The effects of some putative transmitters and biogenic amines on uropod abductor muscle in the crayfish Procambarus clarkii and Cambaroides japonicus

The effects of some putative transmitters and biogenic amines were examined on the uropod ventral abductor exopodite (AbdExV) muscle in two crayfish species Procambarus clarkii and Cambaroides japonicus. Bath application of L-glutamate to the AbdExV muscle caused sustained contract while gamma-aminobutyric acid (GABA) depressed the nerve-evoked contraction of the muscle. Acetylcholine (ACh) had no effect on both the resting tension and the nerve-evoked contraction. Iontophoresis of L-glutamate and GABA onto the surface of the muscle fiber further confirmed that glutamate and GABA are the possible excitatory and inhibitory transmitters respectively at the neuromuscular junction of AbdExV muscle. Bath application of 5-hydroxytryptamine (5-HT) and octopamine (Oct) caused enhancement of the nerve-evoked contraction but dopamine (DA) had no effect on both the resting tension and the nerve-evoked contraction.     

Dendritic properties of uropod motoneurons and premotor nonspiking interneurons in the crayfish Procambarus clarkii Girard

Dendritic properties of uropod motoneurons and premotor nonspiking interneurons of crayfish have been studied using intradendritic recording and current injection. The input resistance of phasic motoneurons (5.20 ± 0.5 M; mean ± standard error) measured by injecting constant hyperpolarizing current was significantly lower than that of tonic motoneurons (10.3 ± 2.6 M; 0.02 < P < 0.05). The membrane time constant of phasic motoneurons (7.3 ± 0.9 ms) was also significantly shorter than that of tonic motoneurons (24.3 ± 2.5 ms; P < 0.001). Both types of motoneurons behaved linearly during hyperpolarization and sub-threshold depolarization. Nonspiking interneurons showed outward rectification upon depolarization. During hyperpolarization, their membrane behaved linearly and showed significantly higher input resistance (19.5 ± 2.5 M) than phasic and tonic motoneurons (P < 0.001). Their membrane time constant (38.0 ± 5.7 ms) was significantly longer than that of phasic motoneurons (P < 0.001) but not than that of tonic motoneurons (P > 0.05). In response to intracellular injection of sinusoidally oscillating current, phasic motoneurons showed one or two spikes per depolarization period irrespective of oscillating frequency ranging from 1 to 16 Hz. Tonic motoneurons showed larger numbers of spikes per stimulus period at lower frequencies. Nonspiking interneurons also showed phase-locked effects on the motoneuron spike activity. The effective frequency range over which injected oscillating current could modulate motoneuron spike activity was similar for tonic motoneurons and nonspiking interneurons.     

The slow muscles in the uropod of crayfish,Procambarus clarkII

• 1.1. The fiber types of uropod muscles of the crayfish, Procambarus clarkii, were determined by the myofibrillar ATP-ase histochemistry and electrophysiology.
• 2.2. The ATP-ase histochemistry was carried out on the sections of the whole tailfan and the slow muscles were identified as being of the slow type on the basis of their low staining intensities.
• 3.3. The location of slow bundles in the mixed type muscles i.e. the dorsal rotator (DRT), the ventral rotator (VRT), and the telson-uropodalis posterior (TUP) was confirmed.
• 4.4. TUP was newly revealed in this study to be a mixed muscle.

     

Dual excitatory innervation through the different ganglionic roots in the uropod adductor muscle of a crayfish,Procambarus clarkii

Dual excitatory innervation of the crayfish (Procambarus clarkii) uropod adductor muscle, adductor exopodite (ADX), through different roots of the sixth abdominal ganglion, was confirmed histologically and electrophysiologically. ADX contraction induced by the excitor in the third root (R3 excitor), showed more rapid fatigue than that by the excitor in the second root (R2 excitor), possibly owing to a more rapid depletion of the transmitter substance in the process of neuromuscular transmission. The size and position of the cell bodies of the two excitors were not necessarily similar, whereas the dendritic fields appeared to be almost overlapping.     

Morphological asymmetry and behavioral laterality in the crayfish, Procambarus clarkii

Lateral asymmetry is found widely among vertebrates, but is scarcely observed in invertebrates. Here, morphological asymmetry and behavioral laterality of a wild crayfish, Procambarus clarkii, was investigated. The carapace morphology of crayfish showed left–right differences; in some, the right side of the carapace was larger than the left side, while in others, the left side was larger. A bimodal distribution in the direction of escape behaviors induced by a tactile stimulus was also observed. Experimental crayfish were definitively divided into two groups: individuals that frequently jumped leftward (right type) and those that jumped rightward (left type). Moreover, carapace asymmetry and lateralized escape responses were significantly correlated. These results suggest that crayfish exhibit left–right dimorphism in natural populations. The ecological advantages and maintenance mechanisms underlying these behaviors are also discussed.     

Nuclear and cytoplasmic differentiation in developing sperm of the crayfish,Cambaroides japonicus

Summary In the present electron microscopic study of spermatogenesis in the crayfish, Cambaroides japonicus, it was possible to clarify several aspects of the unusual differentiation which leads to the production of an aflagellate sperm. The centriole is followed from the metaphase of the second spermatocyte division to the time at which, in the nearly mature sperm, it appears to disintegrate. It has no connection with the acrosome but in the late spermatid and maturing sperm it is found randomly oriented among the convoluted membranes of the filamentous endoplasmic reticulum.There appears to be a close association of mitochondria with the developing acrosomal vesicle. Typical mitochondria, however, are not present after the late spermatid stage of development. It is suggested that the complex lamellar bodies associated with the nuclear envelope in the late stages of spermatogenesis may be related to mitochondria for these lamellar bodies resemble the complex mitochondria found in the adjacent nutritive cells.The development of the acrosome has been traced from an aggregate of dense granules which first appear in the interzonal spindle region and are later segregated at one side of the cell after the second spermatocyte division. As differentiation proceeds, tubular elements appear and disappear within the acrosome, while somewhat later, fibrous elements appear in the matrix. In the mature acrosome, the fibrous elements remain only adjacent to the granular periphery of the acrosome and the core again becomes homogeneous.No typical Golgi complex is found in these cells at any time during their differentiation.In the maturing sperm the development of the arms of the nucleus was studied. Preceding the differentiation of the arms a coarse fibrous material develops in the periphery of the nucleus. It is shown that the fibrillar material in the matrix of the arms is in continuity with the fibrillar material in the matrix of the nucleus proper.Supported in part by Grant No. B 2314 of the National Institute of Neurological Diseases and Blindness, U.S. Public Health Service.Predoctoral Research Fellow of the National Institute of Neurological Diseases and Blindness, U.S. Public Health Service.     

The distribution and form of the endoplasmic reticulum during spermatogenesis in the crayfish,Cambaroides japonicus

Summary The form and differentiation of the endoplasmic reticulum has been studied in the developing sperm of the crayfish, Cambaroides japonicus. Throughout development a relationship between the nuclear envelope and cytoplasmic portion of the endoplasmic reticulum has been shown to exist. Furthermore, large contributions of material from the nuclear envelope to extranuclear cytoplasmic systems has been noted in the development of early spermatids and nearly mature sperm.A sequential predominance of several types of endoplasmic reticulum has been described in the differentiating sperm. An agranular vesicular reticulum is the most common in the early stages although annulate lamellar stacks and rough surfaced stacks are scattered randomly throughout the cytoplasm. Blebs of the nuclear envelope appear to contribute rough surfaced reticulum to the cytoplasmic system in the early spermatid. A fusion of vesicular elements results in the formation of the dense filamentous reticulum which is typical of the nearly mature sperm. Densely packed lamellae develop on the nuclear envelope in the maturing sperm and are connected to both the nuclear envelope and filamentous endoplasmic reticulum. The possible relationships of these lamellar groups to mitochondria or Golgi is discussed.Supported in part by Grant No B-2314 of the National Institute of Neurological Diseases and Blindness, U.S. Public Health Service.Predoctoral Research Fellow of the National Institute of Neurological Diseases and Blindness, U.S. Public Health Service.     

Microinjection of mRNA encoding rat synapsin Ia alters synaptic physiology in identified motoneurons of the crayfish,Procambarus clarkii

Studies of identified neurons have made important contributions to our understanding of cellular neurophysiology. We have developed a technique for modifying gene expression in identified motoneurons of the crayfish Procambarus clarkii in the isolated nervous system as well as in the intact animal through the injection of exogenously synthesized RNAs. mRNA suitable for injection was transcribed in vitro from cDNA templates cloned into a plasmid, pSEM. Initially, mRNAs encoding green fluorescent protein (GFP) and β-galactosidase were injected into the soma of the motor giant neuron (MoG) to determine whether these mRNAs could be successfully translated into protein. Both proteins were expressed. Measurements of GFP fluorescence increase indicated that GFP mRNA was stable and translated into protein for at least 3 days postinjection. We then examined the effects of expression of GFP, AASP-168 (an endogenous crayfish axonal protein), and rat synapsin Ia on MoG synaptic physiology. The mRNA injection procedure did not appear to directly influence synaptic physiology based on the results of the AASP-168 and GFP injections. Injection of mRNA encoding rat synapsin Ia resulted in a significant increase in peak excitatory postsynaptic potential (EPSP) amplitude during repetitive stimulation. These data are consistent with previous studies that have shown that synapsin deficiency reduces synaptic vesicle numbers. The translation of mRNAs with diverse functions and species of origin suggests that this approach will prove useful for studying the function of a wide variety of endogenous and exogenous genes in identified neurons. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 224–236, 1998     

Histological organization of the intestine in the crayfish Procambarus clarkii

Six longitudinal ridges span the length of the intestine in the crayfish Procambarus clarkii. A simple columnar epithelium with tetralaminar cuticle lines the lumen. Folds of the epithelium overlie a dense irregular connective tissue packed with mixed acinar (alveolar) glands. Mucous secretions are probably involved with formation and lubrication of faecal strings; neither the nature nor the role of the serous secretions is immediately apparent. Aggregations of cells with large cytoplasmic vacuoles, called bladder cells, appear in the subepithelial connective tissue near the tops of the intestinal ridges. The bladder cells are suitably positioned to bolster the integrity of the ridges. Striated muscle of the intestine occurs in inner longitudinal and outer circular layers. The inner longitudinal layer consists of six strips, with one strip associated with the base of each intestinal ridge. The outer circular layer is essentially complete, but there are periodic apertures in this layer on the left and right sides of the intestine, providing nerves and haemolymph vessels with access to the interior of the gut. Based on histological features, and consistent with reports on other crayfish, we conclude that the intestine of P. clarkii has a proctodeal (ectodermal) origin.     

Production of farnesoic acid and methyl farnesoate by mandibular organs of the crayfish,Procambarus clarkii

The mandibular organs (MO) of crustaceans secrete methyl farnesoate (MF) and farnesoic acid (FA). To better understand the secretory activity of MO, the kinetics of production and release of both compounds were determined in vitro by following incorporation of [2-¹⁴C]acetate and l-[³H-methyl]methionine into MF and [2-¹⁴C]acetate into FA by MO of Procambarus clarkii. MO released more FA than MF but contained more MF. In medium lacking unlabeled acetate, the percentage incorporation of [¹⁴C]acetate into MF, relative to [³H]methionine, was between 21 and 40%, suggesting that there may be an alternative source of C₂ units.MO produce similar amounts of MF at concentrations of acetate from 0.08 to 10 mM. However, the addition of exogenous unlabelled FA to incubation media did not stimulate the biosynthesis of MF, raising the possibility that unlike JH biosynthesis in insects, the last step in MF production may be rate-limiting. Nonetheless, exogenous FA significantly reduced the incorporation of [¹⁴C]acetate into MF, suggesting that the glands use exogenous FA to synthesize MF. The absence of stimulation of FA production by exogenous FA indicates that there is no feedback effect of this product on the early steps in the biosynthetic pathway.     

Asynchronous swimmeret beating during defense turns in the crayfish, Procambarus clarkii

Swimmeret beating was monitored in freely moving specimens of the crayfish Procambarus clarkii as they exhibited defense turn responses to tactile stimuli. Analysis of videotape records revealed alterations in swimmeret beating during turning responses compared to straight, forward walking. During turns, swimmerets beat with shorter periods and smaller amplitude power strokes than during straight walking. Coordination between swimmerets also changed. Swimmerets on the side toward which the animal turned tended to lag behind their contralateral partners, rather than beat in synchrony as in straight walking, and ipsilateral coordination was loosened relative to straight walking. Asynchronous swimmeret beating accompanied asymmetric motions of the uropods in a manner similar to that observed during statocyst-dependent equilibrium reactions in P. clarkii, but removal of the statoliths did not eliminate turn-associated responses of the swimmerets. The coordinated action of the swimmerets and uropods may contribute to the torque that rotates the animal in the yaw plane. Implications of the observed changes in swimmeret coordination for understanding the underlying neuronal control system are discussed.     

Methyl farnesoate controls adult male morphogenesis in the crayfish, Procambarus clarkii

Adult male crayfish Procambarus clarkii exist in two morphotypes. They continue to molt as adults, switching between Form Is and Form IIs. Form Is are primary reproductive types, with large chelae and spines on the ischiopodites of the third and fourth pair of walking legs. Form IIs are non-reproductive types with smaller chelae and no spines on the ischiopodites. We investigated the hormonal control of these transitions in two ways, by eyestalk ablation and by methyl farnesoate (MF) treatments. Eyestalk ablation accelerates molting and increases MF levels in the blood. MF is a hormone that regulates both reproduction and morphogenesis. MF concentrations were determined in two ways. The hemolymph samples were extracted first, then purified, using normal phase HPLC. The fractions containing MF were collected and analyzed for MF concentration, utilizing both internal and external standards by GC/MS. The other hemolymph samples were analyzed from individual animals by HPLC. The concentrations of ecdysteroids were determined by radioimmunoassay. In the control animals, 4 out of 4 untreated Form I males molted into Form II, while 6 out of 7 Form IIs molted into Form Is. Eight of 8 ablated Form Is molted into Form IIs as expected, while 5 of 5 ablated Form IIs molted into Form IIs, instead of Form Is. MF treatment of intact animals resulted in 6 of 7 Form Is becoming Form IIs and 5 of 6 Form IIs becoming Form IIs. These results were highly significant in comparison of Form I and IIs in each treatment (eyestalk intact, eyestalk ablated and eyestalk intact with MF) by a chi square analysis, P = 0.006, P < 0.0005, and P = 0.013, respectively. MF premolt blood levels suggested that Form IIs were produced in the presence of 1.3 ng/ml MF, while Form Is result from MF levels less than 0.5 ng/ml. Since both eyestalk ablation and MF treatment resulted in the failure of Form IIs becoming Form Is, it was concluded that the control of morphogenesis of primary reproductives (Form Is) depends on a low level of MF prior to the molt, while Form IIs are formed in the presence of increased levels of MF.     

Corazonin promotes tegumentary pigment migration in the crayfish Procambarus clarkii

The undecapeptide corazonin (pGlu-Thr-Phe-Gln-Tyr-Ser-His-Gly-Trp-Thr-AsnNH(2)) elicits a retraction of erythrophore pigment granules and dispersion of leucophore pigment granules in the crayfish Procambarus clarkii. The effects are dose-dependent from 10(-10) to 10(-5)M. Influence on erythrophores is lower than that of Red Pigment Concentrating Hormone (RPCH), which is inactive on leucophores. Corazonin effects are partly blocked by an anti-corazonin antibody, and even less by an anti-RPCH antibody. Corazonin effects are completely suppressed by the calcium chelator BAPTA. Immunoreactive somata and fibers were identified in various regions of the eyestalk (medulla terminalis, medulla interna and medulla externa) with the anti-corazonin antibody. These results suggest the possible existence of a corazonin-like peptide in crustaceans.     

Factors influencing the degradation of photoreceptor membrane in the crayfish,Procambarus clarkii

Summary Light-induced degradation of photoreceptor membrane in the crayfish was studied by quantitative light and electron microscopy. The production of lysosomal organelles within the photoreceptor cells was enhanced by presenting the light stimulus intermittently (i.e., flicker) or by doubling its intensity. The enhancement was seen primarily as an increase in the number and size of multivesicular bodies. As these stimulus conditions are likely to facilitate intracellular Ca⁺⁺ fluxes, the results are compatibl with recent speculations that Ca⁺⁺ ions may regulate membrane degradation. To test the possibility that Ca⁺⁺ acts as a signal coupling receptor stimulation with membrane loss, retinas were incubated in the dark with the ionophore A23187 in the presence or absence of external Ca⁺⁺. The results demonstrate that A23187 produces a Ca⁺⁺-dependent increase in lysosomal organelles, predominantly multivesicular bodies. These data are consistent with a role for intracellular Ca⁺⁺ in the degradative process; however, the exact locus of the effect is unclear.Supported by a grant (BNS 8004587) from the National Science Foundation to G.S.H. The authors gratefully acknowledge the helpful discussions and expert technical assistance of Thomas R. Tokarski     

High prevalence of multiple paternity in the invasive crayfish species,Procambarus clarkii

Reproductive strategy is a central feature of the ecology of invasive species as it determines the potential for population increase and range expansion. The red swamp crayfish, Procambarus clarkii, has invaded many countries and caused serious problems in freshwater ecosystems. However, little is known about the effects of environmental conditions on crayfish paternity and offspring traits in the wild. We studied these reproductive characteristics of P. clarkii in wild populations from two different habitats (ponds and ditches) in three locations with different environmental conditions in China. Genotyping of 1,436 offspring and 30 mothers of 30 broods was conducted by using four microsatellites. An analysis of genotyping results revealed that gravid females were the exclusive mother of the progeny they tended. Twenty-nine of 30 mothers had mated with multiple (2-4) males, each of which contributed differently to the number of offspring in a brood. The average number of fathers per brood and the number of offspring per brood were similar (P > 0.05) among six sampling sites, indicating that in P. clarkii multiple paternity and offspring number per brood are independent of environmental conditions studied. Indirect benefits from increasing the genetic diversity of broods, male and sperm competition, and cryptic female choice are a possible explanation for the high level multiple paternity and different contribution of fathers to offspring in this species.     

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.     

Morphogenesis and pattern formation in the retina of the crayfish Procambarus clarkii

Pattern formation and ommatidial differentiation in the crayfish retina were analyzed using confocal, light and electron microscopy. Optic primordia first appear in the embryo as round elevations covered by a surface epithelial layer. Retinal differentiation begins with a wave of mitotic activity that moves across this epithelium from lateral to medial. Ommatidial cell clusters are visible at the surface along a transition zone, which lies at the interface of the medial undifferentiated retina and the lateral patterned retina. This zone is 8–10 cells wide and composed of small uniform cell profiles. Lateral to the transition zone the initial ommatidial cell clusters form staggered rows across the surface. Each first row cluster contains eight retinula cells surrounded by four cone, two corneagenous and two distal pigment cells. Ommatidial clusters in the first nine rows show significant changes in their organization, which are visible at the surface of the retina. In row 10 the retinula cells recede from the surface and the cone cells close in above them creating a constant cell pattern at the surface. Rhabdome development begins distally and extends downward as the retinula cluster recedes from the surface. Movement of the retinula cells inward and enlargement of the cone and corneagenous cells at the surface creates a two-tiered organization characteristic of each ommatidium. Comparison of retinal pattern formation and differentiation in the crayfish with retinal morphogenesis in Drosophila and other insects show several similarities between the two arthropod groups.     

Behavioral effects of serotonin and serotonin agonists in two crayfish species, Procambarus clarkii and Orconectes rusticus

Exogenous serotonin elicits several behaviors in Procambarus clarkii, including a flexed, elevated posture, reduced locomotion, and changes in aggressive behavior. We conducted experiments to determine if several serotonin agonists mimicked the behavioral effects of serotonin in two crayfish species, P. clarkii and Orconectes rusticus. Drugs tested were 1-(3-Chlorophenyl)-piperazine dihydrochloride (mCPP), Oxymetazoline, 2,5-dimethoxy-4-iodoamphetamine (DOI), CGS-12066A, and (+/-)-8-hydroxy-2-(di-n-dipropylamino) tetralin (8-OH-DPAT). In P. clarkii, mCPP most closely mimicked the effects of serotonin, significantly increasing the performance of the flexed, elevated posture and reducing locomotion; 8-OH-DPAT significantly reduced locomotion as well. Both of these drugs produced significant increases in elevated posture and decreases in locomotion in O. rusticus, and in this species, the drugs at test concentrations were more effective in eliciting these effects than serotonin. The effects of the drugs on behaviors performed during fighting bouts were variable. In both species, only 8-OH-DPAT significantly reduced several agonistic behaviors, and no agonist or 5-HT itself produced significant increases in agonistic behavior.     

Serotonergic regulation of crustacean hyperglycemic hormone secretion in the crayfish, Procambarus clarkii

These studies investigate if crustacean hyperglycemic hormone (CHH) is involved in 5-hydroxytryptamine (5-HT)-induced hyperglycemia. Eyestalk ganglia with intact X-organ-sinus gland complex were dissected from the crayfish Procambarus clarkii and incubated under various experimental conditions. Incubation media were then analyzed for the presence of released hyperglycemic factor using an in vivo bioassay. The results show that 5-HT enhanced release of hyperglycemic factor in a dose-dependent manner. This stimulatory effect of 5-HT was significantly decreased by adding ketanserin or methysergide (both 5-HT receptor antagonists) into incubation of eyestalk ganglia. Further, activity of the 5-HT-released hyperglycemic factor could be eliminated by adsorption of incubation media with anti-CHH serum but not by preimmune or anti-5-HT serum. These results confirm the hypothesis that 5-HT enhances release of CHH, which in turn elicits hyperglycemic responses. It is probable that 5-HT activates an excitation-secretion coupling mechanism by interacting with receptors located on the X-organ neurosecretory cells.