The Structure and Mineralization of the Carapace of the Crab (Cancer pagurus L.) 2. The exocuticle

The exocuticle of the dorsal carapace of the intermoult Cancer pagurus was studied by means of light microscopy, electron microscopy, and contact microradiography. In the exocuticle a 5-15 μm wide highly mineralized external zone was seen where prisms were not present. The cross-sectional shape and diameter of the prisms varied. The prisms were separated by interprismatic areas varying in width and in degree of mineralization. With increasing width of the interprismatic areas the diameter of the prisms decreased. The prism-less external zone may be the result of complete mineralization of the prisms. The pore canals of the exocuticle were circular in cross section and present both within the prisms and within the interprismatic areas. The lamellar system of the exocuticle was built up by layers of horizontal fibrils which were interconnected by vertical or oblique fibrils. The minerals of the external prism-less zone occurred as aggregates of crystalline matter or as large brick-like structures. Crystal edge lengths up to 300 nm were measured. In well mineralized interprismatic areas aggregates of crystals and relatively large crystals were observed. In poorly mineralized interprismatic areas small plate-like crystals occurred. Occasionally a prism-less zone was seen near the exocuticle/endocuticle junction.     

The cuticle of the crabs Cancer pagurus L. and Carcinus maenas (L.)

Fine closely-packed parallel fibres pass obliquely, at an angle of about 10° to the horizontal, through the cuticles of Cancer and Carcinus . They lie on axes parallel to the four faces of an obtuse pyramid, and have no counterpart in the model proposed by Bouligand (1965, 1971, 1972).
Replication of vertically broken surfaces of cuticle on cellulose acetate film shows that the laminae of the cuticle are discrete structural entities which preserve their identity around the angle formed by two vertical faces meeting at right-angles. This does not conform to the requirements of the Bouligand model.     

Carapace repair in the green crab,Carcinus maenas (L.)

Formation of a circular hole 8–10 mm in diameter in the calcified layers of the carapace from crabs in stage C₄ of the molt cycle stimulates the tissue under and adjacent to the injury to deposit a unique calcified cuticular material below the intact membranous layer. Deposition was followed for 69 days using light microscopic histology, histochemistry, and scanning electron microscopy. Quantitative analyses of CaCO₃ were conducted using atomic absorption spectrophotometry and Gran titration. Spatial distribution of CaCO₃ was determined with X-radiography. A scab is formed by day two under the injury. At four days the epithelium changes from squamous to columnar and deposits a PAS-positive layer with an irregular lamellar fine structure, followed by highly organized lamellae structurally similar to normal exocuticle. Histochemically, however, these lamellae resemble normal endocuticle. CaCO₃ is evident external to the outermost lamellae by day eleven as a fused mass of aragonite granules. The lamellar region calcifies proximally from the outer surface and is amorphous CaCO₃. Repair cuticle is approximately 20%CaCO₃ by weight.     

Effect of Extracellular Products of Pseudoalteromonas atlantica on the Edible Crab Cancer pagurus

Previous studies have shown that injection of extracellular products (ECP) of Pseudoalteromononas atlantica isolated from shell disease-infected edible crabs (Cancer pagurus) into healthy crabs causes rapid death. In this study we examined the nature of the active lethal factor(s) in ECP. Injection of ECP into crabs caused a rapid decline in the total number of circulating hemocytes (blood cells), and the crabs died within 60 to 90 min. The individuals that died showed eyestalk retraction, limb paralysis, and lack of antennal sensitivity, suggesting that the active factor(s) targeted the nervous system. Histopathological investigations showed that affected crabs had large aggregates of hemocytes in the gills, and there was destruction of the tubules in the hepatopancreas. The active factor in ECP was not sensitive to heat treatment (100°C for 30 min) and proteinase K digestion. As lipopolysaccharide (LPS) was a potential candidate for the lethal factor, it was purified from whole P. atlantica bacteria or ECP and subsequently injected into crabs. These crabs had all of the external symptoms observed previously with ECP, such as limb paralysis and eyestalk retraction, and they died within 90 min after challenge, although no significant decline in the number of circulating hemocytes was observed. Similarly, in vitro incubation of hemocytes with purified LPS (1 to 20 μg) from P. atlantica did not result in the clumping reaction observed with ECP but did result in a degranulation reaction and eventual cell lysis. Injection of crabs with Escherichia coli or Pseudomonas aeruginosa LPS (1 μg g of body weight⁻¹) did not cause any of the characteristic symptoms observed following exposure to P. atlantica LPS. No mortality of crabs followed the injection of E. coli LPS, but P. aeruginosa LPS caused ca. 80% mortality at 2 h after injection. Overall, these results show that the main virulence factor of P. atlantica for edible crabs is LPS either alone or in combination with other heat-stable factors.     

Deoxyribonucleic acid of the crab Cancer pagurus. 3. Intracellular localization of poly d(A-T) of Cancer pagurus by hybridization in situ

The satellite DNA poly [d(AT) · d(TA)] of the crab Cancer pagurus has been localized in situ by DNA-DNA hybridization in the nuclei of various spermatogenetic, midgut gland, intestinal and tegument cells. The specificity of hybridization was checked by various tests before, during and after hybridization. The nuclear sites revealed by this method were compared with those shown by quinacrine mustard or Giemsa staining. The AT-rich satellite DNA appears to be highly dispersed and does not seem to have any preferential localization inside the crab interphasic nucleus. This situation was compared with that presented by mouse nuclei using similar methods.     

Infection by a Hematodinium-like parasitic dinoflagellate causes Pink Crab Disease (PCD) in the edible crab Cancer pagurus

The edible crab (Cancer pagurus) supports a large and valuable fishery in UK waters. Much of the catch is transported live to continental Europe in specially designed live-well ('vivier') vehicles. During the winter of 2000/2001, many trap-caught crabs from Guernsey, Channel Islands, UK, were reportedly moribund and pink in colour. These crabs generally died before and during vivier transportation. We provide histological, immunological, and molecular evidence that this condition is associated with infection by a Hematodinium-like dinoflagellate parasite similar to that previously reported in C. pagurus and to an infection causing seasonal mass mortalities of the Norway lobster (Nephrops norvegicus). Pathologically, every altered host bore the infection, which was characterised by very large numbers of plasmodial and vegetative stages in the haemolymph and depletion of reserve cells in the hepatopancreas. Due to the hyperpigmentation of the carapace and appendages, we have called this infection 'Pink Crab Disease' (PCD). Similar Hematodinium infections cause 'Bitter Crab Disease' in tanner and snow crabs, which has had a negative effect on their marketability. At present, little is known about the seasonality, transmission, and market impact of this infection in C. pagurus.     

Actin in the acrosome of the spermatozoa of the crab,Cancer pagurus L. (decapoda,crustacea)

The cytoskeletal protein actin was identified in the mature spermatozoon of the European edible crab, Cancer pagurus Linnaeus, by indirect immunofluoresce with monoclonal and polyclonal anti-actin antibodies, fluorescent phalloidin, and DNAase I. The actin was localized in two distinct concentric rings within the acrosome vesicle of the spermatozoon and appeared to correlate with the internal zonation of the vehicle. Modifications of the fluorescent pattern for actin were observed in sperm cells which were undergoing changes associated with the acrosome reaction. In these cases, fluorescent staining was observed in the nucleocytoplasm immediately subjacent to the perforatorial column and sometimes in the perforatorial column within the acrosome vesicle. Equally intense fluorescence was observed in an apical perforatorial projection. SDS-PAGE of C. pagurus sperm confirmed the presence of actin in the cells. A single band of actin (approximately 43 kDa) comigrated with rabbit muscle actin when immunoblotted onto nitrocellulose with mouse monoclonal anti-actin. The actin-associated cytoskeletal proteins α-actinin, tropomyosin, and spectrin were also identified within the spermatozoon of C. pagurus using specific polyclonal antibodies, but their presence was not confirmed by SDS-PAGE and immunoblotting. © 1994 Wiley-Liss, Inc.     

The specificity of a neutral deoxyribonuclease from Cancer pagurus.

The recently isolated neutral deoxyribonuclease from crab (Cancer pagurus) testes has been characterized in its mode of action and its specificity. The enzyme is a typical endonuclease, forming 5'-phosphate oligonucleotides of large average size; after extensive digestion of calf thymus DNA over 75% of the fragments have a size larger than pentanucleotides and mononucleotides are absent. As far as specificity is concerned, thymidine is very abundant in the 5'-penultimate position (approximately 50%) and in the 3'-terminal position (37%) and almost absent in the 5'-terminal position (approximately 1%), the values quoted concerning Escherichia coli digests of average size (Pn) between 50 and 10.     

Exoskeletal proteins from the crab, Cancer pagurus.

Twelve proteins from calcified regions and five from flexible regions (arthrodial membranes) of the exoskeleton of Cancer pagurus have been purified and sequenced. One of the proteins from calcified exoskeleton is identical to one of the arthrodial membrane proteins. Several of the proteins from the calcified regions resemble proteins from corresponding regions of the exoskeleton of the lobster, Homarus americanus, in containing either two or four copies of an 18-residue sequence motif, which so far has been found only in crustacean calcified exoskeletons. The proteins obtained from the flexible arthrodial membranes resemble the proteins from lobster arthrodial membranes, and the similarities are shared with a number of proteins from flexible cuticles in insects, indicating that the common features in these proteins may be important for the mechanical properties of the materials in which they occur.