Neuromuscular system of the causative agent of dicrocoeliosis, Dicrocoelium lanceatum. II. Neuropeptide FMRFamide immunoreactivity in nervous system.

The parasitic flatworm, trematoda Dicrocoelium lanceatum or lancet fluke is the causative agent of a widespread parasite disease of grazing ruminants, dicrocoeliosis. The aim of this work is the study of the presence and localization of neuropeptide FMRFamide immunoreactive elements in the nervous system of D. lanceatum using immunocytochemical technique and confocal scanning laser microscopy. For the first time the data on the presence and distribution of the FMRFamide-immunopositive components in the central and peripheral departments of the nervous system of D. lanceatum has been obtained. FMRFamidergic neurons and neurites were identified in paired brain ganglia, in the brain commissure, longitudinal nerve cords and connective nerve commissures. The innervation of the oral and ventral suckers by peptidergic nerve structures was revealed. The distal part of the reproductive system is innervated by FMRFamide immunopositive neurites. The data obtained suggest that the neuropeptides of FMRFamide family can be involved in the regulation of functions of the attachment organs and the reproductive system in D. lanceatum. The study of neurotransmitters and their functions in flatworms expand our knowledge on the structure and function of the nervous system of trematodes of various taxonomic groups. The results obtained on the morphological organization of D. lanceatum nervous system support the exploitation of the FMRFamidergic components as an anthelmintic target.

The neuro-glandular brain of the Pyramicocephalus phocarum plerocercoid (Cestoda, Diphyllobothriidea): Immunocytochemical and ultrastructural study.

А novel type of a complex neuro-glandular brain structure including both nervous and glandular elements and associated with sensory ones is detected in Pyramicocephalus phocarum plerocercoid (Cestoda: Diphyllobothriidea), parasite of Gadus morua from the White Sea. The brain has two lateral lobes connected by a long cellular median commissure. The brain is tightly surrounded by glandular cells, which receive numerous synapses from the brain neurons. A complex of sensory organs associated with ducts and terminal pores of the frontal glands lies in the scolex tegument. Serotonin, FMRFamide- and GABA-like immunoreactive (IR) neurons are found in the brain, the main nerve cords, and the plexus of the plerocercoid. The innervation of the frontal gland ducts by FMRFamide-IR neurites is detected for the first time proving that they function under control of the nervous system and thus evidencing the eccrine nature of the secretion mechanism. Ultrastructural data show that light, dark and neurosecretory neurons are present in the brain lobes. The median commissure consists of loosely arranged thin parallel axons and several giant and small neurons. The commissure is stratified and penetrated by frontal glandular cells and their processes. Such neuro-glandular morpho-functional brain complex is suggested as a model for Diphyllobothriidae family. Five structural types of sensory organs are described in the scolex of P. phocarum; their colocalization with eccrine gland terminals is supposedly specific for Diphyllobothriidae family. Within the order Diphyllobothriidea, there are significant differences in the architecture of the plerocercoid brain at the family level. We suppose homology of giant commissural neurons among Diphyllobothriidea. Differences between diphyllobothriidean nervous system and that of other cestodes are discussed.

FMRF-NH2 -related neuropeptides in Biomphalaria spp., intermediate hosts for schistosomiasis: Precursor organization and immunohistochemical localization.

Freshwater snails of the genus Biomphalaria serve as intermediate hosts for the digenetic trematode Schistosoma mansoni, the etiological agent for the most widespread form of intestinal schistosomiasis. As neuropeptide signaling in host snails can be altered by trematode infection, a neural transcriptomics approach was undertaken to identify peptide precursors in Biomphalaria glabrata, the major intermediate host for S. mansoni in the Western Hemisphere. Three transcripts that encode peptides belonging to the FMRF-NH2 -related peptide (FaRP) family were identified in B. glabrata. One transcript encoded a precursor polypeptide (Bgl-FaRP1; 292 amino acids) that included eight copies of the tetrapeptide FMRF-NH2 and single copies of FIRF-NH2 , FLRF-NH2 , and pQFYRI-NH2 . The second transcript encoded a precursor (Bgl-FaRP2; 347 amino acids) that comprised 14 copies of the heptapeptide GDPFLRF-NH2 and 1 copy of SKPYMRF-NH2 . The precursor encoded by the third transcript (Bgl-FaRP3; 287 amino acids) recapitulated Bgl-FaRP2 but lacked the full SKPYMRF-NH2 peptide. The three precursors shared a common signal peptide, suggesting a genomic organization described previously in gastropods. Immunohistochemical studies were performed on the nervous systems of B. glabrata and B. alexandrina, a major intermediate host for S. mansoni in Egypt. FMRF-NH2 -like immunoreactive (FMRF-NH2 -li) neurons were located in regions of the central nervous system associated with reproduction, feeding, and cardiorespiration. Antisera raised against non-FMRF-NH2 peptides present in the tetrapeptide and heptapeptide precursors labeled independent subsets of the FMRF-NH2 -li neurons. This study supports the participation of FMRF-NH2 -related neuropeptides in the regulation of vital physiological and behavioral systems that are altered by parasitism in Biomphalaria.

The New Data on the Serotonin and FMRFamide Localization in the Nervous System of Opisthorchis felineus Metacercaria.

BACKGROUND: Trematoda Opisthorchis felineus Rivolta, 1884 is the causative agent of dangerous parasite disease-opisthorchiasis, widespread in the Russian Federation. The details of the neuroanatomical localization of the serotoninergic and FMRFamidergic neurotransmitter elements as well as their functional roles remain not studied enough in both adult and larval forms of O. felineus. The studies in this area are important in term of the development of a new pharmacological strategy of the struggle with the causative agent of opisthorchiasis affecting the neuronal signal substances and the function of its nervous system. PURPOSE: The aim of this work was the immunocytochemical study of the neurotransmitters serotonin (5-HT, 5-Hydroxitryptamine) and neuropeptide FMRFamide localization in the nervous system of the opisthorchiasis causative agent-O. felineus metacercaria. To study the relationship between the detected neurotransmitters and the muscular elements of the parasite, the muscle staining was carried out simultaneously using fluorophore-conjugated phalloidin. METHODS: The localization of 5-HTergic and FMRFamidergic nerve structures was determined by immunocytochemical method. The staining samples were analyzed using a fluorescent and confocal laser scanning microscopies. RESULTS: The new data on the presence and distribution of the serotonin-immunopositive (IP)- and FMRFa-IP components in the central and peripheral departments of the nervous system of  O. felineus metacercaria has been obtained. Besides that a number of the new anatomical details of the nervous system organization and of the innervation of the organs and tissues in the investigated parasite have been revealed. CONCLUSION: The data obtained on the presence and localization of the 5-HTergic and peptidergic (FMRFamide) components in central and peripheral departments of the nervous system of O. felineus metacercaria elaborated and expanded the existing information about the nervous system as well as the innervations of the tissues and organs in the causative agent of opistchorchiasis.

The distribution of cholinergic neurons and their co-localization with FMRFamide, in central and peripheral neurons of the spider Cupiennius salei.

The spider Cupiennius salei is a well-established model for investigating information processing in arthropod sensory systems. Immunohistochemistry has shown that several neurotransmitters exist in the C. salei nervous system, including GABA, glutamate, histamine, octopamine and FMRFamide, while electrophysiology has found functional roles for some of these transmitters. There is also evidence that acetylcholine (ACh) is present in some C. salei neurons but information about the distribution of cholinergic neurons in spider nervous systems is limited. Here, we identify C. salei genes that encode enzymes essential for cholinergic transmission: choline ACh transferase (ChAT) and vesicular ACh transporter (VAChT). We used in-situ hybridization with an mRNA probe for C. salei ChAT gene to locate somata of cholinergic neurons in the central nervous system and immunohistochemistry with antisera against ChAT and VAChT to locate these proteins in cholinergic neurons. All three markers labeled similar, mostly small neurons, plus a few mid-sized neurons, in most ganglia. In the subesophageal ganglia, labeled neurons are putative efferent, motor or interneurons but the largest motor and interneurons were unlabeled. Groups of anti-ChAT labeled small neurons also connect the optic neuropils in the spider protocerebrum. Differences in individual cell labeling intensities were common, suggesting a range of ACh expression levels. Double-labeling found a subpopulation of anti-VAChT-labeled central and mechanosensory neurons that were also immunoreactive to antiserum against FMRFamide-like peptides. Our findings suggest that ACh is an important neurotransmitter in the C. salei central and peripheral nervous systems.

GABA in the nervous system of the cestodes Diphyllobothrium dendriticum (Diphyllobothriidea) and Caryophyllaeus laticeps (Caryophyllidea), with comparative analysis of muscle innervation.

The nervous system (NS) of the cestodes Diphyllobothrium dendriticum (Diphyllobothriidea) and Caryophyllaeus laticeps (Caryophyllidea) was investigated using immunocytochemistry. The GABA neurotransmitter was identified in the NS of both species; GABAergic neurons were detected in the main nerve cords (MC). GABA-like immunoreactive neurons were predominantly unipolar and exhibited more intensive immunoreactivity in the neurite than in the perikaryon. In C. laticeps , GABA-like immunoreactive somas are located in both the MCs and peripheral NS near the longitudinal muscles. The innervation of the body musculature was studied using a combination of antibodies against GABA, serotonin (5-HT), and FMRFamide and with complementary staining of F-actin. In both species, the location of GABAergic neurites is associated with all muscle layers including the subtegumental, longitudinal, transverse, and dorsoventral muscles. The cytomorphology of 5-HTergic motoneurons in the MCs of both species is described and differences in muscle innervation between D. dendriticum and C. laticeps are demonstrated. No evidence for co-localization of GABA with 5-HT or FMRFamide neurotransmitters at any particular neuron was found. Neuropiles in MCs and peripheral NS had separate sets of immunoreactive neurites. A functional role for GABA in muscle innervation is discussed.

Distribution of neuropeptides in the antennal lobes of male Spodoptera littoralis.

Olfaction is an important sensory modality that regulates a plethora of behavioural expressions in insects. Processing of olfactory information takes place in the primary olfactory centres of the brain, namely the antennal lobes (ALs). Neuropeptides have been shown to be present in the olfactory system of various insect species. In the present study, we analyse the distribution of tachykinin, FMRFamide-related peptides, allatotropin, allatostatin, myoinhibitory peptides and SIFamide in the AL of the male Egyptian cotton leafworm, Spodoptera littoralis. Immunocytochemical analyses revealed that most neuropeptides were expressed in different subpopulations of AL neurons. Their arborisation patterns within the AL suggest a significant role of neuropeptide signalling in the modulation of AL processing. In addition to local interneurons, our analysis also revealed a diversity of extrinsic peptidergic neurons that connected the antennal lobe with other brain centres. Their distributions suggest that extrinsic neurons perform various types of context-related modulation.

Distribution of serotonin and FMRF-amide in the brain of Lymnaea stagnalis with respect to the visual system.

Despite serotonin's and FMRF-amide's wide distribution in the nervous system of invertebrates and their importance as neurotransmitters, the exact roles they play in neuronal networks leaves many questions. We mapped the presence of serotonin and FMRF-amide-immunoreactivity in the central nervous system and eyes of the pond snail Lymnaea stagnalis and interpreted the results in connection with our earlier findings on the central projections of different peripheral nerves. Since the chemical nature of the intercellular connections in the retina of L. stagnalis is still largely unknown, we paid special attention to clarifying the role of serotonin and FMRF-amide in the visual system of this snail and compared our findings with those reported from other species. At least one serotonin- and one FMRF-amidergic fibre were labeled in each optic nerve, and since no cell bodies in the eye showed immunoreactivity to these neurotransmitters, we believe that efferent fibres with somata located in the central ganglia branch at the base of the eye and probably release 5HT and FMRF-amide as neuro-hormones. Double labelling revealed retrograde transport of neurobiotin through the optic nerve, allowing us to conclude that the central pathways and serotonin- and FMRF-amide-immunoreactive cells and fibres have different locations in the CNS in L. stagnalis. The chemical nature of the fibres, which connect the two eyes in L. stagnalis, is neither serotoninergic nor FMRF-amidergic.

Temporal and histological evaluation of melatonin patterns in a 'basal' metazoan.

While recent advances suggest functional pleiotropy of melatonin in higher organisms, an understanding of the biological significance of this ancient molecule in early evolutionary groups is lacking. Here, endogenous melatonin production was identified for the first time in the sea anemone Actinia equina, a nonsymbiotic hexacorallian cnidarian. Day/night activity profiles of melatonin in this anemone indicated that melatonin levels oscillate with significant nocturnal peaks. However, dynamic changes in melatonin concentration did not persist under constant dark conditions and therefore were not circadian in nature. Thus, the oscillating pattern of melatonin in A. equina is presumed to be the result of alternative, simpler melatonin control mechanism that likely involves direct regulation by the daily photocycle. As nocturnal melatonin signals still potentially provide 'time-of-day' information and can illustrate the seasonally changing length of the biological night, we hypothesize that melatonin may be relevant to temporal coordination of timed processes also in anthozoans. Spatial patterns of melatonin distribution found in this study indicate abundant melatonin distribution in the endodermal filaments wrapped around gametes. This finding supports the possibility that one of the melatonin-responsive processes in this basal metazoan species may involve reproductive functions.

Analysis of neurotransmitter distribution in brain development of benthic and pelagic octopod cephalopods.

The database on neurotransmitter distribution during central nervous system development of cephalopod mollusks is still scarce. We describe the ontogeny of serotonergic (5-HT-ir) and FMRFamide-like immunoreactive (Fa-lir) neurons in the central nervous system of the benthic Octopus vulgaris and Fa-lir distribution in the pelagic Argonauta hians. Comparing our data to previous studies, we aim at revealing shared immunochemical domains among coleoid cephalopods, i.e., all cephalopods except nautiluses. During development of O. vulgaris, 5-HT-ir and Fa-lir elements occur relatively late, namely during stage XII, when the brain neuropils are already highly differentiated. In stage XII-XX individuals, Fa-lir cell somata are located in the middle and posterior subesophageal mass and in the optic, posterior basal, and superior buccal lobes. 5-HT is predominately expressed in cell somata of the superior buccal, anterior basal, and optic lobes, as well as in the subesophageal mass. The overall population of Fa-lir neurons is larger than the one expressing 5-HT. Fa-lir elements are distributed throughout homologous brain areas of A. hians and O. vulgaris. We identified neuronal subsets with similar cell number and immunochemical phenotype in coleoids. These are located in corresponding brain regions of developmental stages and adults of O. vulgaris, A. hians, and the decapod squid Idiosepius notoides. O. vulgaris and I. notoides exhibit numerous 5-HT-ir cell somata in the superior buccal lobes but none or very few in the inferior buccal lobes. The latter have previously been homologized to the gastropod buccal ganglia, which also lack 5-HT-ir cell somata in euthyneuran gastropods. Among coleoids, 5-HT-ir neuronal subsets, which are located ventrally to the lateral anterior basal lobes and in the anterior middle subesophageal mass, are candidates for homologous subsets. Contrary to I. notoides, octopods exhibit Fa-lir cell somata ventrally to the brachial lobes and 5-HT-ir cell somata close to the stellate ganglia.

The neuro-muscular system in fresh-water furcocercaria from Belarus. I Schistosomatidae.

The neuro-muscular system (NMS) in cercariae of the family Schistosomatidae from Belarus was studied with immunocytochemical methods and confocal scanning laser microscopy. The specimens of Bilharziella polonica were compared with Trichobilharzia szidati and Trichobilharzia franki. The patterns of F-actin in the musculature, 5-HT-immunoreactive (IR), FMRFamide-IR neuronal elements and α-tubulin-IR in sensory receptors and nerves were investigated. No indications of structural differences in the musculature, the 5-HT-IR, FMRF-IR neuronal elements and the general distribution of sensory receptors were noticed between cercariae of Trichobilharzia spp. The number of 5-HT-IR neurons in the cercarial bodies is 16. In cercaria B. polonica, the tail musculature is weaker than in Trichobilharzia spp. A detailed schematic picture of the NMS in the tail of Trichobilharzia spp. cercaria is given. The function of NMS elements in the tail is discussed.

The neuromuscular system in freshwater furcocercaria from Belarus. II Diplostomidae, Strigeidae, and Cyathocotylidae.

The neuromuscular system (NMS) in cercariae of Diplostomum pseudospathaceum, Cotylurus szidati, Australapatemon burti, Holostephanus volgensis, and Paracoenogonimus ovatus was studied with immunocytochemical methods and confocal scanning laser microscopy. The patterns of F-actin in the musculature, 5-HT immunoreactive (-IR), FMRF-amide-IR neuronal elements, and α-tubulin-IR in sensory receptors were investigated. The NMS in the five species studied were compared with each other and with three species of Schistosomatidae studied earlier (Bilharziella polonica, Trichobilharzia szidati, and Trichobilharzia franki). No major structural differences in the musculature, the 5-HT-IR or FMRF-IR neuronal elements were noticed between the cercariae. The minor variations observed in the musculature were related to the size and organization of the muscle fibers. The checked pattern formed by the transverse muscle fibers in the tail stems of D. pseudospathaceum, C. szidati, A. burti, H. volgensis, and P. ovatus was not observed in B. polonica, T. szidati, and T. franki. A trend in the differentiation of the longitudinal muscle fibers in the furca from evenly distributed fibers in H. volgensis and P. ovatus to many bundles in D. pseudospathaceum and two well-organized lateral bundles in C. szidati, A. burti, and Trichobilharzia spp. was observed. The transverse muscle fibers in the furca follow the same trend. The number of 5-HT-IR neurons in the cercarial bodies varied between 10 and 16. In cercariae of H. volgensis and P. ovatus, the central nervous system (CNS) was less centralized compared to the CNS in the other species studied, with only two 5-HT-IR marker neurons in each brain ganglion and the other neurons distributed evenly along the main cords. In the tails of H. volgensis and P. ovatus, many transverse 5-HT-IR comissures were found. In the tails of higher strigeidid cercariae, only a few crosslinks were observed. The number and distribution of sensory receptors on the bodies and tails of the cercarial species differed from each other. A trend in the differentiation of the sensory receptors in the tails was discerned. A process of grouping and decrease in number of ciliated receptors in the stem and in the furca from H. volgensis and P. ovatus to Schistosomatid cercariae took place.

The neuro-muscular system in cercaria with different patterns of locomotion.

The neuro-muscular system (NMS) of cercariae with different swimming patterns was studied with immunocytochemical methods and confocal scanning laser microscopy. Specimens of the continuously swimming Cercaria parvicaudata, Maritrema subdolum and Himasthla elongata were compared with specimens of the intermittently swimming Cryptocotyle lingua and the attached Podocotyle atomon. The patterns of F-actin in the musculature, 5-HT immunoreactive (-IR), FMRFamide-IR neuronal elements, α-tubulin-IR elements in the nervous and sensory systems and DAPI-stained nuclei were investigated. The general plan of the NMS was similar in all cercariae studied. No major structural differences in the patterns of muscle fibres were observed. However, in the tail of C. lingua, transverse muscle fibres connecting the bands of longitudinal muscles were found. No major structural differences in the 5-HT- or FMRFamide-IR nervous systems were observed. The number of 5-HT-IR neurones in the cercarial bodies varied between 12 and 14. The number and distribution of the α-tubulin-IR processes on the cercarial bodies and tails differed from each other. The relation between the number and structure of the α-tubulin-IR processes and the host finding strategy of the cercariae is discussed. A detailed schematic picture of the NMS in the tails of C. lingua and M. subdolum is presented.

The formation of the nervous system during larval development in Triops cancriformis (Bosc) (crustacea, Branchiopoda): An immunohistochemical survey.

We provide data of the development of thenervous system during the first five larval stages of Triops cancriformis. We use immunohistochemical labeling (against acetylated α-tubulin, serotonin, histamine, and FMRFamide), confocal laser scanning microscopy analysis, and 3D-reconstruction. The development of the nervous system corresponds with the general anamorphic development in T. cancriformis. In larval stage I (L I), all brain parts (proto-, deuto-, and tritocerebrum), the circumoral connectives, and the mandibular neuromere are already present. Also, the frontal filaments and the developing nauplius eye are already present. However, until stage L III, the nauplius eye only consists of three cups. Throughout larval development, the protocerebral network differentiates into distinct subdivisions. In the postnaupliar region, additional neuromeres and their commissures emerge in an anteroposterior gradient. The larval nervous system in L V consists of a differentiated protocerebrum including a central body, a nauplius eye comprising four cups, a circumoral nerve ring, mandibular- and postnaupliar neuromeres up to the seventh thoracic segment, each featuring an anterior and a posterior commissure, and two parallel connectives. The presence of a protocerebral bridge is questionable. The distribution of neurotransmitters in L I is restricted to the naupliar nervous system. Over the course of the five stages of development, neurotransmitter distribution also follows an anteroposterior gradient. Each neuromere is equipped with two ganglia innervating the locomotional appendages and possesses a specific neurotransmitter distribution pattern. We suggest a correlation between neurotransmitter expression and locomotion.

Ultrastructural analysis of the dorsal body gland of the terrestrial snail Megalobulimus abbreviatus (Becquaert, 1948) / Análise ultraestrutural da glândula corpo dorsal do caracol terrestre Megalobulimus abbreviatus (Becquaert, 1948)

The ultrastructure of the reproductive gland, dorsal body (DB), of Megalobulimus abbreviatus was analysed. Electron microscope immunohistochemistry was used to detect FMRFamide-like peptides in the nerve endings within this gland. Nerve backfilling was used in an attempt to identify the neurons involved in this innervation. In M. abbreviatus, the DB has a uniform appearance throughout their supraesophageal and subesophageal portions. Dorsal body cells have several features in common with steroid-secreting gland cells, such as the presence of many lipid droplets, numerous mitochondria with tubular cristae and a developed smooth endoplasmic reticulum cisternae. Throughout the DB in M. abbreviatus numerous axonal endings were seen to be in contact with the DB cells exhibiting a synaptic-like structure. The axon terminals contained numerous electron-dense and scanty electron-lucid vesicles. In addition, the DB nerve endings exhibited FMRFamide immunoreactive vesicles. Injection of neural tracer into the DB yielded retrograde labelling of neurons in the metacerebrum lobe of the cerebral ganglia and in the parietal ganglia of the subesophageal ganglia complex. The possibility that some of these retrograde-labelled neurons might be FMRFamide-like neurons that may represent a neural control to the DB in M. abbreviatus is discussed.

Foi analisada a ultraestrutura da glândula reprodutiva corpo dorsal (CD) de Megalobulimus abbreviatus. Imunoistoquímica para microscopia eletrônica foi utilizada para detectar peptídeos relacionados ao tetrapeptídeo FMRFamida nas terminações axonais existentes nessa glândula. Foi utilizada marcação neuronal retrógada com o intuito de localizar os neurônios envolvidos nesta inervação. O CD de M. abbreviatus possui um aspecto uniforme em toda sua extensão, tanto na porção supraesofágica como subesofágica. As células do CD possuem várias características de glândulas esteroidogênicas, tais como a presença de inúmeras gotículas lipídicas, numerosas mitocôndrias com cristas tubulares e cisternas bem desenvolvidas de retículo endoplasmático liso. Por toda a extensão do CD de M. abbreviatus foram encontradas numerosas terminações axonais fazendo contatos estruturalmente semelhantes a sinapses com as células do CD. As terminações axonais continham grande número de vesículas eletrodensas e esparsas vesículas eletrolúcidas. As terminações axonais no CD apresentavam vesículas com conteúdo imunorreativo à FMRFamida. A injeção de traçador neural no CD resultou em marcação retrógrada de neurônios no metacérebro dos gânglios cerebrais e nos gânglios parietais do complexo ganglionar subesofágico de M. abbreviatus. É discutida a possibilidade de que estes neurônios identificados por marcação retrógrada possam representar a via de controle neural do CD de M. abbreviatus, cujo mediador químico seria um neuropeptídeo relacionado à FMRFamida.

Ultrastructural analysis of the dorsal body gland of the terrestrial snail Megalobulimus abbreviatus (Becquaert, 1948).

The ultrastructure of the reproductive gland, dorsal body (DB), of Megalobulimus abbreviatus was analysed. Electron microscope immunohistochemistry was used to detect FMRFamide-like peptides in the nerve endings within this gland. Nerve backfilling was used in an attempt to identify the neurons involved in this innervation. In M. abbreviatus, the DB has a uniform appearance throughout their supraesophageal and subesophageal portions. Dorsal body cells have several features in common with steroid-secreting gland cells, such as the presence of many lipid droplets, numerous mitochondria with tubular cristae and a developed smooth endoplasmic reticulum cisternae. Throughout the DB in M. abbreviatus numerous axonal endings were seen to be in contact with the DB cells exhibiting a synaptic-like structure. The axon terminals contained numerous electron-dense and scanty electron-lucid vesicles. In addition, the DB nerve endings exhibited FMRFamide immunoreactive vesicles. Injection of neural tracer into the DB yielded retrograde labelling of neurons in the metacerebrum lobe of the cerebral ganglia and in the parietal ganglia of the subesophageal ganglia complex. The possibility that some of these retrograde-labelled neurons might be FMRFamide-like neurons that may represent a neural control to the DB in M. abbreviatus is discussed.

Characterization of a novel LFRFamide neuropeptide in the cephalopod Sepia officinalis.

From a single LC-MS/MS analysis, a new C-terminally extended RFamide neuropeptide was characterized in Sepia officinalis. The experimental strategy was based on the specific neutral loss associated with RFamide breakdown. Mass losses of 17 Da (C-terminally amide) and 320 Da (RFamide) have been observed for three known peaks of m/z 581.7 (FLRFamide), 599.8 (FMRFamide), 1096.3 (ALSGDAFLRFamide) and one unknown of m/z 752.8. The primary sequence of the peptide of m/z 752.8 was GNLFRFamide. MS/MS analyses revealed that this novel neuropeptide, called sepFRF1, is largely distributed in the central nervous system of cuttlefish of both sexes. Probably transported in the visceral nerve from the subesophageal mass (the peptide was not detected in the hemolymph), this neuropeptide targeted the rectum in agreement with its peripheral distribution. From concentrations as low as 10(-9)M, sepFRF1 increased the frequency, tonus and amplitude of rectal contractions. SepFRF1 is the first RFamide peptide identified in Sepia officinalis that is not derived from the FaRPs precursor. SepFRF1 could belong to a RFamide subfamily identified in gastropods and may be involved in feeding behavior.

Development of the larval muscle system in the mussel Mytilus trossulus (Mollusca, Bivalvia).

In the present study we examined muscle development throughout the complete larval cycle of the bivalve mollusc, Mytilus trossulus. An immunofluorescence technique and laser scanning confocal microscopy were used in order to study the organization of the muscle proteins (myosin, paramyosin, twitchin, and actin) and some neurotransmitters. The appearance of the muscle bundles lagged behind their nervous supply: the neuronal elements developed slightly earlier (by 2 h) than the muscle cells. The pioneer muscle cells forming a prototroch muscle ring were observed in a completed trochophore. We documented a well-organized muscle system that consisted of the muscle ring transforming into three pairs of velar striated retractors in the early veliger. The striations were positive for all muscle proteins tested. Distribution of FMRFamide and serotonin (5-HT) immunocytochemical staining relative to the muscle ring differed significantly: 5-HT-immunoreactive cells were situated in the center of the striated muscle ring, while Phe-Met-Arg-Phe-NH2 neuropeptide FMRFamid immunoreactive fibers were located in a distal part of this ring. Our data showed clearly that the muscle proteins and the neurotransmitters were co-expressed in a coordinated fashion in a continuum during the early stages of the mussel development. Our study provides the first strong evidence that mussel larval metamorphosis is accompanied by a massive reorganization of striated muscles, followed by the development of smooth muscles capable of catch-contraction.

Presence and distribution of FMRFamide-like immunoreactivity in the cyprid of the barnacle Balanus amphitrite (Cirripedia, Crustacea).

The presence and distribution of FMRFamide-like peptides (FLPs) in the cyprid larvae of the barnacle Balanus amphitrite were investigated using immunohistochemical methods. Barnacles are considered to be one of the most important constituents of animal fouling communities, and the cyprid stage is specialized for settlement and metamorphosis in to the sessile adult condition. FLPs immunoreactive (IR) neuronal cell bodies were detected in both the central and the peripheral nervous system. One bilateral group of neurons somata was immunodetected in the brain, and IR nerve fibers were observed in the neuropil area and optic lobes. Intense immunostaining was also observed in the frontal filament complex: frontal filament tracts leaving the optic lobes and projecting towards the compound eyes, swollen nerve endings in the frontal filament vesicles, and thin nerve endings in the external frontal filament. Thin IR nerve fibers were also present in the cement glands. Two pairs of neuronal cell bodies were immunodetected in the posterior ganglion; some of their axons appear to project to the cirri. FLPs IR neuronal cell bodies were also localized in the wall of the dilated midgut and in the narrow hindgut; their processes surround the gut wall and allow gut neurons to synapse with one another. Our data demonstrated the presence of FLPs IR substances in the barnacle cyprid. We hypothesize that these peptides act as integrators in the central nervous system, perform neuromuscular functions for thoracic limbs, trigger intestinal movements and, at the level of the frontal filament, play a neurosecretory role.

Immunofluorescence analysis of the internal brain anatomy of Nereis diversicolor (Polychaeta, Annelida).

Comparative analyses of neuroanatomical characters can make valuable contributions to the inference of phylogenetic relationships. Whereas investigations in this field are numerous for arthropods, in-depth studies on other protostomes are sparse. Here, we provide a survey of the internal neuroarchitecture of the brain of the aciculate ragworm Nereis diversicolor (Polychaeta, Annelida). Descriptions are based on confocal laser scanning microscope analyses of brain sections labeled with the nuclear marker DAPI and antibodies raised against FMRF-amide, serotonin, and histamine. Autofluorescence of the nervous tissue has been utilized to further elucidate the anatomical structures of the brain. The architecture of two major brain compartments, i.e., the paired mushroom bodies and the central optic neuropil, is described in detail. The findings are compared with existent literature on polychaete neuroanatomy and on arthropod neuroanatomy, and possible phylogenetic implications are outlined.