Multivesicular body formation and function in the light-adapted crayfish retina: a new interpretation

Summary Quantitative TEM refutes the hypothesis of multivesicular body formation at dawn via the degradation of photoreceptor. No significant relationship exists between multivesicular bodies and rhabdom diameter or between multivesicular bodies and coated vesicles. The density of multivesicular bodies is related to the vesicle-producing Golgi bodies. A new theory concerning the formation and possible function of multivesicular bodies is discussed.     

The topological organization of primary afferents in the terminal ganglion of crayfish,Procambarus clarkii

Summary The central projections of primary afferents in the terminal ganglion of the crayfish can be seen when an axonal filling with nickel chloride with subsequent silver intensification was used for identification. We describe here the topological relationships of the projections to the landmark structures of the neuropil.The terminal ganglion has five pairs of sensory nerves associated with the mechanosensory hairs and internal proprioceptors. The projection fields of the primary sensory neurons in the nerves Rl and R2 are almost entirely restricted to the ipsilateral half of the ganglion, whereas those of the nerves R3, R4 and R5 cross the midline to form three sensory commissures, A6SCI, A7SCI and A7SCII. The projection fields are segregated from each other, although all are restricted to the ventral neuropil which lies under the ventral intermediate tract (VIT). The intersegmental projections that ascend via the connective ipsilateral to their origins could be observed. This pattern of projection correlates well with the receptive fields exhibited by several mechanosensory interneurons on the body surface of the final segment.     

Morphology of descending statocyst interneurons in the crayfish Procambarus clarkii Girard

Summary The morphological features of descending interneurons that responded to the artificial bending of statolith hairs were assessed with intracellular recording and staining techniques. Seven statocyst interneurons were identified on the basis of their structure and response characteristics and designated as interneurons S1 to S7. All seven identified interneurons project to the optic lobe, where the optic nerve also projects, and to the dorsal part of the tritocerebrum, where the eyestalk motoneurons originate. All except interneuron S6 also extend their major branches to other neuropilar regions. S2 projects to the dorsal part of the deutocerebrum, where the statocyst nerve terminates, and S3 to the dorsal part of deutocerebrum and the antennal lobe. Four other interneurons (S1, S4, S5, S7) also extend their branches to the parolfactory lobe to which the statocyst nerve projects as well as to the deutocerebrum and antennal lobe. The extensive dendritic projections of S1–S7 suggest that they are complex multimodal interneurons rather than simple relay interneurons, receiving at least visual and statocyst sensory information. The function of the antennal lobe branches, however, has yet to be determined since the functional role of antennal input in equilibrium control is unknown.     

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.     

Multimodal responses of the nonspiking giant interneurons in the brain of the crayfish Procambarus clarkii

Three pairs of nonspiking giant interneurons (NGIs; G1, G2, and G3) of the crayfish brain responded with depolarizing and hyperpolarizing graded potentials to body tilt in roll to the ipsi- and contralateral sides in the dark. The higher and the larger the angle of body tilt, the larger was the amplitude of the geotactic responses. In ipsilaterally statocystectomized animals, all the NGIs responded with hyperpolarizing potentials only to the contralateral side-down tilt, whereas in contralaterally statocystectomized animals, they responded with depolarizing potentials only to the ipsilateral side-down tilt. In bilaterally statocystectomized animals, none of the NGIs responded to body tilt in the dark, but in the presence of an overhead light, they exhibited depolarizing and hyperpolarizing potentials in response to body tilt to the ipsi-and contralateral sides, respectively. All the NGIs responded with depolarizing and hyperpolarizing graded potentials to illumination of the contra- and ipsilateral eyes, respectively. The amplitude of these visual responses, however, varied in association with the amplitude of the geotactic response produced by body tilt. These results indicate that the NGIs integrate the sensory inputs from eyes and statocysts and that the interaction between sensory inputs from the left and right sensory organs with either the same modality or with different modalities enhance the directional sensitivity of NGIs as premotoneurons in the compensatory oculomotor system.     

Cloning and expression of a cDNA encoding a matrix peptide associated with calcification in the exoskeleton of the crayfish

Calcification-associated peptide (CAP)-1 isolated from the exoskeleton of the crayfish, Procambarus clarkii, has anti-calcification activity and chitin-binding ability and is, therefore, considered to be associated with calcification. In this study, a cDNA encoding CAP-1 was cloned and characterized. An open reading frame encoded a pre-propeptide of 99 amino acid residues, which was composed of a signal peptide, a CAP-1 precursor and two-basic amino acid residues at the C-terminus. The dibasic residues were not observed in the natural CAP-1. Expression analyses using Northern blot and RT-PCR revealed that the mRNA encoding CAP-1 was strongly expressed in the epidermal tissue during the postmolt stage, where and when the calcification takes place. These results support that CAP-1 may play an important role in the calcification of the exoskeleton. Based on the nucleotide sequence of the cDNA encoding CAP-1, a recombinant CAP-1 and that carrying the basic residues at the C-terminus were expressed in Escherichia coli. Anti-calcification assay showed that these recombinant peptides were less active than natural CAP-1, indicating that the phosphate group at the 70th residue, Ser, in natural CAP-1 is important for inhibitory activity and that the paired basic residues have some contribution to the elevation of inhibitory activity.     

Synaptic connections between sensory afferents and the common inhibitory motoneuron in crayfish

The sensory inputs to the common inhibitory motoneuron that innervates every leg muscle of the crayfish Procambarus clarkii (Girard) were analyzed by performing intracellular recordings from its neurite within the neuropil of the 5th thoracic ganglion. Two types of sensory inputs involved in locomotion were studied, those from a movement coding proprioceptor (the coxobasal chordotonal organ) and those from sensory neu rons coding contact forces exerted at the tip of the leg on the substrate (the dactyl sensory afferents). Sinusoidal movements applied to the chordotonal organ strand induced a stable biphasic response in the common inhibitory motoneuron that consisted of bursts of spikes during release and stretch of the strand, corresponding to raising and lowering of the leg, respectively. Using ramp movements imposed on the chordotonal strand, we demonstrated that only movement-coding chordotonal afferents produce excitatory post-synaptic potentials in the common inhibitory motoneuron; these connections are monosynaptic. Mechanical or electrical stimulation of the dactyl sensory afferents resulted in an increase in the tonic discharge of the common inhibitory motoneuron through polysynaptic excitatory pathways. These two types of sensory cues reinforce the central command of the common inhibitory motoneuron and contribute to enhancing its activity during leg movements, and thus facilitate the relaxation of tonic muscle fibres during locomotion.Abbreviations ADR anterior distal root - A Lev anterior levator nerve - CB coxo-basipodite joint - CBCO coxo-basal chordotonal organ - CI common inhibitory motoneuron - Dep depressor nerve - DSA dactyl sensory afferents - EPSP excitatory post-synaptic potential - IN interneuron - MN motoneuron - PDR posterior distal root - P Lev posterior levator nerve - Pro promotor nerve - Rem remotor nerve     

Shifts in aquatic macroinvertebrate biodiversity associated with the presence and size of an alien crayfish

To investigate the effects of Procambarus clarkii on macroinvertebrate diversity, we conducted a mesocosm experiment simulating small pools in rice field pads after the rice season. We hypothesized that crayfish predation would negatively impact macroinvertebrate diversity, and the magnitude of this impact should vary with the size of P. clarkii. We conducted a short-term mesocosm experiment to determine macroinvertebrate diversity in the presence of three size classes and in the absence of crayfish, as well as the diet composition of crayfish from the three size classes. At the end of the experiments, the diet of crayfish was composed of the most available taxa (Culicidae, Chironomus, Tanytarsini and Orthocladinae). These results also show evidence that, in confined areas, crayfish are important predators of major rice pests such as rice Chironominae larvae. Macroinvertebrate diversity was negatively affected by crayfish presence, but the effect was inversely proportional to crayfish size. The highest diversity index was obtained in the absence of P. clarkii, and juvenile crayfish significantly reduced macroinvertebrate diversity. Thus, the impact of P. clarkii on aquatic macroinvertebrates is size dependent and may be relevant in small pools formed in rice field pads from early autumn to late winter. Overall, our findings suggest that the negative effects of P. clarkii on macroinvertebrate diversity may be particularly strong in local natural assemblages confined to puddles of water or small ponds in wetland areas.     

Breeding biology of the Louisiana red swamp crayfish Procambarus clarkii Girard in Lake Naivasha,Kenya

This paper describes the breeding biology of the Louisiana red swamp crayfish (Procambarus clarkii Girard) that has been introduced in a tropical lake, Naivasha. Notable differences in crayfish biology are found between the place of origin in Louisiana and lake Naivasha.In Naivasha sexual maturity in females is reached at a larger size (42 mm carapace length (CL), 76 mm full length (FL)) than in males (40 mm CL, 71 mm FL). The corresponding figures for Chalmette area of Louisiana are 31–32 mm CL for both sexes and at Ben Hur Research Centre, Louisiana, 33 mm CL. The females weigh between 15 and 20 g at the onset of egg deposition. The corresponding Louisiana values are from 5 to 10 g. Crayfish in Naivasha as in Louisiana breed both inside burrows which are 10–70 cm deep, and on the sediment in shallow water at a depth of between 0.5 and 4 m. These observations suggest that reproduction is very much a lake edge activity. The number of breeding individuals in Naivasha varies with the trend of the water surface level. Sexually active females (form I) as judged from secondary sexual characters (appearance of basal hooks at the base of 3rd and 4th walking legs) persist throughout the year at no less than 60% of the adult male population. This is confirmed by the appearance of recently hatched juveniles (3.4 to 6.5 mm CL) in the water throughout the year. Similarly berried females in Naivasha are found throughout the year, though with peaks at irregular intervals. In Louisiana breeding takes place only from August through November with a single peak in October. The fecundity of crayfish in Naivasha is 433 (SE ± 21, N = 133) and there is a positive correlation between body length and egg number.     

Molecular characterization of the sarcoplasmic calcium-binding protein (SCP) from crayfish Procambarus clarkii

Sarcoplasmic Calcium-binding Protein (SCP) is believed to function as the invertebrate equivalent of vertebrate parvalbumin, namely to “buffer” cytosolic Ca²⁺. We have cloned and characterized a novel SCP from axial abdominal muscle of crayfish Procambarus clarkii (referred to as pcSCP1), and have examined tissue specific distribution and expression as a function of molting stage in non-epithelial and epithelial tissues. The complete sequence of pcSCP1 consists of 1052 bp with a 579 bp open reading frame, coding for 193 amino acid residues (molecular mass of 21.8 kDa). There is a 387 bp 3′ terminal non-coding region with a poly (A) tail. The deduced pcSCP1 protein sequence matched most closely with published SCP sequences from another crayfish Astacus leptodactylus (92.8%) and from shrimp (78.6–81.2%) and fruit fly (53%). Real-time PCR analysis confirmed that pcSCP1 is ubiquitously expressed in all tissues tested (gill, hepatopancreas, intestine, antennal gland, muscle); however it is most abundant in muscle particularly in the axial abdominal muscle. The real-time PCR analysis revealed that pcSCP1 expression is downregulated in pre- and postmolt stages compared with intermolt. Epithelial (hepatopancreas and antennal gland) SCP expression exhibited a more dramatic decrease than that observed in muscle. Expression trends for pcSCP1 paralleled published trends for sarco/endoplasmic reticular calcium ATPase (SERCA), suggesting that their cellular function in regulating intracellular Ca²⁺ is linked.     

A novel calcium-binding peptide from the cuticle of the crayfish, Procambarus clarkii

A novel peptide named calcification-associated peptide (CAP)-2 was isolated from the exoskeleton of the crayfish, Procambarus clarkii. CAP-2 consists of 65 amino acid residues and has a 44% sequence identity with CAP-1 characterized previously. It has a chitin-binding domain observed in many arthropod cuticle proteins. CAP-2 showed inhibitory activity on calcium carbonate precipitation and chitin-binding ability. A CAP-2 cDNA was cloned using RT-PCR and RACE and the open reading frame encoded a precursor peptide consisting of a signal peptide and CAP-2. RT-PCR revealed that CAP-2 mRNA was exclusively expressed in the epidermal tissue during the postmolt stage, the site and stage being associated with calcification. Calcium-binding assay using recombinant CAP-2 revealed that this peptide had affinity for calcium ions with a Kd value of about 1 mM. All these results suggest that CAP-2 serves as a nucleator or a regulator in the calcification of the exoskeleton.     

Differences in synaptic output between excitatory and inhibitory motoneurons in a crayfish muscle

A pair of antagonistic motoneurons, one excitatory and one inhibitory, innervates the distal accessory flexor muscle in the walking limb of the crayfish Procambarus clarkii. The number and size of synapses formed by these two axons on the muscle fibers (neuromuscular synapses) and on each other (axo-axonal synapses) were estimated using thin-section electron microscopy. Although profiles of nerve terminals of the two axons occur in roughly equal proportions, the frequency of occurrence of neuromuscular synapses differed markedly: 73% were excitatory and 27% were inhibitory. However, inhibitory synapses were 4–5 times larger than excitatory ones, and consequently, the total contact areas devoted to neuromuscular synapses were similar for both axons. Axo-axonal synapses were predominantly from the inhibitory axon to the excitatory axon (86%), and a few were from the excitatory axon to the inhibitory axon (14%). The role of the inhibitory axo-axonal synapse is presynaptic inhibition, but that of the excitatory axo-axonal synapse is not known. The differences in size of neuromuscular synapses between the two axons may reflect intrinsic determinants of the neuron, while the similarity in total synaptic area may reflect retrograde influences from the muscle for regulating synapse number.     

Neuroanatomy of the terminal (sixth abdominal) ganglion of the crayfish,Procambarus clarkii (Girard)

Summary We studied the neuroanatomy of the terminal (sixth abdominal) ganglion in the crayfish Procambarus clarkii with silver-impregnated sections and nickel fills. We describe the fiber tracts, commissures and neuropilar areas, and give the topological relationships of motoneurons and intersegmental interneurons with reference to their neuropilar landmark structures.All five anterior abdominal ganglia have an almost identical number of 600–700 neurons with a similar pattern of distribution. Each contains a single neuromere with a common plan of neuropil organization. In contrast, the terminal ganglion consists of two neuromeres which appear to be derived from the intrinsic sixth abdominal and telson ganglion. The basic organization of each neuromere parallels that of the third abdominal ganglion in the appearance and arrangement of fiber tracts and commissures, although some modifications occur. The fusion of two neuromeres is represented by the duplication of segmentally homologous neurons, MoGs and LGs, whose topological relationships to the neuropil structures are similar to those of the anterior ganglion.We also discuss the origin of the telson and its ganglion (the seventh abdominal neuromere), and dispute the classical theory that the telson derives from a postsegmental region.     

Synaptic connections of the common inhibitory motoneurone within the fifth thoracic ganglion of crayfish

The common inhibitor (CI) has been studied morphologically and electrophysiologically in the fifth thoracic ganglion of crayfish (Procambarus clarkii). It has a large soma and possesses two separate dendritic fields arising from distinct integrative segments.In vitro preparations display motor outputs ranging from tonic activity to fictive locomotion. The CI's tonic firing frequency increases as more excitors are recruited, and displays two peaks of frequency during fictive locomotion, one during stance, the other during swing.Paired intracellular recordings have been used to demonstrate the different central synaptic connections received or made by the CI. At least 27% of the proximal excitors receive monosynaptic connections from the CI corresponding to post-synaptic depolarizations of small amplitude mediated by GABA. However as they do not change the overall activities of the excitors which receive them, they may be used for local inhibition within the dendrites. Besides, electrical synapses between several proximal excitors and the CI may synchronize their activity.The CI receives synaptic connections arising from interneurones. Some are direct either by inhibitory monosynaptic connections or by electrical couplings whereas others arise through polysynaptic pathways. All these connections are functionally significant in the control of the CI firing activity and in its motor coordinations.     

Distribution of synapses on two types of ascending interneurons in the crayfish,Procambarus clarkii

Summary About 60 pairs of ascending interneurons are present in the terminal ganglion of the crayfish Procambarus clarkii (Girard). Some of these interneurons have been impaled intracellularly, characterized physiologically, and then labeled with horseradish peroxidase (HRP) to examine the distribution and ultrastructure of synapses. A close relationship between ultrastructure and physiological properties has been found between two types of interneurons, which either have a pre-motor effect upon motor neurons or have no such effect. In one interneuron with a pre-motor effect (6D2), input and output synapses are intermingled on thicker branches, whereas only input synapses are found on small diameter branches. Only input synapses have been observed on the branches in another interneuron with-out a pre-motor effect (6B1). No differences in branch morphology are found in these two interneurons. Interneuron 6D2 contains large numbers of small round agranular vesicles, but the same type of synaptic vesicles is rarely seen in interneuron 6B1, which has no output synapses. Our results indicate a good correlation between the synaptic distribution and pre-motor effects of interneurons in the terminal ganglion.Abbreviations A6, 7 Sixth and seventh abdominal segment of the terminal ganglion - AVC anterior ventral commissure - DC I dorsal commissure I - DIT dorsal intermediate tract - DMT dorsal medial tract - eLG extra lateral giant interneuron - LVT lateral ventral tract - LG lateral giant interneuron - LVT lateral ventral tract - MDT median dorsal tract - MG medial giant interneuron - MoG motor giant neuron - MVT median ventral tract - PVC posterior ventral commissure - R1s sensory fiber tract of nerve root 1 - R3m motor fiber tract of nerve root 3 - R4–7 nerve roots 4–7 - SC I,II sensory commissure I,II - VC I,III ventral commissure I, III - VIT ventral intermediate tract - VLT ventral lateral tract - VMT ventral medial tract     

Invasion of an occupied niche by the crayfish Orconectes rusticus: potential importance of growth and mortality

We are exploring mechanisms of an invasion that contradicts the oft-cited generalization that species invade vacant niches. In northern Wisconsin lakes, the introduced crayfish Orconectes rusticus is replacing two ecologically similar resident congeners, O. virilis and O. propinquus. In laboratory experiments, we compared growth and mortality of individually maintained crayfish offered one of five ad libitum diets: invertebrates, macrophytes, dentritus, periphyton or all items combined. Mortality was highest for O. virilis and lowest for O. rusticus. Macrophyte diets yielded the highest mortality. All three species grew best on invertebrate and combination diets but grew little or not at all on diets of periphyton, detritus or macrophytes. O. rusticus and O. virilis grew more than O. propinquus. O. rusticus grew more quickly and/or was better able to survive overall than its congeners. Therefore, O. rusticus would probably have advantages over O. virilis and O. propinquus in competitive interactions, reproductive success and avoiding size-selective fish predation. Subtle interspecific differences may interact strongly with other ecological factors and contribute to the displacement of resident species from a well-occupied niche.     

Colonization of freshwater habitats by an introduced crayfish, <Emphasis Type="Italic">Procambarus clarkii</Emphasis>, in Southwest Iberian Peninsula

The introduction of some crustacean species has produced alterations of freshwater environments and declines of native species worldwide. The red swamp crayfish, Procambarus clarkii Girard, was introduced in the Southwest Iberian Peninsula in the 1970’s, producing severe impacts on rice agriculture and on native biota such as macrophytes, gastropods, native crayfish and amphibians. We studied the distribution of P. clarkii in two areas of SW Iberian Peninsula: the Sado River basin (SW Portugal), an area colonized by this species around 1990, and the Parque Natural del Entorno de Doñana (SW Spain), colonized soon after its introduction in the Iberian Peninsula, in the 1970’s. Our main goal was to determine which factors limit crayfish distribution, which could help to identify the most effective management practices to contain its spread. Procambarus clarkii was found in most types of water bodies, including small and shallow ones. Distance to a crayfish source was the single predictor variable explaining crayfish occurrence in most types of habitats and in both areas. The only exception was for the Sado permanent stream points, where crayfish presence was negatively affected by an interaction between elevation and flow velocity. Other habitat characteristics have apparently little or no importance for its successful colonization. Moreover, this study indicated that overland dispersal is apparently a frequent phenomenon in this species. Our findings can be used to determine which habitats are most likely to be colonized by the crayfish and to develop practical measures which may limit its spread and minimize its impacts.     

Dopaminergic Modulation of Neurosecretory Cells in the Crayfish

The main aims of this paper are (a) to locate possible dopaminergic neurons in the eyestalk with anti-tyrosine hydroxylase antibodies, (b) to search for the presence of dopamine (DA) in the nervous structures of the eyestalk, (c) to explore its release, and (d) to test the effect of DA on neurosecretory cells in the eyestalk.Experiments were performed in adult crayfishes Procambarus clarkii, in isolated optic peduncle. Immunocytochemistry was made with the antibody against its precursor synthesizing enzyme tyrosine-hydroxylase. The content and release studies of DA were made using high performance liquid chromatography (HPLC). Extracellular and intracellular recordings were conducted with conventional recording techniques.A large number (2000) of immunopositive somata of different sizes and shapes were identified in various regions of the eyestalk. The majority of somata are of the smallest size (5–25 m diameter). DA content in the eyestalk was 5.6 ± 0.1 pmol per structure; the greatest content is in the MT (over 60%). A basal level release of DA was observed. Incubation of eyestalks in solution containing a high K⁺ concentration increased the DA release (79%). Two effects of DA on the excitability of X-organ neurons were observed; an excitatory effect on neurons of 25 m somata diameter and another inhibitory effect in the group of 35-m somata diameter neurons. The excitation occurs with a depolarization and decrement of membrane conductance in the cell soma while the inhibition occurs with a hyperpolarization and increment of membrane conductance in soma.We concluded the following: (1) Dopamine is present in each optic ganglia of the crayfish eyestalk. (2) There is a basal release of DA from the isolated eyestalk. (3) DA release is enhanced threefold by eyestalk incubation in 40 mM [K⁺] solution. (4) DA selectively excites a population of neurons with low-speed conduction axons, and small somata in the X-organ–sinus gland system, while inhibiting another population characterized by higher axonal conduction speed and large somata. (5) These observations support a role for DA as a neurotransmitter or neuromodulator in the X-organ neurons of the crayfish eyestalk.Dr. Hugo Aréchiga died on September 15th of 2003     

Shape and disposition of clefts,tubules, and sarcoplasmic reticulum in long and short sarcomere fibers of crab and crayfish

Summary The disposition of surface invaginations (clefts, Z and T tubules) and of the sarcoplasmic reticulum has been examined by electron microscopy at three accelerating voltages (100, 200 and 1000 kV) and by phase-contrast light microscopy in crustacean muscles infiltrated by the Golgi stain. In long-sarcomere, tonic type fibers, an extensive system of invaginating clefts has been observed, along with both Z and T tubules. Z and T tubules form interconnections with each other, but only T tubules form specific contacts with the sarcoplasmic reticulum, which in these fibers forms an extended and continuously fenestrated network. In short-sarcomere, phasic type fibers, a ladder-like disposition of an abundant T network is found. Z tubules are absent in these fibers. The sarcoplasmic reticulum forms more frequent junctions with flattened areas of T tubules and with clefts, but has less extensive free surfaces than in the long-sarcomere fibers.We wish to dedicate this paper to the late Graham Hoyle, whose lifetime of work and interest in the study of muscle from a comparative point of view has been an inspiration to us.