Extraretinal photoreceptors in the brain of the crayfish Cherax destructor.

Two clusters of red-brown pigmented cell somata lie among other cell somata along the anterior margin of the cerebral ganglion in the crayfish Cherax destructor. Electron micrographs show these cells to contain round electron dense pigment granules and that the cell membranes of two or more adjacent cells fold together to form rhabdom-like structures. The pigmented cells specifically bind a monoclonal antibody against the major species of opsin in R1-7 retinula cells of the compound eye of Cherax. When stimulated with light, the pigmented cells respond with a receptor potential-like depolarization. The axons of the pigmented cells terminate in the neuropil of the protocerebral bridge, together with neuronal elements that label with antibodies against serotonin and substance P. We suggest that the brain photoreceptors of the crayfish are important in the entrainment of circadian rhythms.     

Immunocytochemistry of GABA and glutamic acid decarboxylase in the thoracic ganglion of the crab Eriphia spinifrons

We have used specific antisera against protein-conjugated -aminobutyric acid (GABA) and rat-brain glutamic acid decarboxylase (GAD) in immunocytochemical preparations to study the distribution of putatively GABAergic neurons in the fused thoracic ganglion of the crab Eriphia spinifrons. In the thoracic neuromeres, about 2000 neurons with somata arranged in clusters or located singly in the cell cortex exhibited both GABA-like and GAD-like immunoreactivity. In addition, more than a hundred cells showed only GABA-like immunoreactivity. Fibrous immunoreactive staining to GAD and GABA was distributed throughout the neuropil of the thoracic ganglion, and several fiber tracts contained immunoreactive processes. Sets of serially homologous neurons exhibited GABA-like and GAD-like immunoreactivity in the thoracic neuromeres. Especially prominent were one medial and four ventro-lateral clusters of somata, together with thirteen individually recognized cells in each neuromere. Six of these cells in the ventro-medial cell cortex may be the somata of inhibitory motoneurons. The leg nerves contained three immunoreactive fibers, corresponding to the previously described common inhibitory motoneuron and the two specific inhibitors. The results present further evidence for GABA being the neurotransmitter of all inhibitory leg motorneurons, and suggest its presence and role as a neurotransmitter in a considerable number of interneurons in the thoracic ganglion of the crab.     

The study of bacteria on artificially incubated noble crayfish eggs

Data on the abundance of heterotrophic bacteria on the surface of artificially incubated noble crayfish (Astacus astacus L.) eggs are presented. The highest abundance of heterotrophic bacteria was observed on crayfish juveniles (stage 1). The abundance of bacteria in the water of the incubator’s system was found to be considerably lower than that on crayfish eggs, and bacteria counts on dead eggs were a 100 times lower than on healthy eggs. The abundance of pigmented bacteria on dead eggs was considerably higher than on healthy eggs. Aeromonas and Pseudomonas bacteria, constituting up to 70% of all bacteria identified, were found to be dominant on noble crayfish eggs.     

Exocytosis and uptake of bacteria by isolated haemocyte populations of two crustaceans: evidence for cellular co-operation in the defence reactions of arthropods

Summary The role of exocytosis in the cellular defence reactions of arthropods was investigated using in vitro cultures of isolated haemocytes (blood cells) from the freshwater crayfish Pacifastacus leniusculus, and the shore crab Carcinus maenas. In both species, activated lysates of those cell types that contain the prophenoloxidase activating system (granular cells of crab and crayfish and semigranular cells of crayfish) were found to induce degranulation (exocytosis) of semigranular and granular cells. A cell lysate, in which the prophenoloxidase system was kept inactive, did not have this effect. Limited degranulation of granular cells of crab was also induced by lipopolysaccharides as has earlier been shown for crayfish semigranular cells. The phagocytic capability of semigranular cells from crayfish was lost after exocytosis induced by the Ca²⁺ ionophore A23187, and under no conditions were the granular cells of crabs or crayfish seen to ingest bacteria in vitro. An opsonic function for the attaching proteins of a 1,3-glucan-activated haemocyte lysate was demonstrated using the phagocytic hyaline cells from crabs. Phenoloxidase appeared to lack opsonic properties.We suggest that, in crustaceans, opsonization takes place through hierarchically stimulated exocytotic release, and biochemical activation of the prophenoloxidase activating system: first from lipopolysaccharide-sensitive cells (semigranular cells of crayfish or granular cells of crabs) and then from granular cells, triggered by the initially released and activated prophenoloxidase system. Finally, sticky proteins of the activated prophenoloxidase system coat the invader, rendering it susceptible to the phagocytes (hyaline cells in both crab and crayfish and, to a lesser extent, semigranular cells of crayfish). These processes would, together, constitute a cellular communication pathway not previously demonstrated for invertebrates.Abbreviations DMSO dimethyl sulfoxide - L-DOPA L-dihydroxy-phenylalanine - GLS granular cell lysate supernatant - HLS haemocyte lysate supernatant - HyLS hyaline cell lysate supernatant - LPS lipopolysaccharide - proPO prophenoloxidase - SGLS semigranular cell lysate supernatant - SITS 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid disodium salt     

Secondary retinae of a primitive jumping spider,Yaginumanis (Arachnida,Araneida, Salticidae)

Summary Retinae of the secondary eyes of a primitive salticid spider, Yaginumanis sexdentatus (Yaginuma 1967) are described at the ultrastructural level. The structures of the anterior lateral, posterior lateral and posterior median eyes are identical. Receptor somata lie in the retinal cups. Each receptor bearing twin rhabdomeres is ensheated by (i) much-divided processes of non-pigmented glial cells whose somata lie distally in the retinal cups; and (ii) four processes of pigmented glial cells whose somata lie basally, below the receptive segments. Pigment granules in the latter are concentrated in the basal retina, and are not present at the level of the rhabdoms. The present findings support the placement of Yaginumanis in a newly erected Subfamily Spartaeinae by Wanless (1984), because of the likelihood of homology in the fine structural organisation of the secondary retinae of this genus and of the genus Portia.     

Golgi study of the anterior dorsal ventricular ridge in a lizard. I. neuronal typology in the adult

Using Golgi techniques we have studied neuronal cell types in the anterior dorsal ventricular ridge (ADVR) of the adult lizard Gallotia galloti. Multipolar, bitufted, and juxtaependymal neuronal forms were found. The multipolar and bitufted neurons are present in both the periventricular and central ADVR zones. Multipolar neurons can be subdivided into multipolar neurons with polygonal somata and four to six main dendritic trunks and multipolar neurons with pyramidal somata and three or more dendritic trunks. The former are the cells most frequently impregnated in the ADVR. In the population of bitufted neurons, we distinguish subtypes I, II, and III according to the number of dendritic trunks that emerge from the somata. Juxtaependymal neurons are restricted to a cell-poor zone, adjacent to ependymal cells. Their dendrites either are orientated parallel to the ventricular surface or extend into the periventricular zone. The dendrites of ADVR neurons have pedunculated spines with knob-like tips. However, such spines do not appear on the somata or on the primary dendritic trunks. The number of spines is scarce or moderate. The periventricular neuronal clusters contain two to five cells. The morphology of these neurons is mainly multipolar, but we also found some bitufted neurons.     

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     

Nest defense and aggressive interactions between a small benthic fish (the johnny darter Etheostoma nigrum) and crayfish

Synopsis Aggression by nest-guarding male johnny darters, Etheostoma nigrum, against intruding crayfish was investigated in laboratoy experiments and field observations. In the laboratory, darter success in chasing crayfish, Orconectes rusticus, from the nest site was inversely related to crayfish size. Small crayfish (less than 15 mm carapace length) were routinely evicted from the nest area by nips directed at the posterior end of the abdomen. Although such aggressive behavior was less successful against larger crayfish, even the largest crayfish tested (carapace length 30–32 mm) were chased from the nest area in 33% of the trials. Those large crayfish that entered nests often remained despite repeated attacks by the male johnny darter and egg predation was observed. In a small Ohio stream, openings to johnny darter nests were generally between 7 and 13 mm. Thus crayfish with a carapace height greater than 13 mm (corresponding to a carapace length greater than 29 mm) would not be able to enter johnny darter nests. In field observations, male Johnny darters successfully defended nests against another crayfish species (Orconectes sanborni, carapace length 12–29 mm). Together, aggressive behavior and small size of nest entrances allow the johnny darter to successfully reproduce in areas with abundant crayfish. The Unit is sponsored jointly by the United States Fish and Wildlife Service, The Ohio Department of NaturalResources, and The Ohio State University.     

Intergenic complementation in chick melanocyte heterokaryons

Melanocytes from chick embryos of the pinkeye (pk/pk) and recessive white (c/c) genotypes do not produce melanin in cell culture. However, aberrant melanogenic organelles are evident when these cells are examined with the electron microscope. Melanocytes of each genotype, previously grown for 5 days in cell culture, were co-cultured for 24 h and then fused with inactivated Sendai virus. Twenty-four hours after fusion faintly pigmented cells could be seen in the culture dishes. These cells were invariably multinucleated. At 48 h post-fusion many darkly pigmented, multinucleated cells could be seen. Pigment-producing cells were found in four separate experiments and occurred at a frequency of approx. 1 per 40 000 cells treated. Co-culturing of the melanocytes without virus treatment failed to elicit pigment production. When one genotype was labeled with [³H]thymidine prior to fusion, autoradiograms showed that the pigmented cells contained at least one labeled and one unlabeled nucleus. Electron micrographs of the pigmented cells confirmed that cell fusion was complete and showed normal pigment granules with welldefined matrices and deposited melanin. The results show that recessive white and pinkeye can complement as heterokaryons. This indicates that each mutation affects a different melanogenic function and that the expression of the normal function of each does not require nuclear integration. The simplest hypothesis is that the two mutations affect structural genes and that the complementing cytoplasms contain functional gene products. The hypothesis that one or both mutants have altered control functions cannot be ruled out, however.     

Absence of the crayfish plague pathogen (Aphanomyces astaci) facilitates coexistence of European and American crayfish in central Europe

1. Most European crayfish species are strongly threatened, mainly as a result of the introduced pathogen, Aphanomyces astaci, transmitted by invasive North American crayfish. Long‐term coexistence of American and European crayfish species is therefore regarded as almost impossible, even though some coexisting populations have been observed. 2. In this study, crayfish were collected from presently coexisting populations of the introduced spiny‐cheek crayfish (Orconectes limosus) and the native noble crayfish (Astacus astacus) from nine standing waters in central Europe. Our aim was to resolve whether the coexistence resulted from reduced virulence in local strains of A. astaci, increased immunity in the native crayfish or an absence of the pathogen in these populations. We used highly sensitive A. astaci‐specific real‐time PCR to evaluate the crayfish latent carrier status, combined with transmission experiments to further validate the molecular results. 3. From the total of 523 crayfish tested (490 spiny‐cheek crayfish, 33 noble crayfish), none positive for A. astaci was detected. Transmission experiments confirmed these results: No abnormal mortality or behavioural changes were seen in noble crayfish kept together with American crayfish from the coexisting populations. If we assume a very low prevalence of A. astaci of 10% in a carrier population, there is a 98% probability of disease being absent in five of the nine coexisting populations tested. Hence, a consistent absence, or an extremely low prevalence, of A. astaci seems to allow the coexistence of European and American crayfish in these central European populations. 4. The results are important for native crayfish conservation and management and demonstrate that disease transmission risk may vary substantially between the different populations of spiny‐cheek crayfish in central Europe.     

Comparative morphological studies on the visual systems in a binocular and a multi-ocular species of freshwater planarian

The visual systems of Bdellocephala brunnea Ijima & Kaburaki, a species with two eyes, and Polycelis sapporo (Ijima & Kaburaki), a species with multiple eyes, were investigated using light and electron microscopy. The eye of the binocular species consisted of 40–50 photoreceptor cells and 6–12 pigmented eyecup cells. The eye of the multi-ocular species was smaller and consisted in most specimens of one photoreceptor cell and one pigmented eyecup cell. The ultrastructure of the photoreceptor cells and of the pigmented cells was similar in the two species. Despite differences in numbers of constitutive cells, the arrangement of functional elements in the ocelli of these planarians is the same.     

Glial patterning during postembryonic development of central neuropiles in the brain of the honeybee

 Glial cells are involved in several functions during the development of the nervous system. To understand potential glial contributions to neuropile formation, we examined the cellular pattern of glia during the development of the mushroom body, antennal lobe and central complex in the brain of the honeybee. Using an antibody against the glial-specific repo-protein of Drosophila, the location of the glial somata was detected in the larval and pupal brain of the bee. In the early larva, a continuous layer of glial cell bodies defines the boundaries of all growing neuropiles. Initially, the neuropiles develop in the absence of any intrinsic glial somata. In a secondary process, glial cells migrate into defined locations in the neuropiles. The corresponding increase in the number of neuropile-associated glial cells is most likely due to massive immigrations of glial cells from the cell body rind using neuronal fibres as guidance cues. The combined data from the three brain regions suggest that glial cells can prepattern the neuropilar boundaries. Received: 3 November 1996 / Accepted: 7 February 1997     

Misidentification of OLGA-PH-J/92, believed to be the only crustacean cell line

Continuous cell lines from aquatic invertebrate species are few and the development of crustacean cell lines remains an elusive goal. Although a crayfish cell line derived from neural ganglia of Orconectes limosus was reported in 2000, this cell line OLGA-PH-J/92 failed to be authenticated as such. In this report, we describe our attempts to identify the taxonomic identity of the cell line through immunological and molecular techniques. Immunohistochemical screening for the expression of a suite of invertebrate neuropeptides gave negative results, precluding an invertebrate neural origin. PCR amplification and DNA sequencing for the mitochondrial cytochrome c oxydase I, and 18S ribosomal RNA genes that had been widely used to confirm species identity, could not confirm the OLGA-PH-J/92 cells as originating from crayfish. Subsequent attempts to identify the cells provided moderate homology (82%) to Gephyramoeba sp. (AF293897) following PCR amplification of an 18S rDNA fragment after a BLAST search. A literature search provided morphological evidence of the similarity of OLGA-PH-J/92 to the Gephyramoeba distributed by the American Type Culture Collection as ATCC 50654, which also had been misidentified and was renamed Acramoeba dendroida (Smirnov et al., Eur J Protistol 44:35–44, 2008). The morphology of the OLGA-PH-J/92 cells which remains identical to the original report (Neumann et al., In Vivo 14:691–698, 2000) and matched corresponding micrographs that were available from the ATCC before the cell line was dropped from their catalog (ATCC CRL 1494) is very similar to A. dendroida and could thus belong to the Acramoebidae. These results unequivocally indicate that the OLGA-PH-J/92 cell line is not derived from the crayfish O. limosus, and the search for an immortal crustacean cell line continues.     

Ultrastructural and immunocytochemical observations of the nervous systems of three macrodasyidan gastrotrichs

The nervous systems of three macrodasyidan gastrotrichs, Dactylopodola baltica, Macrodasys caudatus and Dolichodasys elongatus, were investigated using immunocytochemistry and electron microscopy. Labelling of neural structures against serotonin revealed the presence of two pairs of cerebral cells, a dorsal cerebral connective, and paired ventral nerve cords in D. baltica. In M. caudatus and D. elongatus serotonin immunoreactivity was present in a single pair of dorsal cerebral cells and the ventral nerve cords; the dorsal connective of D. elongatus was also immunoreactive to serotonin and acetylated α‐tubulin. The presence of paired, serotonin‐like immunoreactive cells in D. baltica and other species may represent the plesiomorphic condition in Macrodasyida. The fine structure of the photoreceptors in D. baltica was also investigated to explore the potential ground pattern for eyes in the Macrodasyida. The pigmented photoreceptors of D. baltica contain a unicellular pigment cup, sheath cell and sensory receptor. The pigment cup contains numerous osmiophilic granules that presumably function to shield the eyes from downwelling light in the red part of the spectrum. Projecting into the pigment cup and sheath cell are numerous microvilli from a bipolar sensory cell. A single sensory cell may represent the plesiomorphic condition in Macrodasyida, with multiplication of sensory cells representative of more derived taxa.     

Light- and electron-microscopic immunocytochemistry of a molluscan insulin-related peptide in the central nervous system of Planorbarius corneus

Summary Two groups of cerebral dorsal cells of the pulmonate snail Planorbarius corneus stain positively with antisera raised against synthetic fragments of the B- and C-chain of the molluscan pro-insulin-related prohormone, proMIP-I, of another pulmonate snail, Lymnaea stagnalis. At the light-microscopic level the somata of the dorsal cells and their axons and neurohemal axon terminals in the periphery of the paired median lip nerves are immunoreactive with both antisera. Furthermore, the canopy cells in the lateral lobes of the cerebral ganglia are positive. In addition, MIP_B-immunoreactive neurons are found in most other ganglia of the central nervous system. At the ultrastructural level, pale and dark secretory granules are found in somata and axon terminals of the dorsal cells. Dark granules are about 4 times as immunoreactive to both antisera as pale granules. Release of anti-MIP_B- and anti-MIP_C-immunopositive contents of the secretory granules by exocytosis is apparent in material treated according to the tannic acid method. It is concluded that the dorsal and canopy cells synthesize a molluscan insulin-related peptide that is packed in the cell body into secretory granules and that is subsequently transported to the neurohemal axon terminals and released into the hemolymph by exocytosis. Thus, MIP seems to act as a neurohormone on peripheral targets. On the basis of the analogy between the dorsal cells and the MIP-producing cells in L. stagnalis, it is proposed that the dorsal cells of P. corneus are involved in the control of body growth and associated processes.     

Immunocytochemical identification of structures containing putative red pigment-concentrating hormone in two species of decapod crustaceans

Summary By use of an antiserum raised against the Nterminal sequence pGlu-Leu-Asn-Phe..., common to red pigment-concentrating hormone (RPCH) of Pandalus borealis and three structurally similar insect neuropeptides, putative RPCH-immunopositive structures were revealed in the eyestalks of Carcinus maenas and Orconectes limosus and in the brain and thoracic ganglion (TG) of C. maenas. In the eyestalks, complete neurosecretory pathways were demonstrated, consisting of perikarya, axons and terminals in the neurohemal organ, the sinus gland (SG). In C. maenas approximately 20 small RPCH cells are present as a distinct group adjacent to the medulla terminalis ganglionic X-organ (MTGXO, XO). They are morphologically different from the larger XO perikarya, which contain the crustacean hyperglycemic hormone (CHH). The occurrence of both neuropeptides in distinct neurosecretory pathways was ascertained by immunologic double staining (PAP/gold) or by analysis of consecutive sections. In addition, a group of two to four larger RPCH cells is located in the proximal part of the MT. In O. limosus, RPCH cells are found in the XO. Cells corresponding to the proximal MT cells of C. maenas were not found. In both species, a few more weakly staining immunopositive perikarya were observed in clusters of cell somata of the optic ganglia. It is uncertain whether these are connected to the SG.In the brain of C. maenas, several smaller and three larger perikarya were consistently observed in the dorsal lateral cell somata adjacent to the olfactory lobes. In the optic nerve, two axons that project into the eyestalk were stained. Some axons were also observed in the ventral median neuropil of the brain. In the TG, RPCH cells were found in small numbers in median positions, i.e., in clusters of somata between the ganglia of the appendages.HPLC analysis of the red pigment-concentrating activity from the SG of C. maenas revealed that the retention time of the neuropeptide is similar but not identical to that of Pandalus borealis RPCH.     

Fine structure of the eyes of Pseudoceros canadensis (Turbellaria,Polycladida)

Summary The cerebral and epidermal ocelli of the Müller's larva and the cerebral and tentacular eyes of the adult turbellarian Pseudoceros canadensis were studied by electron microscopy. The right cerebral ocellus of the larva consists of one cup-shaped pigmented cell and three sensory cells that bear microvilli. The left cerebral eye of the larva has the above named cells plus a sensory cell with many cilia. Evolutionary significance is attributed to the presence of both ciliary and microvillar photoreceptors in an eye of a flatworm. The one epidermal ocellus of the larva is composed of two cells: a cup-shaped pigmented one bearing flattened cilia, the presumed photoreceptors, and a cell above the cup that adds a few nonciliary lamellae to the stack of ciliary ones from the pigmented cell. The adult eyes contain only microvillar receptors; cilia were not observed.     

Morphological convergence in a Mexican garter snake associated with the ingestion of a novel prey

Morphological convergence is expected when organisms which differ in phenotype experience similar functional demands, which lead to similar associations between resource utilization and performance. To consume prey with hard exoskeletons, snakes require either specialized head morphology, or to deal with them when they are vulnerable, for example, during molting. Such attributes may in turn reduce the efficiency with which they prey on soft‐bodied, slippery animals such as fish. Snakes which consume a range of prey may present intermediate morphology, such as that of Thamnophiine (Natricinae), which may be classified morphometrically across the soft–hard prey dietary boundary. In this study, we compared the dentition and head structure of populations of Thamnophis melanogaster that have entered the arthropod–crustacean (crayfish)‐eating niche and those that have not, and tested for convergence between the former and two distantly related crayfish specialists of the genus Regina (R. septemvittata and R. grahamii). As a control, we included the congener T. eques. Multivariate analysis of jaw length, head length, head width, and number of maxillary teeth yielded three significant canonical variables that together explained 98.8% of the variance in the size‐corrected morphological data. The first canonical variable significantly discriminated between the three species. The results show that head dimensions and number of teeth of the two Regina species are more similar to those of crayfish‐eating T. melanogaster than to non‐crayfish‐eating snakes or of T. eques. It is unclear how particular head proportions or teeth number facilitates capture of crayfish, but our results and the rarity of soft crayfish ingestion by T. melanogaster may reflect the novelty of this niche expansion, and are consistent with the hypothesis that some populations of T. melanogaster have converged in their head morphology with the two soft crayfish‐eating Regina species, although we cannot rule out the possibility of a morphological pre‐adaptation to ingest crayfish.     

Distribution of glutamate decarboxylase-like immunoreactivity in the sixth abdominal ganglion of the cockroach Periplaneta americana

Summary An antiserum against glutamate decarboxylase (GAD) of the rat brain was used to locate GAD activity in sections of the nervous system of the cockroach, Periplaneta americana. The sixth abdominal ganglion was chosen because electrophysiological evidence suggests the presence of GABAergic inhibitory synapses in the cereal-giant interneuron system. Groups of somata and numerous fibres and tracts were positively labelled by the GAD antiserum. A posterior group of labelled somata could be identified close to the entry of the cereal nerves. A line of somata clusters lay along a ventro-lateral furrow. Another discrete row of GAD-like cells was located dorso-laterally. Some small cells among the dorsal unpaired neurons were labelled. A small central group appeared under these cells. An abundance of GAD-like processes and transversal tracts were found within the neuropile. The different systems of GABAergic inhibitors in the ganglion are discussed; in particular we show that the fibres of cereal nerve X are not labelled. This demonstrates that the latter act on the giant fibres via interneurons. We suggest that the group that sends axons into the overlapping region between the cereal nerve and the giant fibre could be the inhibitory interneurons involved in this system.