Substance P-like immunoreactivity in the stomatogastric nervous systems of the crab Cancer borealis and the lobsters Panulirus interruptus and Homarus americanus

Summary The distribution of substance P-like immunoreactivity in the stomatogastric nervous systems of three decapod crustacean species, Cancer borealis, Homarus americanus, and Panulirus interruptus, was studied. The stomatogastric ganglion showed dense staining in the neuropil, but none in the somata. A single neuron stained in the esophageal ganglion. Lucifer yellow backfills and intracellular injections followed by incubation with the substance P antibody showed that the axons of this neuron project into the inferior esophageal nerves towards the paired commissural ganglia. The commissural ganglia showed a pronounced projection from a large bundle of fibers in the anterior medial portion of the circumesophageal connective. Additionally, less dense neuropil and stained somata were seen in the commissural ganglia. Staining was completely blocked by preabsorption with authentic substance P, physalaemin, eledoisin, and substance K. These data suggest that in the nervous system of crustacean species a molecule with C-terminal homology to substance P and other tachykinins is released as a neuroregulator in the stomatogastric ganglion.     

Mitotic and meiotic chromosomes of the American lobster Homarus americanus (Nephropidae, Decapoda)

The chromosomes of H. americanus have been characterised by C-banding, fluorochrome banding and restriction endonuclease banding. Thanks to these techniques, it has been possible to identify mitotic and meiotic figures clearly and to study the distribution and structure of heterochromatic regions. Moreover, we have identified small supernumerary chromosomes, variable in number and often asynaptic in first meiotic metaphase.     

Neuromuscular synapses on the dactyl opener muscle of the lobster Homarus americanus

The crustacean dactyl opener neuromuscular system has been studied extensively as a model system that exhibits several forms of synaptic plasticity. We report the ultrastructural features of the synapses on dactyl opener of the lobster (Homarus americanus) as determined by examination of serial thin sections. Several innervation sites supplied by an inhibitory motoneuron have been observed without nearby excitatory innervation, indicating that excitatory and inhibitory inputs to the muscle are not always closely matched. The ultrastructural features of the lobster synapses are generally similar to those described previously for the homologous crayfish muscle, with one major distinction: few dense bars are seen at the presynaptic membranes of these lobster synapses. The majority of the lobster neuromuscular synapses lack dense bars altogether, and the mean number of dense bars per synapse is relatively low. In view of the finding that the physiology of the lobster dactyl opener synapses is similar to that reported for crayfish, these ultrastructural observations suggest that the structural complexity of the synapses may not be a critical factor determining synaptic plasticity.This work was supported by funds from the University of Virginia Research and Development Committee.     

Immunoreactivity to molluskan neuropeptides in the central and stomatogastric nervous systems of the earthworm, Lumbricus terrestris L.

Polyclonal antisera against two related command neuropeptides (CNP2 and CNP4) described in neurons of the terrestrial snail Helix were used in a study of the nervous system of the earthworm Lumbricus. The CNP-like peptides belong to the same neuropeptide subfamily and bear a C-terminal signature sequence Tyr-Pro-Arg-X. The distribution patterns of immunoreactive (IR) neurons were studied in the central nervous system (CNS), skin, and stomatogastric nervous system of the earthworm. IR neurons were found in all CNS ganglia, the patterns being similar for both antibodies used. Several clusters of IR cells were observed in the cerebral and subesophageal ganglia. In the ventral cord ganglia, the number of IR cells decreased in the rostro-caudal direction, and the IR cells sent their fibers mostly into the median fiber bundle. Segmental nerves contained no IR fibers. After injury of the worm body, the number of IR neurons in the CNS significantly increased. In the skin, IR sensory neurons were present in sensory buds. The stomatogastric ganglia only contained IR fibers. Numerous scattered IR neurons were found in the inner subepithelial layer of the esophagus and formed the enteric plexus in which the cell bodies displayed a segmentally repeated pattern. Possible involvement of CNP-like-IR neurons in central integratory processes, sensory processes, and the regulation of feeding is discussed.This work was supported by INTAS (grant 01-2117), CRDF (grant RB1-2321-MO-02), and the Russian Foundation for Basic Research (grants 05-04-48724 and 03-04-48179).     

Central nervous system projections to and from the commissural ganglion of the crab Cancer borealis

Higher-order inputs provide important regulatory control to motor circuits, but few cellular-level studies of such inputs have been performed. To begin studying higher-order neurons in an accessible model system, we have localized, in the supraesophageal ganglion (brain), neurons that are candidates for influencing the well-characterized motor circuits in the stomatogastric nervous system (STNS) of the crab Cancer borealis. The STNS is an extension of the central nervous system and includes four ganglia, within which are a set of motor circuits that regulate the ingestion and processing of food. These motor circuits are locally regulated by a set of modulatory neurons, most of which are located in the paired commissural ganglia (CoGs). These modulatory neurons are well-positioned to receive input from brain neurons because the circumesophageal commissures (CoCs) connect the brain with the CoGs. We have performed a series of CoC backfills to localize the brain neurons that are likely to innervate the CoGs and are, therefore, candidates for influencinng the STNS motor patterns. CoC backfill-labeled neuronal somata within the brain are clustered around a subset of anatomically defined neuropil regions. We have concomitantly localized many CoG neurons that project into the brain. This latter pathway presumably includes neurons that provide feedback regarding ongoing STNS activity. Interestingly, nearly all of these brain and CoG neurons project through the medial aspect of the CoC. This work provides an initial framework for future studies to determine the way that higher-order input regulates rhythmic motor patterns. This work was supported by a grant from the National Institute of Neurological Disorders and Strokes (NS42813 to M.P.N.) and a National Science Foundation Fellowship (DGE9616278 to M.S.K.).     

Winter movements and activity of signal crayfish Pacifastacus leniusculus in an upland river,determined by radio telemetry

Radio-telemetry was used to study the late autumn and winter movements of twenty adult signal crayfish Pacifastacus leniusculus (32.9–63.8 mm carapace length) an introduced exotic crayfish species, in the upland River Wharfe, northern England. The distances moved during the study varied greatly between individuals (0–328 m). Movements were generally sporadic; crayfish would remain in one position for several weeks and make occasional movements to new locations. Total distances travelled, linear range and ranging area did not differ significantly between males and females. The distance travelled in upstream and downstream directions did not differ significantly and there was no correlation between distance travelled and crayfish size. Several high flow events occurred during the study, but these did not cause any mortality or apparent displacement of crayfish downstream, suggesting that this is not a significant factor in downstream dispersal or mortality of adults of this invasive crayfish species in winter. A marked reduction in large-scale movements occurred in mid-December which coincided with a decline in water temperature. There was a less distinct pattern in local activity which was strongly correlated with water temperature and varied before and after mid-December.     

Formation of sarcomeres in the embryonic heart of the lobster

Summary Cardiomyoblasts in the myocardium of embryonic lobsters at 3–4 weeks and 6 months of development were examined with the transmission electron microscope in order to describe the events in the formation of sarcomeres in a neurogenic cardiac system.Thick and thin myofilaments appear first in the cell periphery near the sarcolemma. They align in parallel in a sequential fashion to form consecutive sarcomeric units. Well-defined A and I bands appear before any semblance of a Z line is present. The initial sarcomere is anchored to the sarcolemma by the insertion of thin myofilaments into a region of electron dense material associated intimately with the sarcolemma. Myofibrils grow outward in several planes away from the electron-dense regions of membrane that serve as focal points for myofibril formation.     

Histamine immunoreactivity in the central nervous system of the spider Cupiennius salei

In this study, immunohistochemistry on Vibracut sections is used to demonstrate anti-histamine immunoreactivity in the brain of the spider, Cupiennius salei (Keys.) (Ctenidae). We describe a system of histamine-immunoreactive neurons within the central nervous system that consists of six omnisegmental neurons. These histamine-immunoreactive neurons form two subgroups: a dorsal system with two cells per hemisphere and a ventral system with only one cell per hemisphere. The cells have extended arborizations in the motor and sensory areas of all neuromeres in the suboesophageal ganglionic mass. We have also found histamine immunoreactivity in the photoreceptors of C. salei and suggest that histamine is a neurotransmitter of photoreceptors in all arthropods, since it is also known to occur in the photoreceptors of the other main arthropod taxa (Merostomata, Crustacea, and Insecta).     

Competition and parasitism in the native White Clawed Crayfish Austropotamobius pallipes and the invasive Signal Crayfish Pacifastacus leniusculus in the UK

Many crayfish species have been introduced to novel habitats worldwide, often threatening extinction of native species. Here we investigate competitive interactions and parasite infections in the native Austropotamobius pallipes and the invasive Pacifastacus leniusculus from single and mixed species populations in the UK. We found A. pallipes individuals to be significantly smaller in mixed compared to single species populations; conversely P. leniusculus individuals were larger in mixed than in single species populations. Our data provide no support for reproductive interference as a mechanism of competitive displacement and instead suggest competitive exclusion of A. pallipes from refuges by P. leniusculus leading to differential predation. We screened 52 P. leniusculus and 12 A. pallipes for microsporidian infection using PCR. We present the first molecular confirmation of Thelohania contejeani in the native A. pallipes; in addition, we provide the first evidence for T. contejeani in the invasive P. leniusculus. Three novel parasite sequences were also isolated from P. leniusculus with an overall prevalence of microsporidian infection of 38% within this species; we discuss the identity of and the similarity between these three novel sequences. We also screened a subset of fifteen P. leniusculus and three A. pallipes for Aphanomyces astaci, the causative agent of crayfish plague and for the protistan crayfish parasite Psorospermium haeckeli. We found no evidence for infection by either agent in any of the crayfish screened. The high prevalence of microsporidian parasites and occurrence of shared T. contejeani infection lead us to propose that future studies should consider the impact of these parasites on native and invasive host fitness and their potential effects upon the dynamics of native-invader systems.     

Identification of different types of serotonin-like immunoreactive olfactory interneurons in four infraorders of decapod crustaceans

Summary An antiserum raised against serotonin (5HT) was applied to the brains of representatives of four different infraorders of decapod crustaceans, and revealed two morphological classes of olfactory interneurons. They were classified by the position and size of their cell bodies, and by their connection pattern. One class consisted of giant olfactory interneurons and the other of globuli cells. They were regarded as input and intrinsic interneurons, respectively, because of their morphology. The two classes displayed a similar pattern in two of the infraorders, whereas only one class appeared in the other two infraorders.     

The potential use of PIT telemetry for identifying and tracking crayfish in their natural environment

A method for tracking crayfish and other benthic animals in rivers and streams, based on passive integrated transponder (PIT) technology, using a portable detector was investigated. The effect of implanting crayfish with PIT tags and the efficiency of the PIT tag detector system at locating tags is described. In a laboratory study 30 signal crayfish Pacifastacus leniusculus (>33.7 mm carapace length) were internally implanted with PIT tags (12-mm long×2.1-mm diameter) and 30 crayfish matched for size and sex were kept as controls and maintained for 6 months. Tagging had no significant effect on survival, moulting or growth of crayfish, and tag retention was 100%. The reader unit consists of an antenna coil mounted on a pole and designed to be moved over the streambed to search for tagged crayfish. Efficiency testing indicated that more than 80% of tags were identified and located when hidden within different stream microhabitats.     

The 76 kD cell-adhesion factor from crayfish haemocytes promotes encapsulation in vitro

Summary Semigranular cells from the crayfish, Pacifastacus leniusculus, were separated by Percoll gradient centrifugation and were used to study the encapsulation of foreign particles. The semigranular cells were found strongly to encapsulate glass beads coated with haemocyte lysate in which the prophenoloxidase-activating system had been activated with laminarin or with a low concentration of calcium ions. The granular cells only weakly encapsulated these particles. The encapsulationpromoting factor was purified from haemocyte lysates and found to be a 76 kD protein which was recognized by an antiserum to the previously described 76 kD cell-adhesion factor. After the last step in purification (Con A-Sepharose chromatography), the flowthrough consisted of several proteins, which had some, but less, encapsulation-promoting activity and contained a 30 kD band that was also recognized by the antiserum to the 76 kD cell-adhesion factor. If the haemocyte lysate prepared in low [Ca²⁺] was incubated with a -1,3-glucan prior to purification, no 76 kD protein could be isolated but only a 30 kD protein. The 30 kD protein thus seems to be a degradation product of the 76 kD cell-adhesion factor. We conclude that the 76 kD protein which is released from degranulating haemocytes, and to a lesser extent its 30 kD fragment, can promote encapsulation. Phenoloxidase did not have any encapsulation-promoting activity.     

Cellular organization of the embryonic lobster heart

Summary The cellular organization of the embryonic heart of the lobster Homarus americanus was examined in 6-week and 6-month-old animals. The heart wall consists of an outer adventitial layer of fibroblast cells and an inner layer of transversely striated myocardial cells. Present in close association with the myocardium are cardiac neurons, hemocytes and so-called storage cells.Adjacent fibroblasts form fasciae adhaerentes and gap junctions. Adherent junctions also occur between fibroblasts and myocardial cells. Intercalated discs and differentiated membrane regions of close apposition (4 nm) occur between adjacent myocardial cells.The cardiac neurons form a ganglion that contains four small and five large somata. Regions of neuropil are present. Motor axons arising from the cardiac ganglion form neuromuscular synapses with the myocardial cells.The storage cells contain large inclusions and form gap junctions with the myocardial cells. They may supply nutritive material to the developing myocardium.The heart at 6 weeks is about 200 m long and 160 m wide. At 6 months, it is about 300 m long and 250 m wide. The myocardium at 6 weeks is one cell layer thick, and the cells are from 2–6 m in maximum width. At 6 months the myocardium is 2–4 cells thick, and the cells are from 6–12 m in width. Therefore, the myocardium grows by an increase in the number and size of the myocardial cells.     

Changes in haemolymph ion concentrations of Astacus astacus L. and Pacifastacus leniusculus (Dana) after exposure to low pH and aluminium

During exposure to soft water, acidified to pH 4.0, the haemolymph concentrations of Na⁺, K⁺, and Cl^– decreased whereas the Ca²⁺ concentration fluctuated in Astacus astacus. The haemocyte content of K⁺ decreased from 9% to 2% of the total haemolymph K⁺ content after exposure to pH 3.7 for 3 days. Within 14 days, 250 µg Al³⁺ l^–1, as Al₂(SO₄)₃ at pH 5.0, reduced the haemolymph Na⁺ content in Astacus astacus and Pacifastacus leniusculus, however, the effects were less pronounced than earlier reported for fish. Disturbed ion regulation, mainly depending on low pH, is thought to contribute to the absence of these species in acid waters.     

Localization of pigment-dispersing hormone (PDH) immunoreactivity in the central nervous system of Carcinus maenas and Orconectes limosus (Crustacea), with reference to FMRFamide immunoreactivity in O. limosus

Summary By use of antisera raised against synthetic pigment-dispersing hormone (PDH) of Uca pugilator and FMRFamide, the distribution of immunoreactive structures in the central nervous system (CNS) of Carcinus maenas and Orconectes limosus was studied by light microscopy. In both species, a total of 10–12 PDH-positive perikarya occur amongst the anterior medial, dorsal lateral and angular somata of the cerebral ganglion (CG). In C. maenas, one PDH-perikaryon was found in each commissural ganglion (COG) and several more in the thoracic ganglion. In O. limosus, only four immunopositive perikarya could be demonstrated in the ventral nerve cord, i.e., two somata in the anterior and two in the posterior region of the suboesophageal ganglion (SOG). PDH-immunoreactive tracts and fiber plexuses were present in all central ganglia of both species, and individual axons were observed in the connectives. FMRFamide-immunoreactivity was studied in O. limosus only. Neurons of different morphological types were found throughout the entire CNS, including numerous perikarya in the anterior medial, anterior olfactory, dorsal lateral and posterior cell groups of the CG. Four perikarya were found in the COG, six large and numerous smaller ones in the SOG, and up to eight cells in each of the thoracic and abdominal ganglia. In each ganglion, the perikarya form fiber plexuses. Axons from neurons belonging to the CG could be traced into the ventral nerve cord; nerve fibers arising from perikarya in the SOG appeared to project to the posterior ganglia. In none of the structures examined colocalization of PDH- and FMRF-amide-immunoreactivity was observed.Dedicated to Prof. K.-E. Wohlfarth-Bottermann on the occasion of his 65th birthday     

The artificial incubation of crayfish eggs: review and report from an experimental study concerning the effects of offspring origin (maternal or artificial incubation) on the survival and growth of juvenile signal crayfish (Pacifastacus leniusculus, Astacidae)

The development of artificial incubation techniques in astacid crayfish has attracted attention from scientists in many countries ever since the nineteenth century. It is only in the last few years that these techniques, along with studies on egg storage and transport, have provided reliable options for improving the reproductive phase in farming. The juveniles produced need to be reared until they reach a sufficient size both for restocking and for growing purposes. In view of the current level of knowledge of rearing juvenile astacids, two 80-day experiments were carried out under controlled conditions to compare the survival and growth of Stage 2 juvenile signal crayfish (Pacifastacus leniusculus) from two origins: maternal or artificial incubation. In the first experiment, three treatments were tested: juveniles from artificially incubated eggs with formaldehyde treatments, juveniles from maternal incubation and a mixture from both origins (50% each). Survival rates ranged from 87.8% to 93.3% with no significant differences among treatments. Crayfish from artificial incubation grew significantly faster (11.47 mm carapace length (CL), 373.80 mg weight) than crayfish from maternal incubation. In the second experiment, a bifactorial design included four treatments: the crayfish was derived from artificial or from maternal incubation and was fed once a day or twice a day. Final survival rates ranged from 68.89% to 77.78%, with no significant differences among treatments. Crayfish from artificial incubation grew significantly faster than crayfish from maternal incubation. The highest CL (14.54 mm) and weight (780.13 mg) were reached by the juveniles from artificial incubation that were fed once a day. No significant differences were found between the two feeding frequencies. Results showed that artificial incubation with formaldehyde treatments had no harmful effects and made it feasible to get a better performance from the juveniles obtained.     

Distribution of peptidyl-glycine alpha-amidating monooxygenase immunoreactivity in the brain,pituitary and islet organ of the anglerfish (Lophius americanus)

Peptidyl-glycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is an enzyme that catalyzes conversion of glycine-extended peptides to alpha-amidated bioactive peptides. Two peptides that are processed at their carboxyl-termini by this enzyme are neuropeptide Y and anglerfish peptide Y, both of which possess a C-terminal glycine that is used as a substrate for amidation. Results from previous reports have demonstrated that neuropeptide Y-like and anglerfish peptide Y-like immunoreactivities are present in the brain of anglerfish (Lophius americanus). Furthermore, neuropeptide Y-like peptides, namely anglerfish peptide Y and anglerfish peptide YG (the homologues of pancreatic polypeptide) are present in the islet organ of this species. Neuropeptide Y has also been localized in the anterior, intermediated and posterior lobes of the pituitary gland in a variety of species. In order to learn more about the distribution of the enzyme responsible for alpha amidation of these peptides in the brain and pituitary and to specifically investigate the relationship of this enzyme to peptide synthesizing endocrine cells of the anglerfish islet, we performed an immunohistochemical study using several antisera generated against different peptide sequences of the enzyme. PAM antisera labeled cells in the islet organ, pituitary and brain, and fibers in the brain and pituitary gland. The PAM staining pattern in the brain was remarkably similar to the distribution of neuropeptide Y immunoreactivity reported previously. Clusters of cells adjacent to vessels in the anterior pituitary displayed punctate PAM immunoreactivity while varicose fibers were observed in the pituitary stalk and neurohypophysis. Endocrine cells of the islet organ were differentially labeled with different PAM antisera. Comparison of the staining patterns of insulin, glucagon, and anglerfish peptide Y in the islet organ to PAM immunoreactivity suggests a distribution of forms of PAM enzyme in insulin and anglerfish peptide Y-containing cells, but no overlap with glucagon-producing cells. The results also indicate that PAM immunoreactivity is widely distributed in the brain, pituitary and islet organ of anglerfish in cells that contain peptides that require presence of a C-terminal glycine for amidation.     

An historical overview of the Republic of Ireland's lobster (Homarus gammarus Linnaeus) fishery,with reference to European and North American (Homarus americanus Milne Edwards) lobster landings

Historical developments of the Republic of Ireland's Homarus gammarus fishery are reviewed and compared in the context of landings in Europe and the North American H. americanus fishery. H. gammarus fishing has provided essential income for over a century to many Irish coastal communities. There have been significant fluctuations (greater than 460%) in recorded landings over this time and the relative importance of H. gammarus fishing has changed with the establishment of ancillary crustacean fisheries. In 1994, H. gammarus landings reached an all-time high of 714 tonnes, with a landed value of IR £6.3 million; in 1999 landings were 597 tonnes valued at IR £6.15 million. Current Republic of Ireland lobster fishery regulations are: (a) minimum carapace length of 85 mm, (b) ban on landing lobsters that have been `V-notched' or have a mutilated tail fan, and (c) prohibition of capturing lobsters by SCUBA diving. Chronological details on recent legislation, regulations and landings relating to Ireland's lobster fishery sector are reviewed. In the last 40 years landings of H. gammarus in the Republic of Ireland exhibit signs of an overexploited stock characterised by a reduction in catch per unit effort and there has been a general decline of total landings for H. gammarus throughout Europe. This contrasts with North America, where many regulations govern the H. americanus fishery and landings appear to be in a healthier state.