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.     

The mandibular muscle receptor organ of Homarus gammarus (L.) (crustacea,decapoda)

Summary A muscle receptor organ is present in the mandible of macruran decapods. The mandibular muscle receptor organ (Mand. MRO) of Homarus gammarus (L.) consists of a ribbon of muscle innervated at its ventral insertion by 10–20 multiterminal sensory neurones. The sensory cells have a small number of dendritic processes.The receptor muscle exhibits some structural properties of both fast and slow muscle. The mean sarcomere length is similar to that of the slow abdominal MRO but the receptor muscle in cross section has a punctate distribution of myofibril bundles more typical of fast muscle.This work was supported by Science Research Council grants B/SR/1871 and BR/G/585.     

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.     

Polymorphic nuclear microsatellite loci for studies of brown crab, Cancer pagurus L

Twelve polymorphic microsatellite DNA loci were isolated from the brown crab, Cancer pagurus L., by construction of microsatellite-enriched genomic libraries. Genotyping of 40 individuals from Norfolk (UK) revealed variable levels of locus polymorphism with an average of 7.75 alleles per locus (range 2-22). The observed and expected heterozygosities per locus ranged from 0.025 to 0.868 and from 0.025 to 0.947, respectively. No evidence of linkage disequilibrium was detected between pairs of loci and genotype proportions at all loci conformed to Hardy-Weinberg equilibrium expectations. The microsatellite loci developed constitute a suite of genetic markers applicable to numerous areas of C. pagurus research.     

Carbohydrate metabolism during osmoregulation in Chasmagnathus granulata dana, 1851 (crustacea,decapoda)

• 1.1. Since glucose is one of the main energetic substrates for general metabolic processes in crustaceans, analysis of carbohydrate levels can furnish information on the energy metabolism of intact animals during osmoregulation.
• 2.2. Different groups of Chasmagnathus granulata were transferred to different salinities (0 and 40%), and the glucose and glycogen concentrations in blood, gills, muscle and hepatopancreas were determined at the beginning of the experiment and 24, 72, 168 and 360 hr after the salinity changes.
• 3.3. Differences in tissues carbohydrate levels were observed between summer and winter, that reflected differences in reserve mobilization.
• 4.4. In the summer, hypo- and hyperosmotic shocks induced an increase in carbohydrate levels in almost all tissues studied, indicating gluconeogenesis.
• 5.5. In the winter, a carbohydrate mobilization occurred only in the gills and hepatopancreas after both osmotic shocks.
• 6.6. Thus, the substrate reserve used for energy production required for osmoregulation seems to be dependent on the season and tissues.

     

Locations, ultrastructural morphology, and putative functions of the branchial podocytes of the fresh water crab Potamon niloticus - Savigy (crustacea, decapoda, potamonidae)

The gills of the African fresh water crab, Potamon niloticus, were investigated by transmission electron microscopy to verify the presence, outline the location(s) of, and describe the ultrastructural attributes of the branchial podocytes (BPs). Topographically, the cells were diffusely distributed in the gills. They were found at the arterial ends of the intralamellar spaces (ILSs), in the efferent hemolymphatic vessel (EHV), in the gill shaft, and in the marginal and central ILSs. In the EHV and the terminal ends of the ILSs, the BPs occurred in cohesive clusters of from three to eight large cells which were affixed to the vessel wall by small fibrocytic cells and bands of myofibrils. In the clusters, the BPs attached directly across interspersed junctional complexes (separated by wide intercellular spaces) and indirectly over a common basement membrane. Abundant heteromorphic, variably electron-dense vacuoles were scattered in the cytoplasm, apparently displacing the nuclei peripherally. The plasmalemma of the BPs were amplified into feet processes (pedicels) which inserted onto a basement membrane. The feet were joined by a thin unit membrane (diaphragm), leaving subcisternal spaces which contained flocculent to granular electron-dense material. The general ultrastructural morphology of the BPs of Potamon was similar to that of other crustaceans. However, atypically, a labyrinth of intercellular spaces (reckoned to be filtratory channels) was observed in the cell clusters. An ultrafiltration role was attested by the characteristic specializations of the plasmalemma and a phagocytic one inferred from the conspicuous intracytoplasmic vacuolation, presence of phagosomes, and overt necrosis and desquamation of the outlying cells of the clusters. The topographic location of the BPs in the EHV and at the terminal ends of the ILSs was perceived to be a strategic arrangement for promoting the detoxification and destruction of harmful materials and invasive agents which pass through the gills, an organ that presents an extensive surface area that interfaces with the ambient medium.     

pH regulation in the stomatogastric ganglion of the crab Cancer pagurus

The regulation of intracellular neuronal pH and pH from the extracellular space was studied in the isolated stomatogastric ganglion of the crab Cancer pagurus. Intracellular neuronal pH was found to be 0.3–0.4 pH units more acidic than the standard bath pH of 7.4 and surprisingly, the extracellular space pH was found to be around 0.1 pH units more alkaline than the bath pH. Extracellular space pH shifts in response to bath pH changes decreased as a function of the depth of the recording site within the ganglion, suggesting the existence of restrictions in the free diffusion of H⁺. The amplitude of these pH_e shifts increased in Na⁺-free saline or with amiloride, suggesting Na⁺-dependent regulation of the extracellular space pH. In Na⁺ free saline or in the presence of amiloride, intracellular pH recovery from an NH₄Cl induced acidosis was reduced, and the H⁺ muffling capacity (cf. Thomas et al. 1991) of the extracellular space was markedly reduced. Changes of bath pH had only small effects on the rhythm generating properties of one of the central pattern generators of the stomatogastric ganglion, while NH₄Cl-induced intraganglionic pH changes markedly altered this rhythm.Abbreviations CPG central pattern generator - ECS extracellular space - LP lateral pyloric neuron - NMDG N-methyl-D-glucamine - PD pyloric dilator neuron - pH_e extracellular space pH - pH_i intracellular pH - pH_o bath pH - STG stomatogastric ganglion - V_ref reference potential     

Effect of air exposure on nitrogen metabolism in the crab Cancer pagurus.

In C. pagurus exposed to air for 18 h, blood ammonia content decreased within the 2 first hours, then increased at a relatively constant rate (25 microM/h); blood urate content increased at a lower rate (10 microM/h) and a classical blood acidosis was observed. In the cheliped muscle, a transient 22% decrease in GDH activity for ammonia formation and a 48% increase in GDH activity in the reverse reaction (glutamate synthesis) occurred following 6 and 12 h of emersion, respectively. Changes in LDH activity, used as an indicator of anaerobic potential of muscle, were not observed, except for an 18% increase in crabs exposed to air for 24 h. The increase in blood urate content, not known as a response to emersion in decapods, appeared to be different from that observed in response to hypoxia. The relatively low blood ammonia overload and the GDH increased activity for glutamate synthesis suggested that part of the produced ammonia was stored under a bound form in some tissues. The response of C. pagurus to air exposure is discussed on account of the Storey and Storey ('90) theory.     

An electron microscope study on the developing oocytes of the crab Cancer pagurus L. with special reference to yolk formation (Crustacea)

Summary The developing oocytes of the crab Cancer pagurus L. were studied with the light and electron microscope.Protein yolk formation was found to take place in two different ways. Yolk precursors of type 1 accumulate within the cisternae of an extensively developed granular endoplasmic reticulum. Also further growth and transformation into the definite yolk body occur within the reticular membranes. There is no structural indication that any other cell organelle contributes to the synthesis of this type of yolk building.Protein yolk formation of type 2 involves accumulation and transformation of material within a limiting membrane of the smooth type. The enclosed material is presumably derived from micropinocytosis, enclosed cellular elements and vesicles originating from the Golgi complex.It thus appears that the cell organelles play an important role in the process of drotein yolk formation in the growing oocytes of Cancer pagurus.     

In vivo regulation of the mandibular organ in the edible crab, Cancer pagurus

Considerable evidence indicates that methyl farnesoate (MF) production by the crustacean mandibular organs is negatively regulated by neuropeptides from the sinus gland (SG) in the eyestalk. In the crab Cancer pagurus, two neuropeptides (MO-IH-1 and -2) have been isolated from the SG that inhibit MF synthesis by mandibular organs of female crabs in vitro. To test their activity in vivo, we treated eyestalk-ablated male crabs with SG extracts (SGEs) or MO-IH-1 and -2. SGEs reduced haemolymph levels of MF by 60-80%, while MO-IH-1 and -2 had little effect. Protease treatment of SGEs destroyed the in vivo activity, suggesting that the extract contains an additional peptide responsible for the in vivo activity. When separated by reversed-phase high performance liquid chromatography (HPLC), the in vivo activity eluted in fractions prior to MO-IH-1 and -2. When mandibular organs were removed from animals previously treated in vivo with these active fractions, they had reduced levels of MF synthesis and activity of farnesoic acid O-methyl transferase compared with mandibular organs from animals treated with saline. Together, these results indicate that the regulation of the crustacean mandibular organ is complex and may involve several SG compounds. Some of these compounds (i.e., MO-IH-1 and -2) act directly on the tissue while others affect the mandibular organ indirectly.     

A histological study of shell disease syndrome in the edible crab Cancer pagurus.

Shell disease syndrome is characterised by the external manifestation of black spot lesions in the exoskeletons of crustaceans. In the present study, gills, hepatopancreas and hearts from healthy (<0.05% black spot coverage) and diseased (5 to 15% coverage) edible crabs, Cancer pagurus, were examined histologically to determine whether this disease can cause internal damage to such crabs. There was clear evidence of cuticular damage in the gills of diseased crabs leading to the formation of haemocyte plugs termed nodules. Nephrocytes found within the branchial septa of the gills showed an increase in the accumulation of dark material in their vacuoles in response to disease. In the hepatopancreas, various stages of tubular degradation were apparent that correlated with the severity of external disease. Similarly, there was a positive correlation between the number of viable bacteria in the haemolymph and the degree of shell disease severity. Approximately 21% of the haemolymph-isolated bacteria displayed chitinolytic activity. Overall, these findings suggest that shell disease syndrome should not be considered as a disease of the cuticle alone. Furthermore, it shows that in wild populations of crabs shell perforations may lead to limited septicaemia potentially resulting in damage of internal tissues. Whether such natural infections lead to significant fatalities in crabs is still uncertain.     

Oogenesls in the terrestrial hermit crab,coenobita clypeatus (decapoda,anomura): II. Vitellogenesis

Oocytes from the land hermit crab, Coenobita clypeatus, in various stages of vitellogenesis were examined by light and electron microscopy. Early vitellogenic oocytes are characterized by accumulations of discrete vesicles of endoplasmic reticulum in the perinuclear cytoplasm. As oocytes develop, the endoplasmic reticulum becomes abundant, and numerous Golgi complexes are seen. There is a well developed Golgi-endoplasmic reticulum interaction. Within the confines of the reticulum are discrete intracisternal granules, which can be seen coalescing into electron-dense yolk bodies. Lipid accumulation is seen throughout the cytoplasm. Coincident with the burst of intra-oocytic metabolism are oolemma modifications and micropinocytosis, which provide ultrastructural evidence for extra-oocytic yolk production. The mature oocyte contains numerous yolk and lipid vesicles of varying electron density that comprise both intra- and extra-oocytic substrates.     

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.     

Autoproteolytic stability of a trypsin from the marine crab Cancer pagurus

Autoproteolytic stability is a crucial factor for the application of proteases in biotechnology. In contrast to vertebrate enzymes, trypsins from shrimp and crayfish are known to be resistant against autolysis. We show by characterisation of a novel trypsin from the gastric fluid of the marine crab Cancer pagurus that this property might be assigned to the entire class of crustaceans. The isolated and cloned crab trypsin (C.p.TryIII) exhibits all characteristic properties of crustacean trypsins. However, its overall sequence identity to other trypsins of this systematic class is comparatively low. The high resistance against autoproteolysis was determined by mass spectrometry, which revealed a low susceptibility of the N-terminal domain towards autolysis. By homology modelling of the tertiary structure, the elevated stability was attributed to the distinctly different pattern of autolytic cleavage sites, which is conserved in all known crustacean trypsin sequences.     

Fine structure of the mature spermatozoon of rhynchocinetes typus,crustacea decapoda

Rhynchocinetes typus spermatozoa obtained from the vas deferens have the shape of a round-headed nail. The head measures 30 μm in diameter and 14 μm of height. At the center of the flat face of the head emerges a single rigid spike of 53 μm in length. Cross sections of this spike show that it has a wall of 0.4 μm in thickness and a core of 0.6 to 0.8 μm. The outer surface of the spike has a longitudinal striation. When the spermatozoa are placed in sea water it is possible to observe the unfolding of rays. The number of rays in different spermatozoa of the same individual varies from 9 to 13. Each ray is formed by a channel-like sheath that contains a rigid rod that occupies about 1/3 the length of the ray. This rod has a transverse striation with a periodicity of 185A. The rays are bound among them by a thin membranous sheet that is highly folded in vas deferens spermatozoa. At the distal end of each ray there is a rigid spine of 50 μm in length. The nucleus is coplanar to the radial plane and it extends through the rays. The structure and ultrastructure of R typus spermatozoa depart from that reported for spermatozoa of other Caridea species.     

Use of 2-mercaptoethanol during chromatography of crab (Cancer pagurus) metallothionein on DEAE cellulose

The chromatographic properties of crab metallothionein appear particularly sensitive to oxidation, and this oxidation does not take place when 2-mercaptoethanol is present in the buffers. The chromatographic changes brought about by oxidation can be reversed by subsequent addition of 2-mercaptoethanol.     

The morphology and physiology of CAP organs in Jasus novaehollandiae (crustacea,decapoda, reptantia,macrura)

The location and external anatomy of the CAP organs of Jasus novaehollandiae were examined and found to be similar to those in Homarus gammarus (Laverack, 1978a). Histological examination of the organs showed threads or filaments arising from the internal surface of the spines to run through canals in the cuticle and join with dendrites of CAP sensory cells in the region of the hypodermis. The CAP neuron cell bodies lie in the nearby chordotonal organ strand. It is demonstrated that flexion of the limb causes the articulating membrane to deflect the spines of the sensillae distally. A variety of experimental techniques used to investigate the physiology of the organs reveals why previously reported attempts to record from the receptors failed. Direct stimulation of the sensillae with an analogue of the membrane and summation of many traces revealed phase constant responses at points corresponding to the covering and uncovering of the sensillae.     

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.     

Age,growth, mortality and sex ratio of the inshore population of the edible crab,Cancer pagurus (Linnaeus 1758) in South Wales (UK)

Age, growth, and mortality of the edible crab, Cancer pagurus, were determined for the native population in South Wales (UK). Sampling was carried out on a monthly basis between February 2001 and September 2002. Carapace width ranged between 10.4 and 163 mm. Based on the carapace width frequency distribution, the Swansea and Gower population was composed mainly of males belonging to the first and second age‐class (1 and 2), and of females belonging to the third and fourth age‐class (3 and 4). Sex ratio was 1.126 ± 0.27 in favour of males. Carapace width frequency distributions and weight‐at‐age data were used to estimate the von Bertalanffy growth equation parameters. For the population as a whole, these were: L∞ = 199 mm, W∞ = 1179.56 g, K = 0.24 year^?1, t₀ = ?0.1004 years. The overall carapace width–weight relationship was: W = 0.38(CW^2.69). Analysis of covariance indicated a significant difference in the carapace width–weight relationship between males and females in the study area. Total mortality Z and natural mortality M rates for combined sexes were 1.245 year^?1 and 0.567 year^?1, respectively. The exploitation ratio E was estimated to be 54.43%.     

Ultrastructure of a possible new type of crustacean cuticular strain receptor in Carcinus maenas (crustacea,decapoda)

A hitherto unknown sensillum type, the “intracuticular sensillum” was identified on the dactyls of the walking legs of the shore crab, Carcinus maenas. Each sensillum is innervated by two sensory cells with dendrites of “scolopidial” (type I) organization. The ciliary segment of the dendrite is 5–6 μm long and contains A-tubules with an electron-dense core and dynein arm-like protuberances; the terminal segment is characterized by densely packed microtubules. The outer dendritic segments pass through the endo- and exocuticle enclosed in a dendritic sheath and a cuticulax tube (canal), which is suspended inside a slit-shaped cavity by cuticular lamellae. The dendrites and the cavity terminate in a cupola-shaped invagination of the epicuticle. External cuticular structures are lacking. Three inner and four to six outer enveloping cells are associated with each intracuticular sensillum. The innermost enveloping cell contains a large scolopale that is connected to the ciliary rootlets inside the inner dendritic segments by desmosomes. Scolopale rods are present in enveloping cell 2. Since type I dendrites and a scolopale are regarded as modality-specific structures of mechanoreceptors, and since no supracuticular endorgan is present, the intracuticular sensilla likely are sensitive to cuticular strains. The intracuticular sensilla should be regarded as analogous to insect campaniform sensilla and arachnid slit sense organs.