Computer-based 3-dimensional reconstruction of major organ systems of a new aeolid nudibranch subspecies, Flabellina engeli lucianae, from Brazil (Gastropoda: Opisthobranchia)

Obtaining accurate and comprehensive anatomical informationfrom small opisthobranch specimens has been a major problem.Computer-based 3-dimensional reconstruction from serial histologicalslides applying AMIRA (TGS Graphics) software is shown to bean efficient and fully reproducible way to analyse tiny andcomplex organ systems in their true relative positions and proportions;this method is herein applied to nudibranchs for the first time.We used Flabellina engeli lucianae n. subsp., a small aeolid(up to 8 mm body preserved length) from subtropical southernBrazil, to illustrate all major organ systems including nervoussystems, and discuss them comparatively. Flabellina engeli lucianaediffers externally from congeners by having a translucent bodywith opaque white and iridescent blue spots, orange ceratalbands, and by the special branching of cerata forming distinctgroups on common peduncles. External and internal differencesfrom the apparently geographically and hydrographically separatedCaribbean specimens of F. engeli engeli Marcus & Marcus,1968 are discussed in detail. (Received 9 January 2006; accepted 10 July 2007)     

Towards a phylogeny and evolution of Acochlidia (Mollusca: Gastropoda: Opisthobranchia)

The Acochlidia are unique among opisthobranch gastropods in combining extremely high morphological and ecological diversity with modest species diversity. The phylogeny of acochlidians has never been addressed by cladistic means, as their evolution has remained unknown. This study gives a first overview on more than 150 biological and morphological characters that are potentially useful for phylogenetic analysis. Based on 107 characters, a parsimony analysis (PAUP) was performed for all 27 valid acochlidian species together with 11 (plus two) outgroup taxa. The resulting strict consensus tree shows a moderate overall resolution, with at least some bootstrap support for most resolved nodes. The Acochlidia are clearly monophyletic, and originate from an unresolved basal opisthobranch level. The Acochlidia split into the Hedylopsacea (Tantulum (Hedylopsis (Pseudunela (Strubellia (‘Acochlidium’, ‘Palliohedyle’))))) and Microhedylacea (Asperspina (Pontohedyle, ‘Parhedyle’, ‘Microhedyle’, (Ganitus, Paraganitus))). The formerly enigmatic Ganitidae, resembling sacoglossan opisthobranchs by having dagger‐like rachidian radular teeth, are likely to be highly derived microhedylids. The paraphyly of some of the traditionally recognized family level taxa induced a preliminary reclassification. From the phylogenetic hypothesis obtained, we conclude that the acochlidian ancestor was marine mesopsammic. The colonization of limnic systems occurred twice, independently: first in the Caribbean (with the development of the small interstitial Tantulum elegans), and second in the Indo‐Pacific, with a radiation of large‐sized benthic acochlidian species. The evolution of extraordinary reproductive features, such as hypodermic impregnation by a complex copulative aparatus in hedylopsaceans, cutaneous insemination via spermatophores in microhedylaceans, and gonochorism in Microhedylidae s.l. (including Ganitidae), is discussed. © 2010 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 158 , 124–154.     

Three-dimensional evaluation of the spinal local neural network revealed by the high-voltage electron microscopy: a double immunohistochemical study

Three-dimensional (3-D) analysis of anatomical ultrastructures is important in biological research. However, 3-D image analysis on exact serial sets of ultra-thin sections from biological specimens is very difficult to achieve, and limited information can be obtained by 3-D reconstruction from these sections due to the small area that can be reconstructed. On the other hand, the high-penetration power of electrons by an ultra-high accelerating voltage enables thick sections of biological specimens to be examined. High-voltage electron microscopy (HVEM) is particularly useful for 3-D analysis of the central nervous system because considerably thick sections can be observed at the ultrastructure level. Here, we applied HVEM tomography assisted by light microscopy to a study of the 3-D chemical neuroanatomy of the rat lower spinal cord annotated by double-labeling immunohistochemistry. This powerful methodology is useful for studying molecular and/or chemical neuroanatomy at the 3-D ultrastructural level.     

Preparation of serial sections of arthropods using 2,2-dimethoxypropane dehydration and epoxy resin embedding under vacuum

Improved methods are described for anatomical investigation of small insects and other arthropods using serial semithin sections. The specimens were dehydrated with acidified 2,2-dimethoxypropane and embedded in ERL 4206 epoxy resin under vacuum. This procedure ensures good resin impregnation of thin, long body compartments and appendages. Furthermore, it produces excellent overall preservation of the specimen and its fragile anatomical structures. This procedure saves time and gives excellent results when sectioning difficult arthropod material. A continuous recording of serial semithin sections is possible when diamond knives are used.     

Preparation of serial sections of arthropods using 2,2-dimethoxypropane dehydration and epoxy resin embedding under vacuum.

Improved methods are described for anatomical investigation of small insects and other arthropods using serial semithin sections. The specimens were dehydrated with acidified 2,2-dimethoxypropane and embedded in ERL 4206 epoxy resin under vacuum. This procedure ensures good resin impregnation of thin, long body compartments and appendages. Furthermore, it produces excellent overall preservation of the specimen and its fragile anatomical structures. This procedure saves time and gives excellent results when sectioning difficult arthropod material. A continuous recording of serial semithin sections is possible when diamond knives are used.     

Organisation,Systematik und Biologie vonMicrohedyle cryptophthalma nov. spec. (Gastropoda,Opisthobranchia) aus dem Brandungsstrand des Mittelmeeres

Microhedyle cryptophthalma nov. spec. was found in the mesopsammon of different sandy beaches of the Mediterranean Sea (Tunisia, Italy, France and Lebanon). The slugs inhabit a small, particularly exposed region of the surf-beach generally covered by waves except in the sampling locality in Italy where maximum abundance occured about 2 m above the surfedge. The vertical distribution extends down to a substrate depth of 15 cm. Distribution was studied as a function of average sediment particle size, but no definite relationship could be established. The new species is placed in the family Microhedylidae because of its size, external morphology, male sexual organs, form of the tentacles and shape of the midgut gland. The epidermal enclosures are almost identical to those described byKowalevsky (1901) inParhedyle tyrtowii. ButMicrohedyle cryptophthalma is a clearly distinct species by the formula of the asymmetrical radula (1.1.2×n), the form of the teeth, and the almost invisible eyes.Parhedyle is a junior synonym ofMicrohedyle and must be eliminated. The only species,P. tyrtowii, shows no significant differences and is, therefore, considered to belong to the genusMicrohedyle. Sexes inM. cryptophthalma are separate; sperm are transferred through simple spindle-shaped spermatophores. Spermatophores, body size, extremely small eyes, and lack of pigment are recognized as typical “Lebensform”-characteristics of the interstitial sand fauna.     

Distribution of 5HT-immunoreactivity in the pedal ganglion of Mytilus galloprovincialis

Summary Serotonergic cell bodies and fibers were identified in the pedal ganglia of Mytilus gattoprovincialis with a serum raised against serotonin and the unlabelled peroxidase-antiperoxidase pre- and post-embedding methods. Examination of Vibratome and serial semithin sections showed that most reactive perikarya are located in the ganglionic cortex, being mainly concentrated at the medial aspect of the postero-dorsal portion of the ganglia. Immunoreactive fibers form a dense network in the neuropil, extend throughout the commissure and run parallel in the nerves and connective tracts. The morphology of serotonin-positive cells compared with that of Golgi-impregnated neurons allows the identification of a main population of unipolar, probably projecting neurons and of smaller multipolar cells likely representing local circuit elements. The ultrastructure of labelled neurons is comparable to that of serotonergic cells described in both vertebrate and invertebrate nervous systems.Supported by Ministero Pubblica Istruzione (40%)     

Licht- und fluoreszenzmikroskopische Darstellung spezifischer Zellen und Strukturen des Binde- und Nervengewebes von Gastropoden im Semidünnschnitt

Zusammenfassung Im Binde- und Nervengewebe von Gastropoden, welche in Kunstharze eingebettet wurden, lassen sich nach Entfernung des jeweiligen Einbettungsmediums mit licht- und fluoreszenzmikroskopischen Färbeverfahren (Paraldehydfuchsin, Aldehydthionin, Alcianblau, Pseudoisocyanin, RF 500) mit hoher histochemischer Spezifität Zellen und Strukturen darstellen. Neben verschiedenen Zelltypen mit unterschiedlichen Funktionsstadien im Bindegewebe können vor allem die neurosekretorischen Zellen und ihre Fortsätze in den Cerebralganglien der untersuchten Schneckenarten bis in ihre Neurohämalbereiche differenziert angefärbt werden. Mit AT und PAF konnten dabei neurosekrethaltige Axone in der Cerebralkommissur von Planorbarius corneus erstmals sicher lichtmikroskopisch nachgewiesen werden. Die von uns verwendeten Verfahren erlauben eine gezielte elektronenmikroskopische Analyse der im Semidünnschnitt licht- und fluoreszenzoptisch selektiv histochemisch identifizierten Zellen und Strukturen.

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Light- and fluorescence microscopic investigations on specific cells and structures in semithin sections of the connective- and nervous tissues of gastropods

Summary In the present study specific cells and structures in the connective-and nervous tissues of some gastropods were demonstrated by light- and fluorescence techniques, performed on semithin sections of plastic embedded material. After dissolving the embedding medium (Epon or Styrene-methacrylate), the semithin sections are stainable by Gomori's Paraldehyde-Fuchsin, Aldehyde-Thionin, Alcian Blue and the fluorochromes Pseudoisocyanin and RF 500. Best results are obtained optically by staining with Pseudoisocyanin, because all fluorescing substances (i. a. the neurosecretory cells and fibres in the ganglia) appear on a practically dark background. For the first time, with Aldehyde-Thionin and Paraldehyde Fuchsin—but not with Pseudoisocyanin—neurosecretory fibres has been demonstrated in the cerebral commissure of Planorbarius corneus.The histochemical identifying of specific cells and structures in semithin sections by light- and especially fluorescence microscopic highly selective reactions gives the possibilities to analyse these target cells by electron microscopic investigations.

     

Methods for imaging labeled neurons together with neuropil features in Drosophila.

We describe staining protocols for serial semithin sections of Drosophila central ganglia that allow visualization of gene expression in particular neurons with counterstaining to display the ganglion architecture. Green fluorescent protein (GFP), expressed in a subset of sensory neurons from a selected enhancer trap line, is visualized by conventional immunohistochemistry with a peroxidase-linked antibody, and neural architecture is revealed by reduced silver staining. This makes visible in histological sections the same GFP-labeled cells seen with confocal microscopy, but with the especial advantage that neuropil structures are also revealed at the level of individual cells and neuron processes. Not only does this allow the physical relationships among intracellularly labeled neurons to be determined by reference to specific features in the neuropil but it also enables a function to be ascribed provisionally to particular regions of neuropil. These methods have particular utility for mapping morphological information on specific neurons in the context of central nervous system architecture, both in adult Drosophila and during development.     

Morphology of the bourgueticrinid and isocrinid aboral nervous system and its possible phylogenetic implications (Echinodermata, Crinoidea)

Bohn, J. M. & Heinzeller, T. 1999. Morphology of the bourgueticrinid and isocrinid aboral nervous system and its possible phylogenetic implications (Echinodermata, Crinoidea). — Acta Zoologica (Stockholm) 80: 241-249.
On the basis of semithin serial sections the aboral nervous system within the calyx of five bourgueticrinid and one isocrinid species was reconstructed using the computer programm NIH Image. The aboral nervous system of all bourgueticrinids belongs to a common type which is proposed to be called ' Bathycrinus -type' (B-type), that differs from the ' Isocrinus -type' (I-type). The morphology of the B-type and the I-type are described and differences are discussed. Together with the B-type there are now six aboral nervous system types well established and the consequences regarding phylogeny are discussed. Conclusions: 1) The morphology of the aboral nervous system may be a useful tool for the clarification of relationships between crinoid groups; 2) All known aboral nervous system types can be derived from the I-type, which seems to be the most primitive one; 3) The B-type aboral nervous system is probably a synapomorphic feature charac-terizing a monophyletic group within the Bourgueticrinida.     

Gastrin-cholecystokinin-like immunoreactivity in the central nervous system of the milkweed bug,Spilostethus pandurus. Ultrastructural aspects of the reactive A cells of the brain

Summary— All the ganglia belonging to the central nervous system of adults of the milkweed bug Spilostethus pandurus (Hemiptera) were screened immunohistochemically for vertebrate gastrin-cholecystokinin (CCK-8(s))-like peptides. Several large reactive perikarya are present in the median part of the protocerebrum, their processes extending to the dorsal ‘aorta’. These cell bodies are also paraldehyde fuchsin-positive, ie they are A-type cells. In the lateral part of the protocerebrum, in the deutocerebrum and tritocerebrum, and in the suboesophageal, prothoracic and abdominal ganglia, a few immunoreactive cell bodies send axonal processes into their respective neuropiles. The A-type cells reactive to CCK antiserum were identified, at the ultrastructural level, by combining paraldehyde fuschin staining of semithin sections with a post-embedding immunogold technique carried out on adjacent ultrathin sections. The neurosecretory cells contain numerous vesicles of elevated electron density. These data suggest that members of the CCK peptide family are present in the central nervous system of Spilostethus pandurus.     

The nervous system of the tube-worm Chaetopterus variopedatus (Polychaeta)

Silver impregnation of serial histological sections of the tubeworm Chaetopterus variopedatus revealed the presence of a subepidermal nervous system. The anterior nervous system is delimited by the first 11 segments and comprises (1) two dorsolateral cerebral ganglia and lateral instead of ventral nerve cords which are widely separated and thus connected by unusually long commissures, (2) a pharyngeal ganglion in the fourth segment which is connected to the cerebral ganglia by pharyngeal nerves and constitutes along with the pharyngeal plexus a stomatogastric or enteric nervous system, and (3) small, presumably segmental ganglionic swellings along the lateral nerve cords from which emerge commissures and parapodial nerves. No subesophageal ganglion or periesophageal connective could be identified. The lateral nerve cords converge toward the midline in the 12th segment to form the posterior nervous system comprising a pair of ventromedian nerve cords with their repetitive segmental ganglia from which emerge numerous short commissures and three segmental nerves coursing toward the dorsal and ventral regions of parapods and toward the neuropod. Light and electron microscopic investigations of cerebral and segmental ganglia showed an arrangement of inner neuropile and of unipolar neuron somata at the periphery. The neuropile comprises numerous neurites ranging in diameter from 0.5 to 10 μm and making polarized or symmetrical synaptic junctions with each other. The pharyngeal ganglion consists of a similar neuropile and of a large mass of cell bodies which is traversed by an elaborate network of sinuses and harbors three types of neurosecretory cells in addition to the conventional neuron somata. These findings are interpreted in the framework of the highly specialized morphological features and habits of Chaetopterus, and the welldeveloped stomatogastric system is considered to be related to control of the feeding activities.     

Spatial organization of the follicles of the thyroid gland in newborn infants

By means of multilayered plastic reconstruction using serial semithin epoxide sections, it has been demonstrated that besides typical follicles there are some other ones in the thyroid gland of newborns: follicles that have a local thickening on one side, follicles that have reniform proliferates with a cavity, and also follicles with two cavities. The variants mentioned are considered as separate stages of the process for newly formed follicles from the wall proliferates of the preceding ones. While studying 150 interfollicular islets in serial semithin sections, as well as by means of plastic reconstruction, it has been found that they are sections of the follicular wall cut tangentially, and as independent structures in the thyroid gland, they are absent.     

Mapping stimulus-induced nervous activity in small brains by [3H]2-deoxy-D-glucose

Summary Nervous activity may be localized in anatomical sections of brain tissue by the autoradiographic deoxyglucose technique. The method provides sufficient structural preservation and spatial resolution for detailed functional investigation of complex but small-sized nervous systems when the original technique is modified as follows: (i) use of ³H instead of ¹⁴C as radioactive label, (ii) application of labeled deoxyglucose in concentrations close to physiological glucose levels rather than in trace amounts, (iii) stimulation for 4–9 h after deoxyglucose application instead of 20–45 min, (iv) subsequent preparation avoiding aqueous phases at all stages from fixation to autoradiography, and (v) plastic embedding of the tissue such that serial semithin sections of good structural preservation may be routinely cut. Brief aqueous fixation and dehydration at room temperature as has been described for vertebrates apparently cannot preserve stimulus-induced distribution of radioactive label in the brain of the fly Drosophila melanogaster. Aspects of the results that illustrate the potential and some limitations of the present technique are discussed.     

The ultrastructural characteristics of the corticotropic cells of the rat adenohypophysis

Ultrastructural characteristics of corticotropic cells have been studied on serial semithin and ultrathin sections of the hypophysis from intact rats by the immunofluorescence and electron microscopic methods. It is shown that the population of corticotropic cells is heterogeneous and consists of degranulated, moderate granulated, hypergranulated cells and transitional forms among them. Ultrastructurally corticotropic cells correspond to Siperstein-Nakayama's ACTH cells. Kurosumi-Kobayashi "corticotrophs" fail to react with the antiserum to ACTH, so they have not regard for its production.     

Electron microscope analysis of tissue components identified and located by computer-assisted 3-D reconstructions: ultrastructural segmentation of the developing human proximal tubule

A method is described for ultrastructural analysis of renal tubules after precise identification of tubule segments by computerized 3-D reconstruction at the light microscope level. Semithin serial sections were cut of entire nephrons and 3-D coordinate information was obtained by digitization of tubule cross sections in the semithin sections. With the aid of the computer the tubule axis was traced from one section to the other. Precise lengths and positions of the tubules in three dimensions were calculated and stereoscopic images generated. The method was used to analyze the 3-D structure of developing human nephrons, and the ultrastructural development of the proximal tubule. Ultrastructural segmentation of the proximal tubule was demonstrated in the human fetal nephron in developmental stage IV.     

Neurogenesis in the mossy chiton,Mopalia muscosa (Gould) (Polyplacophora): evidence against molluscan metamerism

Neurogenesis in the chiton Mopalia muscosa (Gould, 1846) was investigated by applying differential interference contrast microscopy, semithin serial sectioning combined with reconstruction techniques, as well as confocal laser scanning microscopy for the detection of fluorescence-conjugated antibodies against serotonin and FMRFamide. The ontogeny of serotonergic nervous structures starts with cells of the apical organ followed by those of the cerebral commissure, whereas the serotonergic prototroch innervation, pedal system, and the lateral cords develop later. In addition, there are eight symmetrically arranged serotonergic sensory cells in the dorsal pretrochal area of the larva. FMRFamide-positive neural elements include the cerebral commissure, specific "ampullary" sensory cells in the pretrochal region, as well as the larval lateral and pedal system. In the early juvenile the cerebral system no longer stains with either of the two antibodies and the pedal system lacks anti-FMRFamide immunoreactivity. Outgroup comparison with all other molluscan classes and related phyla suggests that the cord-like, nonganglionized cerebral system in the Polyplacophora is a reduced condition rather than a primitive molluscan condition. The immunosensitivity of the pedal commissures develops from posterior to anterior, suggesting independent serial repetition rather than annelid-like conditions and there is no trace of true segmentation during nervous system development. Polyplacophoran neurogenesis and all other available data on the subject contradict the idea of a segmented molluscan stem species.     

Morphological reinvestigation and phylogenetic relationship of Acanthobdella peledina (Annelida,Clitellata)

Summary In recently collected specimens of Acanthobdella peledina the nervous system, the genital organs and the coelomic organisation were reinvestigated after complete serial sections. These anatomical results are schematically represented. In addition, the integument, the chaetae and the peripheral muscle layer were investigated by electron microscopy. In general, the results confirm Livanow's classic monograph (1906), with the exception of a few details. The body apparently possesses neither a prostomium nor an achaetous buccal region (peristomium). The number of 29 true segments is concluded from the number of segmental ganglia. The five anteriormost segments, each with four pairs of hookshaped chaetae arranged around the mouth opening, are considered to be functionally equivalent to an anterior sucker. The ultrastructure of the integument and the chaetae generally conforms to the typical annelidan pattern. The muscle cells are of the typical hirudinean type. The outer male genital pore is positioned in segment 10; the female organs open in segment 11 directly behind the septum between segments 10 and 11. The main emphasis is laid on the evaluation of the position of the taxon within the Clitellata, including a discussion of the Branchiobdellida, and the cladograms presented show the Acanthobdellida to be the sister group of the Euhirudinea. Characters shared by the Branchiobdellida and Hirudinea (including A. peledina) are considered to be convergently evolved.