Effects of Microphallus papillorobustus (Platyhelminthes: Trematoda) on serotonergic immunoreactivity and neuronal architecture in the brain of Gammarus insensibilis (Crustacea: Amphipoda).

The larval flatworm Microphallus papillorobustus encysts in the protocerebrum of its intermediate host, Gammarus insensibilis, and changes the gammarid's responses to mechanical and photic stimuli. The resulting aberrant escape behaviour renders infected gammarids more susceptible to predation by birds, the definitive hosts of the parasite. We used immunocytochemical methods to explore the mechanisms underlying these subtle behavioural modifications. Whole mounts of gammarid brains were labelled with fluorescent anti-serotonin and anti-synapsin antibodies and viewed using confocal microscopy. Two types of change were observed in infected brains: the intensity of the serotonergic label was altered in specific regions of the brain, and the architecture of some serotonergic tracts and neurons was affected. A morphometric analysis of the distribution of the label showed that serotonergic immunoreactivity was decreased significantly (by 62%) in the optic neuropils, but not in the olfactory lobes, in the presence of the parasite. In addition, the optic tracts and the tritocerebral giant neurons were stunted in parasitized individuals. Published evidence demonstrates changes in serotonin levels in hosts ranging from crustaceans to mammals infected by parasites as diverse as protozoans and helminths. The present study suggests that the degeneration of discrete sets of serotonergic neurons might underlie the serotonergic imbalance and thus contribute to host manipulation.

Serotonin depletion by 5,7-dihydroxytryptamine alters deutocerebral development in the lobster, Homarus americanus.

The olfactory and accessory lobes constitute prominent histological structures within the larval and mature lobster deutocerebrum, and both are associated with a dense innervation from paired serotonergic nerve cells, the dorsal giant neurons (DGNs). During development, the cell bodies of the DGNs are the first central somata to express serotonin (5-HT), and the onset of their 5-HT immunoreactivity coincides with the beginning of accessory lobe formation. In contrast, the olfactory lobe anlagen emerge much earlier and grow in the apparent absence of serotonin. The role of serotonergic input for the development of these brain structures was investigated in lobster embryos after serotonin had been depleted pharmacologically with the neurotoxin 5,7-dihydroxytryptamine. A approximately 90% reduction of serotonin was confirmed in eggs using high-performance liquid chromatography with electrochemical detection. Morphometric analyses suggested that serotonin depletion dramatically slowed the growth of olfactory and accessory lobes, although glomeruli differentiated at the normal time in both areas. The toxin exhibited a high degree of specificity for serotonergic neurons and associated target regions, and serotonin depletion persisted for at least 2 months following treatment. The goal of future experiments is to determine which of the cell types that innervate the olfactory and accessory lobes are affected by toxin treatment, thereby resulting in the retarded growth of these areas.

Amines and peptides in the brain of the American lobster: immunocytochemical localization patterns and implications for brain function.

The distributions of serotonin- (5HT-), substance P- (SP-), small cardioactive peptideb- (SCPb-), and histamine- (HA-) like immunoreactivities were examined in the adult lobster supraesophageal ganglion. Vibratome sections were labeled using avidin-biotin-peroxidase immunocytochemical methods. The localization patterns for each substance were assessed in 21 regions within the median protocerebrum, deutocerebrum, and tritocerebrum. Each immunoreactivity has a unique distribution within the brain; however, most regions are immunoreactive for more than one neurotransmitter. Of particular interest are SP-immunoreactive protocerebral neurons that contact olfactory projection neurons and appear homologous to those found in other crustaceans. Regional differences in immunolabeling within the deutocerebral olfactory and accessory lobes suggest that specific areas within individual olfactory lobe glomeruli serve distinct functions in olfactory processing, and that subpopulations of accessory lobe glomeruli are innervated by different groups of neurons. This detailed comparison of the labeling patterns also has allowed us to define the anatomical connectivity between several cell body clusters, fiber tracts, and neuropil areas in the lobster brain.

Glomerular organization in developing olfactory and accessory lobes of American lobsters: stabilization of numbers and increase in size after metamorphosis.

Olfactory glomeruli are columnar and radially arranged at the periphery of the primary chemosensory areas, the olfactory lobes (OLs), in the American lobster Homarus americanus. The number of olfactory glomeruli reaches nearly 100/lobe in midembryonic life, increases rapidly during larval life, and stabilizes at about 200 in juvenile and adult lobsters. The accessory lobes (ALs), higher order integration areas, are composed of cortical columns and of spherical glomeruli. Two populations of spherical glomeruli are defined, the cortical glomeruli located at the bases of the columns, and the medullary glomeruli scattered throughout the ALs. Both cortical columns and spherical glomeruli are seen for the first time in the second larval stage. There are about 1000 cortical columns and 1700 glomeruli/AL in the postlarva and these numbers remain constant during the life of the lobster. In both OLs and ALs, it is the size of the interglomerular spaces and of the glomeruli themselves that increases. Therefore, the data suggest that in both OLs and ALs the glomeruli were already generated when the lobster metamorphoses (stage III to IV) and switches from a planktonic to a benthic existence, and that the new sensory neurons that are formed at each molt in the antennulae grow into existing olfactory glomeruli. Stability of the glomerular population in the primary olfactory centers, once the full complement of glomeruli is acquired, has also been reported in insects, fish, and mammals.

Dopamine in the lobster Homarus gammarus: II. Dopamine-immunoreactive neurons and development of the nervous system.

Dopamine-immunoreactive neurons were revealed in lobster embryos, larvae, and postlarvae, and staining patterns were compared to neuronal labeling in the juvenile lobster nervous system (Cournil et al. [1994] J. Comp. Neurol. 344:455-469). Dopamine immunoreactivity is first detected by midembryonic life in 35-40 neuronal somata located anteriorly in brain and subesophageal ganglion. When the lobsters assume a benthic life during the first postlarval stage, an average of 58 cell bodies are labeled. The acquisition of dopamine in lobster neurons is a protracted event spanning embryonic, larval, and postlarval life and finally reaching the full complement of roughly 100 neurons in juvenile stages. Some of the dopaminergic neurons previously identified in the mature nervous system, such as the paired Br cells, L cells, and mandibular cells, are labeled in embryos and persist throughout development. In contrast, other neurons stain transiently for dopamine during the developmental period, but, by the adult stage, these neurons are no longer immunoreactive. Such transiently labeled neurons project to the foregut, the thoracic dorsal muscles, the neurohormonal pericardial plexus, and the pericardial pouches. It is proposed that these neurons are alive and functioning in adult lobster but that dopamine levels have been abolished, providing that neurotransmitter status is a dynamic, changing process.

Development of the olfactory and accessory lobes in the American lobster: an allometric analysis and its implications for the deutocerebral structure of decapods.

The allometric changes characterizing the growth of the deutocerebrum (midbrain) of the American lobster (Homarus americanus) are studied using computerized three-dimensional reconstructions of serial brain sections. During the embryogenesis of the midbrain, the paired accessory lobes (higher order processing areas) appear later than the paired olfactory lobes (primary olfactory centers), but the former grow faster from their emergence until metamorphosis. The accessory lobes, as they enlarge, shift progressively from a medial to a posterior position in the lateral deutocerebrum. In early juvenile stages the accessory lobes are one of the largest neuropils of the brain. However, these lobes stop growing in adult animals, whereas the brain and olfactory lobes continue to enlarge, albeit at a slow rate. The overall shape of the brain and the relative proportions and locations of the deutocerebral neuropils and associated cell clusters of various lobster ontogenetic stages are similar to those of selected adult decapods. In addition, the relation between deutocerebral organization and brain size seem parallel during lobster development and across crustacean species. Measurements of the brains of 13 species of decapods (illustrated in Sandeman et al. [1993] J. Exp. Zool. 265:112, plus Homarus) indicate the following trends: Small brains possess olfactory lobes but no accessory lobes, larger brains possess accessory lobes that are medial and small relative to the olfactory lobes, and the largest brains contain relatively voluminous posterior accessory lobes. These observations indicate that some differences in the organization of the deutocerebrum are related to absolute brain size in crustaceans and suggest that ontogenetic scaling of proportions may apply to the deutocerebral neuropils of decapods. Peramorphosis and paedomorphosis in the evolution of the decapod brain are considered.

Dopamine in the lobster Homarus gammarus. I. Comparative analysis of dopamine and tyrosine hydroxylase immunoreactivities in the nervous system of the juvenile.

As a catecholamine, dopamine belongs to a class of molecules that have multiple transmitter and hormonal functions in vertebrate and invertebrate nervous systems. However, in the lobster, where many central neurons have been identified and the peripheral innervation pattern is well known, the distribution of dopamine-containing neurons has not been examined in detail. Therefore, immunocytochemical methods were used to identify neurons likely to contain dopamine and tyrosine hydroxylase in the central nervous system of the juvenile lobster Homarus gammarus. Approximately 100 neuronal somata stain for the catecholamine and/or its synthetic enzyme in the brain and ventral nerve cord. The systems of neurons labeled with dopamine and tyrosine hydroxylase antibodies have the following characteristics: 1) the two systems are nearly identical; 2) every segmental ganglion contains at least one pair of labeled neurons; 3) the positions and numbers of cell bodies labeled with each antiserum are similar in the various segmental ganglia; 4) six labeled neurons are anatomically identified; two interneurons from the brain project within the ventral cord to reach the last abdominal ganglion, two neurons from the commissural ganglia are presumably neurosecretory neurons, and two anterior unpaired medial abdominal neurons project to the hindgut muscles; and 5) no cell bodies are labeled in the stomatogastric ganglion, but fibers and terminals in the neuropil are stained. The remarkably small numbers of labeled neurons and the presence of very large labeled somata with far-reaching projections are distinctive features consistent with other modulatory aminergic systems in both vertebrates and invertebrates.

Comparative brain ontogeny of the crayfish and clawed lobster: implications of direct and larval development.

The freshwater crayfish Cherax destructor and the lobster Homarus americanus have many similarities including life style, body form, and neural organization. However, the ontogenic history is very different in the two species. The development of Cherax is short and direct whereas the development of Homarus comprises three pelagic larval stages and takes more than twice as long from extrusion to benthic stages at constant temperature. In order to determine the progression of maturation of the nervous system in each species and the potential implications of pelagic forms on brain structure, the timing of appearance of 22 general and neural developmental events clearly identifiable in both species was compared. The onset of serotonin antigenicity in the different parts of the brain was chosen as one marker of neural development. During the first month of embryogenesis the timing of morphological, physiological, and neural events is similar in the two species. Morphological development is then accelerated in the crayfish near hatching time and over the two postembryonic stages before the advent of the independent benthic stage. Such heterochronic processes can at least partly account for the different developmental patterns in the two decapods. Among the characters showing similar timing in the two species is the formation of glomeruli (presumptive zones of synaptic contact) in the olfactory lobes of the deutocerebrum, although this event is embryonic in Homarus but postembryonic in Cherax. In contrast, glomerular formation in the accessory lobes is heterochronic: in both species, the glomeruli of the accessory lobes are acquired postembryonically, that is, 3 to 4 months earlier in Cherax than in Homarus. These data suggest that the development of the glomeruli in the olfactory lobes may depend primarily on internal developmental signals, whereas the triggering of glomerular formation in the accessory lobes may depend on external cues. The fact that, in Homarus, only the postlarval stages show mature accessory glomeruli may be a reflection of the functional requirements of benthic life.

Aspects of the embryology and neural development of the American lobster.

It is feasible to study the anatomical, physiological, and biochemical properties of identifiable neurons in lobster embryos. To exploit fully the advantages of this preparation and to lay the foundation for single-cell studies, our recent goals have been to 1) establish a quantitative staging system for embryos, 2) document in detail the lobster's embryonic development, 3) determine when uniquely identifiable neurons first acquire their transmitter phenotypes, and 4) identify particular neurons that may serve developmental functions. Behavioral, anatomical, morphometric, and immunocytochemical studies have led to a detailed characterization of the growth and maturation of lobster embryos and to the adoption of a percent-staging system based upon the eye index of Perkins (Fish. Bull., 70:95-99, 1972). It is clear from these studies that the lobster nauplius molts at approximately 12% embryonic development (E12%) into a metanauplius, which subsequently undergoes a complete molt cycle within the egg. This molt cycle climaxes with the emergence of the first-stage larva shortly after hatching. Serotonin and proctolin, neurohormones widely distributed in the lobster nervous system, appear at different times in development. Serotonin immunoreactive neurons begin to appear at approximately E10%, with the adult complement being established by E50%. In contrast, proctolin immunoreactive neurons appear later and attain their full complement over a protracted period including larval and juvenile stages. The development of serotonergic deutocerebral neurons and their targets, the olfactory and accessory lobes in the brain, are also examined. The olfactory lobes are forming by E10% and have acquired their glomerular organization by E50%, whereas the formation of the accessory lobes is delayed; the early rudiments of the accessory lobes are seen by E50%, and glomeruli do not form until the second larval stage.

Embryonic Development of the American Lobster (Homarus americanus): Quantitative Staging and Characterization of an Embryonic Molt Cycle.

The growth of a single brood of lobsters (Homarus americanus Milne-Edwards 1837) maintained at constant temperature is studied from the naupliar stage to hatching, and the sequence of appearance of morphological, anatomical, and behavioral characteristics observed. A percent-staging system based upon Perkins' eye index (1972) is presented, and ten equally spaced embryonic stages are illustrated and characterized at different levels of resolution: whole eggs, dissected embryos, antennulae and telsons. The tegumentary and setal changes in the telson show that a complete molt cycle takes place in the egg starting at about 12% embryonic development (E12%) with the molt of the nauplius into the metanauplius and ending just after hatching when the metanauplius molts into a first stage larva (L1, first zoea). At E30%, the cuticle begins to separate from the setae in the telson; this signals the start of Drach's (1939) stage D0 of the metanaupliar embryonic molt cycle. At that time, the first sign of organogenesis of the L1, the formation of the endopod of the antennulae, becomes visible; presumed sensory neurons and their axons are observed at the tip of the exopod of the antennulae where a giant sensillum is differentiating. During D0 the setae of the first larval stage are forming proximally and medially in the bilobed telson under the metanaupliar cuticle. At E90%, these setae are retracting, and the embryo has entered stage D1. After hatching (E100%), the telson of the free metanauplius (prelarva) shows the characteristics of stage D2-3 and ecdysis soon follows. The arrested development observed at constant temperature in the experimental brood occurred at stage D0 of the metanaupliar molt cycle, whereas development was resumed as the embryos entered stage D1. These changes in developmental pace from D0 to D1 in the embryonic molt cycle are parallel to those occurring in older lobsters (Aiken, 1973). The quantitative staging of lobster development from extrusion to hatching, and the description of the embryonic molt cycle will facilitate future investigations on particular aspects of the embryogenesis of Homarus such as neural differentiation.

Patterns of appearance of serotonin and proctolin immunoreactivities in the developing nervous system of the American lobster.

Serotonin (5-HT) and proctolin, neurohormones widely distributed in the lobster nervous system, have been implicated in a variety of behaviors and also are known to coexist in large pairs of identified neurons in the fifth thoracic (T5) and first abdominal ganglia (A1) of adults (Siwicki, Beltz, and Kravitz, 1987). Earlier studies also have shown that these paired neurons already contain 5-HT in embryos approximately halfway through development, whereas proctolin immunoreactivity does not appear in these cells until near the time of hatching (Beltz and Kravitz, 1987a). In the current studies, the brain and ventral nerve cord have been screened for the appearance of serotonin and proctolin immunoreactivities using immunocytochemical and biochemical methods, in order to determine whether the late appearance of proctolin in the paired T5 and A1 cells is a general feature of development in other neurons as well. In embryos approximately halfway through development, the adult complement of 5-HT-staining cells is already present. In several cases, embryonic serotonin cells are proportionally very large and prominent, suggesting possible developmental roles. In contrast to serotonin, fewer than 10% of the proctolin-staining neurons of juvenile animals are seen in embryos halfway through development. The number of immunoreactive cells gradually increases, but even by the sixth larval stage only half the number of cells that will eventually stain for proctolin are observed. Therefore, the developmental appearance of proctolin in lobster neurons, assayed using immunocytochemical methods, is relatively late and protracted compared to the appearance of serotonin. Quantitative measurements for 5-HT in lobster larvae were performed using high pressure liquid chromatography (HPLC) with dual electrochemical detection and for proctolin using radioimmunoassay. A gradual, probably growth-related increase in the amounts of serotonin and proctolin were seen during larval development. The implications of the biochemical data, in light of the immunocytochemical studies, are discussed.

[Host-parasite relations of the trematode Microphallus papillorobustus (Rankin 1940). III Factors involved in the behavioral changes of the Gammarus, intermediate hosts and predator tests]. / Relations hôtes-parasites du trématode Microphallus papillorobustus (Rankin, 1940). III--Facteurs impliqués dans les modifications du comportement des Gammarus, hôtes intermédiaires et tests de prédation.

The responses of infected and uninfected Gammarus to certain stimuli are experimentally studied. Under the influence of the cerebral metacercaria of Microphallus papillorobustus , the light preferendum of the Gammarids is shifted towards an area of higher illumination, the phototactism becomes strongly positive, the geotactism is reversed from positive to negative, the responses to mechanical disturbances are altered. Predator tests show that infected Gammarids are more vulnerable than uninfected ones to predation by one of the definitive host of the Trematode, Larus cachinnans . Thus the transmission is favoured.

[Host-parasite relations of the trematode Microphallus papillorobustus (Rankin, 1940). II-Changes in the behavior of Gammarus intermediate hosts and localization of metacercaria]. / Relations hôtes-parasite du trematode Microphallus papillorobustus (Rankin, 1940) II--Modifications du comportement des Gammarus hôtes intermédiaires et localisation des métacercaires.

The aberrant behaviour observed in some Gammarids is correlated with the presence, in their brain, of mature metacercriae of Microphallus papillorobustus. Young cerebral larvae and mature thoracic or abdominal cysts of this Trematode do not induce alterations of behaviour in their hosts. Gammarus insensibilis and Gammarus aequicauda show different patterns in the distribution of M. papillorobustus larvae. Whereas many metacercariae are found in the thorax or abdomen of G. aequicauda adult, very few are present in this same location in G. insensibilis. Field data and experimental infections show that the post-cercariae encyst in the cerebroid ganglions in juveniles of both species as well as in adults of G. insensibilis; the post-cercariae encyst in the thorax or in the abdomen in adult G. aequicauda. The larvae could be attracted to the Amphipods' brain by a neurosecretion characteristic of young stages; this neurosecretion would still be produced at a high rate in adult G. insensibilis.

[Larval development of Paricterotaenia porosa (Cestoda: Cyclophyllidea, Dilepididae) in dipterans of the genus Chironomus, experimental hosts. On comparative larval forms of dilepidid flatworms (author's transl)]. / Développement larvaire de Paricterotaenia porosa (Cestoda: Cyclophyllidea, Dilepididae) chez des diptères du genre chironomus, hotes expérimentaux. Etude comparée des formes larvaires de Dilepididae.

The authors describe the larval evolution of Paricterotaenia porosa (Rud., 1810) Fuhrmann (1932) (Cestoda: Cyclophyllidea, Dilepididae) in three species belonging to the genus Chironomus (Diptera, Nematocera) used as experimental hosts. The protoscolex of the infestant larva is retracted into a cystic vesicule, the cysticercoïd is surrounded by a fragmented cercomer and a capsule. P. porosa in contrary to P. paradoxa (Rud., 1802) do not present asexual reproduction in its intermediate host. P. porosa and Anomotaenia constricta (Molin, 1858) Cohn (1900) show a similar evolution, though the cercomer appears sooner in the former. These cysticercoïds are regarded as Monocercus Villot (1883) the characteristics of which are given. The Monocercus is specific to the Dilepididae. The other larval types described in this family i.e. Cercoscolex and Plerocercus (Jackera, 1970) Plerocercoïd (Freeman, 1973) ans Strobilo cysticercoïd (Freeman, 1973) are compared to the larva of other families: Proteocephalidae, Mesocestoididae and Amabiliidae respectively.