Antigenic characterization of Nephrops norvegicus (L.) hepatopancreas cells

During this work structural, differentiation and proliferation antigenic markers developed for mammals were applied in paraffin sections of Nephrops norvegicus (L.) hepatopancreas. The purpose was to establish standards for the characterization of invertebrate cells in vitro. Antibody concentration was optimized for quantification of cell proliferation. There are no antibodies specific for crustaceans on the market. An avidin–biotin immunoperoxidase method was used to visualize cell antigen expression. The immunocytochemical results indicate that the epithelium in the Nephrops hepatopancreas digestive tubules does express cytokeratins and proliferating cell nuclear antigen. The results of this work indicate that some mammalian antibodies cross‐react with crustacean epitopes. This may facilitate cell characterization of cell types cultured in vitro. Copyright © 1999 John Wiley & Sons, Ltd.     

A parasitic scuticociliate infection in the Norway lobster (Nephrops norvegicus)

A histophagous ciliate infection was discovered in a number of Norway lobsters (Nephrops norvegicus) from the Clyde Sea Area, Scotland. Silver-carbonate staining of cultured ciliates revealed an oral apparatus and additional structural features that are morphologically similar to scuticociliates in the genus Mesanophrys, which are known to parasitize crustaceans. However, ribosomal DNA sequences (ITS1/5.8S/ITS2) of the ciliate were identical to Orchitophyra stellarum, a parasitic scuticociliate of sea stars with a different morphology from Mesanophrys spp. and to the ciliate from N. norvegicus. Associated pathology included degeneration and necrosis of the myocardial heart muscle, and large numbers of ciliates in the gill filaments.     

Detection of the parasitic dinoflagellate Hematodinium in the Norway lobster Nephrops norvegicus by ELISA

Norway lobsters Nephrops norvegicus from the coastal waters of Scotland are seasonally infected by a parasitic dinoflagellate of the genus Hematodinium. An enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of the parasite in the haemolymph of N. norvegicus. The ELISA is simple to perform with a detection limit of 5 x 10(4) parasites ml(-1) haemolymph. The ELISA is currently being used to study the prevalence and seasonality of Hematodinium infection in N. norvegicus and other crustacean hosts.     

Molecular detection of Hematodinium spp. in Norway lobster Nephrops norvegicus and other crustaceans

The Norway lobster Nephrops norvegicus (L.) from the coastal waters of Scotland is seasonally infected by a parasitic dinoflagellate of the genus Hematodinium. Methods used to detect infection include a morphological index (pleopod diagnosis) and several immunoassays. The present study describes the development and application of a set of Hematodinium-specific polymerase chain reaction (PCR) primers and DNA probes based on Hematodinium ribosomal DNA (rDNA). In the PCR assay, a diagnostic band of 380 bp was consistently amplified from total genomic DNA isolated from Hematodinium-infected N. norvegicus. The sensitivity of the assay was 1 ng DNA, which is equivalent to 0.6 parasites. The primer pair also detected Hematodinium DNA in preparations of the amphipod Orchomene nanus, indicating that the amphipod may be infected with the same Hematodinium sp. infecting N. norvegicus. DNA probes detected Hematodinium parasites in heart, hepatopancreas and gill tissues from N. norvegicus, and hepatopancreas and gill tissues from Carcinus maenas, confirming Hematodinium infection in the latter.     

The reactions of the Norway lobster,Nephrops norvegicus (L.), to water currents

Observations were made on the reactions of the Norway lobster, Nephrops norvegicus, to water currents in a sea‐water flume tank. Blind animals were used to prevent visually‐guided behaviour. Nephrops adopted a downstream orientation, and usually walked downstream, in response to water current speeds in the range of 0.07 to 0.20 ms^?1. Patterns of water flow around the body revealed that it was most effectively streamlined when the animal adopted a downstream orientation. Direct measurements of the forces acting on the body revealed that animals with a downstream orientation experienced the least hydrodynamic drag and the greatest downforce.     

Mitochondrial DNA variation in Northeast Atlantic and Mediterranean populations of Norway lobster, Nephrops norvegicus

Analysis of the genetic structure of the Norway lobster (Nephrops norvegicus), a marine crustacean with high commercial value, was undertaken to gain information regarding the differentiation of Atlantic from Mediterranean populations of marine invertebrates. Restriction fragment length polymorphism analysis of two mitochondrial DNA segments, 3.6 kilobases in total, was performed. Twelve populations from the North Sea, Irish Sea, Portuguese coast and Aegean Sea were analysed. Low levels of differentiation were found among them (F(ST) = 0.018, P < 0.001) and there were no signs of an Atlantic-Mediterranean divide or of an isolation-by-distance scheme of differentiation. Possible reasons for these low levels of differentiation can be found in the recent expansion of N. norvegicus populations. This is supported by the mismatch distribution of pairwise haplotype differences, as well as by the high mean haplotype diversity (h = 0.93) combined with medium nucleotide diversity (pi = 0.0057) (in comparison to values for marine crustaceans or teleosts) found in this study. This combination of high levels of haplotype diversity with moderate to low levels of nucleotide diversity has also been frequently attributed to a recent time of divergence for various marine species. No evidence was found for a Mediterranean refugium during glaciation periods, separate from the Atlantic, as has been reported for some marine species. The Irish Sea population was the most differentiated as a result of reduced levels of diversity. Results are also discussed in the light of future management of N. norvegicus stocks.     

Identification and partial characterisation of metalloproteases secreted by a Mesanophrys-like ciliate parasite of the Norway lobster Nephrops norvegicus

A ciliate parasite, tentatively identified as Mesanophrys sp. of Norway lobsters Nephrops norvegicus, is demonstrated to secrete several proteases into the culture medium (modified Nephrops saline). Analyses using substrate-impregnated sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed 12 activity bands differing greatly in mobility in the gels. The complete inhibition of proteolytic activity by 1,10-phenanthroline indicated that the proteases are of the metallo class. The proteases were active at the physiological temperature (8 degrees C) and haemolymph pH (7.8) of the host. The proteases were selective in the degradation of several host proteins, including the myosin heavy chain, which is a major structural component of lobster muscle. Consequently, these proteases may have important roles in several aspects of the host-parasite interaction including invasion, nutrient uptake by the ciliate, and pathogenesis.     

Histochemical heterogeneity of fibers in the abdominal superficial flexor muscles of the Norway lobster, Nephrops norvegicus (L.).

The superficial flexor muscle in the abdomen of the Norway lobster Nephrops norvegicus (L.), comprises medial and lateral bundles with distinct fiber type composition. Fibers of the medial bundle have long sarcomeres (> 9 microns) and a thick fringe of subsarcolemmal mitochondria. In histochemical tests they have a low total myofibrillar ATPase activity, a pH-stable isoform of myosin ATPase, and a high level of oxidative enzyme activity. A few fibers of the lateral bundle also display these morphological and histochemical properties. However, the majority of lateral fibers have shorter sarcomeres (< 8 microns), no subsarcolemmal mitochondria, but a well-developed tubular system. They also have a higher total myofibrillar ATPase activity, a pH-labile isoform of myosin ATPase, and a low level of oxidative enzyme activity. The heterogeneous pattern of different fiber types in the lateral bundle of this muscle is similar but not identical in the different abdominal segments and in different individuals.     

Effects of emersion and re-immersion on physiological and immunological variables in creel-caught and trawled Norway lobster Nephrops norvegicus

Immune defence in creel-caught and trawled Nephrops norvegicus was investigated to assess a possible relationship between phenoloxidase (PO) activation and the total haemocyte count (THC). Capture, capture method and emersion evoked physiological and immunological responses that may have implications for the ability of N. norvegicus to survive the effects of such stressors. Haemolymph THC was always negatively related to PO activity in the trawled samples, suggesting a decreased level of the plasma serine proteinase inhibitors which reportedly regulate the ProPO system (Le Moullac et al. 1998; Fish shellfish Immunol 8:621-629). In contrast, creel-caught samples showed increased levels of both PO and THC (cf. control N. norvegicus), after a 12 h emersion period. Trawling and emersion evoked progressive and significant increases (p < 0.05) in the mean levels of haemolymph L-lactate, glucose and total ammonia. The evidence of overt activity and measured haemolymph parameters suggest that creel fishing yields N. norvegicus that are more likely to survive post-harvest treatments than those that are trawled.     

Internal anatomy and ultrastructure of the male reproductive system of the Norway lobster Nephrops norvegicus (Decapoda: Astacidea)

The Norway lobster (Nephrops norvegicus) is economically important in Europe. However, apart from the female reproductive system, very little is known about its internal anatomy. This article focuses on studying the internal anatomy and ultrastructure of the male reproductive system. This system follows the general pattern found among decapod crustaceans, with several peculiarities. Testes are composed of lobular sperm ducts in which the spermatozoa are fully constituted. The spermatozoa present three lateral arms and a long acrosome, which gives a false appearance of flagellated spermatozoa. The two testes form a double H under the heart, and the vas deferens (VD) arise from each side at the posterior edge of the double H. The main characteristic of the VD is the presence of a sphincter in the enlarged area of the distal end of the middle VD. The MVD here shows an increase in musculature of the wall as compared to the VD, which regulates the passage of the sperm cord to the distal VD (DVD) and thence to the thelycum of the female. The wall of the spermatophore is formed in the distal part of the proximal VD, which surrounds the unique sperm cord present in the VD. Isolated spermatophores are not observed in the VD. The sperm cord is pinched off during copulation by the musculature of the DVD. Then, a portion of the sperm cord is transferred from each VD to form the isolated spermatophores. The wall of the spematophores and the spermatozoa that are observed inside the thelycum have the same morphology as those observed in the VD. J. Morphol., 2012. © 2012 Wiley Periodicals, Inc.     

The effect of temperature on the bacterial load and microbial composition in Norway lobster (Nephrops norvegicus) tail meat during storage

Aims: The aim of this study was to update and extend our knowledge of the bacterial load and microbial composition in Norway lobster (Nephrops norvegicus) under commercially relevant storage conditions to optimize handling procedures. Methods and Results: Total viable counts were performed at different storage temperatures (0, 4, 8, 10, 12 or 16°C) and after different storage times (1–7 days). Storage at 16°C was found to be most detrimental, and storage at 0°C was found to be optimal. 16S‐rRNA sequencing was utilized to determine the composition of the bacteria within the microflora. In this way, Photobacterium isolates, especially Photobacterium phosphoreum, were identified as the main specific spoilage organisms. The abilities to reduce trimethylamineoxide (TMAO) and to produce H₂S were analysed in a selection of bacterial isolates. The higher the incubation temperature during storage, the more isolates were found to reduce TMAO and produce H₂S. Conclusions: Nephrops norvegicus possesses an unusually high initial microbial load when fresh. Storage temperature is the most crucial factor affecting microbial growth, microbial activity and spoilage potential in N. norvegicus produce. Spoilage can be attributed mainly to P. phosphoreum. Significance and Impact of the Study: This study presents significant new findings with regard to the progression and causative agents of spoilage in N. norvegicus. Based on the results, we can recommend that N. norvegicus tails should be stored in a 0°C environment immediately after catch. Stored this way, the growth and spoilage activity of the microflora may be reduced significantly and an extension of shelf life might be attained.     

An analysis of swimming performance in the Norway lobster, Nephrops norvegicus L. infected by a parasitic dinoflagellate of the genus Hematodinium

Various components of swimming performance were measured in uninfected Norway lobsters (Nephrops norvegicus) and compared to animals at different stages of infection by a parasitic dinoflagellate (Hematodinium sp.). Animals showed a progressive decline in overall swimming performance as infection severity increased, with reductions in the number of tail-flips performed, the number of swimming bouts and the total distance travelled by swimming. The velocity of the first (giant-fibre mediated) tail flip and average velocity over the swimming bout were also significantly reduced in infected lobsters. Possible reasons for this decreased swimming performance are suggested and the implications of this for predator avoidance of infected lobsters in the benthic habitat, and for capture of Nephrops by trawl rigs are discussed.     

Intercellular junctions in myriapods

Tissue from the intestinal tract of myriapods, including millipedes, centipedes and pauropods were examined in tracer-impregnated sections and freeze-fracture replicas. The foregut and hindgut of all three classes exhibit pleated septate junctions; these display undulating intercellular ribbons in thin sections. In replicas they show discrete intramembranous particle (IMP) arrays aligned in rows in parallel; with one another. The tissues of the hindgut also possess scalariform junctions, characterized by cross-striated intercellular clefts in sections and IMP-enriched membranes in replicas. Gap junctions occur in all groups, but they are atypical in replicas in that their component IMPs do not always fracture onto the E face, as is characteristic of other arthropods; some IMPs cleave to the P face and others to the E face. The midgut of these organisms exhibits smooth septate junctions with conventional straight septal ribbons and occasional interseptal columns. However the intramembranous appearance in replicas is variable, particularly in centipedes, in that the rows of IMPs in chemically-unfixed propanecryofixed tissues, are prominent and adhere preferentially to the E face, with complementary P face grooves, while in fixed tissues the IMPs are much less distinct and fracture to either P face or E face. They tend not to protrude far beyond the mid-plane of the membrane bilayer and lie in rows which commonly take on the form of a network. Individual rows of the network sometimes curve to run beside a second row, over a short distance, before bending away into another part of the network. The aligned particle rows, which are much more prominent in millipedes, where they frequently lie in close parallel appositions, do not fuse into ridges as often occurs in insect tissues. The myriapod junctions, therefore, are of the same general kind as are found in the gut tract of other arthropod groups, but differ with respect to the subtleties of their intramembranous organization and disposition.     

Survival of discarded plaice (Pleuronectes platessa) from Norway lobster (Nephrops norvegicus) otter‐trawl fishery

The introduction of landing obligations in the European Union common fisheries policy, has increased the focus on discard survival. Since species with scientifically proven high survival rates may be exempted from the landing obligations, the economic significance for the fishing industry is large. Landing obligations include individuals below the minimum conservation reference size (MCRS), which are not allowed to be sold for human consumption. The Norway lobster (Nephrops norvegicus) fishery is a high value fishery, but with a substantial amount of bycatch of plaice (Pleuronectes platessa) below the MCRS. In the present study, observations were made on the short‐term survival of plaice at or below the MCRS, discarded from a small otter board trawler targeting Nephrops in Skagerrak (ICES 3a). The average short‐term survival of plaice was 15% at haul level, ranging from 0% to 39%, after 10 days of captive observation. Survival significantly decreased with time on deck and the retention of debris in the codend (p < 0.0001), while survival was not correlated to size. Vitality assessments, using a combined reflex impairment and injury score, were tested as predictor of survival and were significantly associated to survival (p < 0.0001) both for individual fish and when grouped by hauls. Seabird predation behaviour was assessed and it was estimated that seabirds predated or pursued 85% of discarded plaice below the MCRS.     

Reproduction in Nephrops norvegicus (Decapoda: Nephropidae)

During the period October 1969 to April 1972 a comprehensive study of the reproductive biology of the Norway lobster, Nephrops norvegicus (L. 1758), was made in the Irish Sea. Where possible comparison has been made with data from other areas. The results of the present investigation show that most females are berried annually, egg-laying taking place in August and September, and hatching in April to June of the following year. Moulting and copulation occur in mature females during May to August. In contrast males do not show a marked reproductive cycle, spermatogenesis occurring throughout the year. The method of fertilization is discussed, and it is suggested that this may be internal. Copulatory and egg-hatching behaviour is described, together with details of egg-laying and incubation in the laboratory. A pre-zoeal larval stage is described for the first time, bringing the total number of larval instars to four. The terminology of the larval stages is revised.     

Intercellular junctions in nerve-free hydra

Summary Epithelial cells of nerve-free hydra contain septate and gap junctions. In thin sections the gap junctions are characterized by a gap of 3–4 nm. Freeze-fracture demonstrates the presence of septate junctions and two further types of structures: (i) the E-type or inverted gap junctions with particles in an en plaque conformation appearing as a raised plateau on the E-face or as a depression on the P-face; (ii) structures morphologically similar to gap junctions in rat liver, containing particles on the P-face and corresponding pits on the E-face, both having hexagonal packing with a lattice constant of 8 nm. We propose that these structures are also gap junctions.     

Gut bacteria associated with different diets in reared Nephrops norvegicus

The impact of different diets on the gut microbiota of reared Nephrops norvegicus was investigated based on bacterial 16S rRNA gene diversity. Specimens were collected from Pagasitikos Gulf (Greece) and kept in experimental rearing tanks, under in situ conditions, for 6 months. Treatments included three diets: frozen natural (mussel) food (M), dry formulated pellet (P) and starvation (S). Gut samples were collected at the initiation of the experiment, and after 3 and 6 months. Tank water and diet samples were also analyzed for bacterial 16S rRNA gene diversity. Statistical analysis separated the two groups fed or starved (M and P vs. S samples). Most gut bacteria were not related to the water or diet bacteria, while bacterial diversity was higher in the starvation samples. M and P samples were dominated by Gammaproteobacteria, Epsilonproteobacteria and Tenericutes. Phylotypes clustering in Photobacterium leiognathi, Shewanella sp. and Entomoplasmatales had high frequencies in the M and P samples but low sequence frequencies in S samples. The study showed that feeding resulted in the selection of specific species, which also occurs in the natural population, and might be associated with the animal's nutrition.     

The effects of the metal ions Mn2+ and Co2+ on muscle contraction in the Norway lobster Nephrops norvegicus (L.)

The effects of the metal ions manganese and cobalt on force production by the abdominal superficial flexor muscle of the Norway lobster, Nephrops norvegicus, have been studied in response to both neuronal stimulation and electrical field stimulation applied to an isolated neuromuscular preparation, and by selectively blocking synaptic transmission with ivermectin. In response to both forms of stimulation, low concentrations of manganese added to the standard N. norvegicus saline increased the contractile force produced by the muscle, whereas higher concentrations of manganese inhibited both responses in a dose-dependent manner, until force was completely abolished at concentrations above 2.9 mM manganese. Cobalt ions produced similar effects, and no significant difference was found between the concentration of the two ions at 50% force inhibition (K_m) or between the two stimulation methods (manganese: 1.22 mM; cobalt: 1.29 mM, P = 0.86). This suggests that they have a similar mode of action, and a postsynaptic site of inhibition. These K_m values are considerably higher than the concentrations of these ions known to accumulate in the haemolymph of N. norvegicus under eutrophic conditions, and it therefore seems unlikely that accumulations of manganese or cobalt ions under such conditions would cause any significant inhibition of muscle contraction force. Accepted: 28 April 1999