the larger and faster growing species, P. leniusculus .
3. Access to limited shelter was generally determined by aggressive dominance, although species-specific preference also influenced the outcome of competition for shelter. Austropotamobius torrentium had a higher preference for experimental shelters and often defended these even against larger P. leniusculus .
4. In accordance with theoretical models of animal conflicts, agonistic interactions between equally sized contestants were more severe than between animals of different size.
5. The P. leniusculus used in the present experiment were infected with the crayfish plague, Aphanomyces astaci , to which the animals are resistant. The crayfish transmitted the disease to non-resistant A. torrentium which died ≈ 2 weeks after contact with P. leniusculus .     

Invasive crayfish and crayfish plague on the move: first detection of the plague agent Aphanomyces astaci in the Romanian Danube

Native European crayfish, such as Astacus leptodactylus, are threatened, among other factors, by the crayfish plague agent Aphanomyces astaci, dispersed by invasive North American crayfish. Two of these invaders, Pacifastacus leniusculus and Orconectes limosus, have extended their distribution in the River Danube catchment; the latter was detected for the first time in Romania in 2008. We monitored, at monthly intervals for over 2 yr, occurrence of native A. leptodactylus and invasive O. limosus at 6 sites on the Romanian Danube and checked for the invasive species in 4 of its tributaries. Between January 2009 and March 2011, the relative abundances of O. limosus steadily increased with time, while the native A. leptodactylus dramatically decreased in abundance. O. limosus expanded downstream at a rate of ca. 15 km yr-1; in August 2011, it was already present in the upper 105 km of the Romanian Danube. An agent-specific real-time PCR analyses demonstrated the presence of A. astaci DNA in at least 32% of the analysed invasive (n = 71) and 41% of the native (n = 49) crayfish coexisting in the Danube. Furthermore, A. astaci was also detected in A. leptodactylus captured about 70 km downstream of the O. limosus invasion front (at the time of sampling). Assuming a steady rate of expansion, O. limosus may invade the sensitive Danube delta area in the mid-2060s, even without long-distance dispersal. The crayfish plague agent, however, may reach the delta substantially earlier, through dispersal downstream among populations of native crayfish.     

Phenotypical characterization of indigenous freshwater crayfish populations

The morphology of an animal is known to reflect both genetic variation and adaptation to the environment. Thus, phenotypic criteria have been used to characterize indigenous crayfish populations. Twenty-one morphometric parameters were measured on crayfish from 25 waterbodies in the Austrian and Italian parts of Tyrol. They were analysed with hierarchical cluster analysis to obtain the population structure based on morphological similarity between and within the freshwater crayfish species Astacus astacus (noble crayfish), Austropotamobius torrentium (stone crayfish) and Austropotamobius pallipes (white-clawed crayfish). Furthermore, a stepwise discriminant analysis was applied to the morphometric data to test their differentiating power between populations. Both analyses resulted in a clear differentiation of species and populations, and reflected geographic separations. Thus, the developed morphometric methods were shown to be applicable to characterize the phenotype in freshwater crayfish and seem appropriate to be used for stock identification and the effective characterization of management units in decapod crustaceans.     

Phylogeny and species composition of five European species of Branchiobdella (Annelida: Clitellata: Branchiobdellida) reflect the biogeographic history of three endangered crayfish species

Branchiobdellid annelids are usually found as commensal symbionts associated with crayfish populations, but knowledge of their dispersion and ecology in Europe is generally scarce. We hypothesized that their geographic extension of species and populations may mirror the distribution history of their hosts. We analysed potential host specificities and the geographic distribution of species from the Italian and Austrian Tyrol and Carinthia by characterizing the morphological and genetic features. On the three indigenous crayfish species Astacus astacus, Austropotamobius pallipes and Austropotamobius torrentium , we identified four branchiobdellid species based on morphological characteristics: Branchiobdella hexodonta, Branchiobdella pentodonta, Branchiobdella balcanica and Branchiobdella parasita . In contrast to the morphological classification, phylogenetic analysis using mitochondrial cytochrome c oxidase I (CO-I) sequences identified five main lineages: B. balcanica, B. hexodonta, Branchiobdella italica, B. parasita and B. pentodonta . The arrangement of branchiobdellid species corresponded generally to the geographical distribution of their crayfish hosts' locations but also confirmed previous assumptions of crayfish translocations. Our study provides the first application of ideas on the association of freshwater crayfish and their ectosymbionts to be used for discussing the biogeography of crayfish populations. The phenotypical and genotypical analysis also demonstrated so far ignored effects of human activities at both macro-ecological and micro-ecological levels.     

Metabolic responses in two species of crayfish (Parastacus defossus and Parastacus brasiliensis) to post-hypoxia recovery

The period of post-hypoxia recovery is essential for the rapid replenishment of energy reserves and for the removal of metabolic end products formed during hypoxia. Periods of post-hypoxia recovery were analyzed in two crayfish species, where Parastacus defossus is a fossorial species, and Parastacus brasiliensis lives in lotic environments with higher oxygen levels. After 4 h of hypoxia (2 mg O(2)/L), groups of animals were placed in tanks with oxygenated water and were then removed at intervals of 1, 3, 6, and 9 h. Hemolymph and tissues (hepatopancreas, muscle, and anterior and posterior gills) were extracted for the determination of glucose, lactate, free glucose, glycogen, total proteins, total lipids, arginine phosphate, and arginine. As expected, lactate levels were restored more rapidly in P. defossus than in P. brasiliensis. P. defossus restored its glycogen reserves of the hepatopancreas and muscle tissue. Free glucose was quickly restored in all tissues of both species. In relation to arginine phosphate reserves, P. defossus showed a greater ability to restore this metabolite in the hepatopancreas. Both species recovered their arginine phosphate reserves, but they also used this metabolite in longer periods of recovery. Mainly in P. brasiliensis the reserves of total lipids seem to be an important source of energy during the recovery period. The animals developed various metabolic strategies to post-hypoxia recovery, mainly P. defossus which restored its reserves more completely and more rapidly than did P. brasiliensis.     

A comparative study of the physiological condition of native and invasive crayfish in Croatian rivers

The physiological condition of two invasive crayfish species identified in Croatian rivers (Orconectes limosus and Pacifastacus leniusculus) was compared with that of native crayfish populations (Astacus astacus and A. leptodactylus). Hepatosomatic, gonadosomatic and abdominal muscle indices were examined for all four species and within a specific invadernative pair. The results demonstrate that invasive crayfish had better overall physiological condition when compared to native species, with a higher energy content of the hepatopancreas, abdominal muscle and gonads in invasive than in native species, with the exception of A. leptodactylus females which demonstrated an increased ability to allocate energy to reproduction in comparison with other species. These results also indicate that A. astacus is physiologically more unstable than A. leptodactylus and thus more likely to be outcompeted and replaced by non-indigenous crayfish species.     

Detection of Aphanomyces astaci in North American crayfish by polymerase chain reaction

We present a PCR based method to detect Aphanomyces astaci in North American crayfish. Primers were designed to specifically amplify parts of the internal transcribed spacer (ITS) regions and the 5.8 rRNA gene of A. astaci. A single round and a semi-nested assay were tested for their sensitivity and specificity. Specificity of the PCR assays was tested against several closely related Aphanomyces species, other Oomycetes and some non-A. astaci DNA that might be found in or on crayfish. The single round assay was fully specific against all DNA tested. In the semi-nested assay, cross-reaction was seen when the equivalent of 40,000 or more genomic units of A. invadans or A. frigidophilus were entered into the PCR reaction. The lower detection limit of both assays lies around 1 genomic unit of A. astaci. Investigation of various parts of the exoskeleton of 3 North American crayfish species revealed that for O. limosus and P. leniusculus the telson and soft abdominal cuticle yielded a positive PCR reaction most frequently. For the third species, Procambarus clarkii, only 1 individual tested positive, so no conclusion as to preferred infestation site(s) could be drawn.     

Effect of water temperature on the immune response and infectivity pattern of white spot syndrome virus (WSSV) in freshwater crayfish

The susceptibility of two species of freshwater crayfish, Pacifastacus leniusculus and Astacus astacus, to white spot syndrome virus (WSSV) by intramuscular injection was compared and the results show that both species are susceptible to WSSV. The effect of water temperature on the development of white spot disease in crayfish was also studied. Crayfish were exposed to different temperatures after WSSV injection or oral exposure and the mortalities were recorded over a period of 45 days. No mortality was observed when crayfish were held at 4+/-2 degrees C or 12+/-2 degrees C and reached 100% when these crayfish transferred to 22+/-2 degrees C. The mortalities of nearly moribund crayfish at 22+/-2 degrees C with WSSV could be delayed after transfer to temperature below 16 degrees C. These results clearly show that low temperature affects the WSSV pathogenicity in crayfish. Moreover, haemocyte counts, phenoloxidase activity, mRNA levels of prophenoloxidase (proPO) and the lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) in crayfish exposed to various water temperatures were studied. Total haemocyte and granular cell counts of crayfish held at different temperatures were not significantly (P>0.05) different, except for the total haemocyte number at 18 degrees C was significantly (P<0.05) higher than in crayfish at 4 degrees C. The percentage of granular cells in crayfish held at 4 degrees C was the highest compared to crayfish maintained at other temperatures. The phenoloxidase activities in haemocyte lysate supernatant (HLS) of crayfish at all temperature groups remained similar. The amount of proPO-mRNAs in haemocytes was much higher than the amount of LGBP-m RNAs in all the experimental groups. However, there was no change in the level of pro PO-mRNA at the tested temperatures. Interestingly, the level of LGBP-mRNA of crayfish kept at 22 degrees C was much lower than in those held at lower temperatures. Proliferation of the haematopoietic tissues was higher at high temperatures which may support replication of WSSV, and explain the high mortality of crayfish with WSSV infection at high temperature. Based on these studies it is concluded that crayfish might act as a carrier of WSSV at low water temperature and could develop white spot disease if the water temperature is increased.     

Effects of starvation and - or -alanine administration on the free - and -alanine levels in the muscle and hepatopancreas of the crayfish, Procambarus clarkii

Changes of - and -alanine and other free amino acids were examined in muscle and hepatopancreas of the crayfish Procambarus clarkii before and after a 1-month starvation in freshwater, 50% seawater, and seawater. Total free amino acids and - and -alanine in both tissues decreased during starvation. The largest decrease was found in the animals starved in freshwater. In 50% seawater and full-strength seawater, the ratio of -alanine to total alanine increased in both tissues, although the level was reduced a little. A large dose of -alanine administered into the tail muscle of freshwater crayfish was converted to -alanine and -alanine returned to the control level within a week. A large amount of -alanine was transported from muscle to hepatopancreas. -Alanine injected into freshwater crayfish was converted to -alanine, but was not transported to the hepatopancreas. In the case of -alanine injection into the muscle of crayfish acclimated to 50% seawater, large amounts of - and -alanine were maintained in the muscle during the experimental period of 4 days. These data indicate that -alanine is metabolized actively in crayfish tissues and accumulated as an important osmolyte during osmotic stress.     

C-type lectin from red swamp crayfish Procambarus clarkii participates in cellular immune response

Lectins are potential immune recognition proteins. In this study, a novel C-type lectin (Pc-Lec1) is reported in freshwater crayfish Procambarus clarkii. Pc-Lec1 encodes a protein of 163 amino acids with a putative signal peptide and a single carbohydrate recognition domain. It was constitutively expressed in various tissues of a normal crayfish, especially in the hepatopancreas and gills. Expressions of Pc-Lec1 were up-regulated in the hepatopancreas and gills of crayfish challenged with Vibrio anguillarum, Staphylococcus aureus, or the white spot syndrome virus. Recombinant mature Pc-Lec1 bound bacteria and polysaccharides (peptidoglycan, lipoteichoic acid, and lipopolysaccharide) but did not agglutinate bacteria. Pc-Lec1 enhanced hemocyte encapsulation of the sepharose beads in vitro, and the blocking of beads by a polyclonal antibody inhibited encapsulation. Pc-Lec1 promoted clearance of V. anguillarum in vivo. These results suggest that Pc-Lec1 is a pattern recognition receptor and participates in cellular immune response. Pc-Lec1 performs its function as an opsonin by enhancing the encapsulation or clearance of pathogenic bacteria.     

A successful crayfish invader is capable of facultative parthenogenesis: a novel reproductive mode in decapod crustaceans

Biological invasions are impacting biota worldwide, and explaining why some taxa tend to become invasive is of major scientific interest. North American crayfish species, particularly of the family Cambaridae, are prominent invaders in freshwaters, defying the "tens rule" which states that only a minority of species introduced to new regions become established, and only a minority of those become invasive and pests. So far, success of cambarid invaders has largely been attributed to rapid maturation, high reproductive output, aggressiveness, and tolerance to pollution. We provide experimental evidence that females of one cambarid species particularly widespread in Europe, the spiny-cheek crayfish Orconectes limosus, are capable of facultative parthenogenesis. Such reproductive mode has never before been recognized in decapods, the most diverse crustacean order. As shown by analysis of seven microsatellite loci, crayfish females kept physically separated from males produced genetically homogeneous offspring identical with maternal individuals; this suggests they reproduced by apomixis, unlike those females which mated with males and had a diverse offspring. Further research is needed to clarify what environmental conditions are necessary for a switch to parthenogenesis in O. limosus, and what role it plays in natural crayfish populations. However, if such reproductive plasticity is present in other cambarid crayfish species, it may contribute to the overwhelming invasive success of this group.     

Occurrence of Alanine Racemase in Crustaceans and the Changes of the Properties During Seawater Acclimation of Crayfish

Alanine racemase activity was detected in the muscle and hepatopancreas of six macruran species. Optimal pH was around 8.5 irrespective of species and tissues. Apparent Michaelis constants ranged from 48 to 157 mM for muscle enzyme and 35 to 239 mM for hepatopancreas enzyme. The enzyme specifically catalyzed the racemization of d- and l-alanine. The enzyme did not require pyridoxal 5′-phosphate as well as FAD as a cofactor. Pyruvate and l-alanine showed strong inhibition in the direction of d to l.During seawater acclimation of crayfish Procambarus clarkii, alanine racemase activity increased about twice in muscle and 1.5 times in hepatopancreas. Michaelis constant, on the other hand, decreased 33% for muscle enzyme and 65% for hepatopancreas enzyme, suggesting the increase of substrate affinity during seawater acclimation. The activity in the physiological pH range (6.5–7.5) also increased with increasing salinity.     

Responses of free radical metabolism to air exposure or salinity stress, in crabs (Callinectes danae and C. ornatus) with different estuarine distributions

The swimming crabs Callinectes danae and C. ornatus are found in bays and estuaries, but C. danae is more abundant in lower salinities, while C. ornatus remains restricted to areas of higher salinity. Experimental crabs of both species were submitted to: air exposure (Ae, 3h), reimmersion in 33‰ (control) sea water (SW) (Ri, 1h) following air exposure; hyposaline (Ho, 10‰ for 2h) or hypersaline (He, 40‰ for 2h) SW, then return to control 33‰ SW (RHo and RHe, for 1h). Hemolymph was sampled for osmolality and chloride determinations. Activity of antioxidant enzymes [glutathione peroxidase (GPX), catalase, glutathione-S-transferase] and levels of carbonyl proteins and lipid peroxidation (TBARS) were evaluated in hepatopancreas, muscle, anterior and posterior gills. In Ho groups, hemolymph concentrations were lower in both species, compared to He groups. C. danae displayed higher control activities of GPX (hepatopancreas and muscle) and catalase (all four tissues) than C. ornatus. C. ornatus presented increased activities of catalase and GPX in Ae, Ri, and He groups. Increased TBARS was seen in C. ornatus tissues (He group). The more euryhaline species displayed higher constitutive activities of antioxidant enzymes, and the less euryhaline species exhibited activation of these enzymes when exposed to air or hyper-salinity.