Insights into Germline Development and Differentiation in Molluscs and Reptiles: The Use of Molecular Markers in the Study of Non-model Animals.

When shifting research focus from model to non-model species, many differences in the working approach should be taken into account and usually methodological modifications are required because of the lack of genetics/genomics and developmental information for the vast majority of organisms. This lack of data accounts for the largely incomplete understanding of how the two components-genes and developmental programs-are intermingled in the process of evolution. A deeper level of knowledge was reached for a few model animals, making it possible to understand some of the processes that guide developmental changes during evolutionary time. However, it is often difficult to transfer the obtained information to other, even closely related, animals. In this chapter, we present and discuss some examples, such as the choice of molecular markers to be used to characterize differentiation and developmental processes. The chosen examples pertain to the study of germline in molluscs, reptiles, and other non-model animals.

Emergence of a cholecystokinin/sulfakinin signalling system in Lophotrochozoa.

Chordate gastrin/cholecystokinin (G/CCK) and ecdysozoan sulfakinin (SK) signalling systems represent divergent evolutionary scenarios of a common ancestral signalling system. The present article investigates for the first time the evolution of the CCK/SK signalling system in a member of the Lophotrochozoa, the second clade of protostome animals. We identified two G protein-coupled receptors (GPCR) in the oyster Crassostrea gigas (Mollusca), phylogenetically related to chordate CCK receptors (CCKR) and to ecdysozoan sulfakinin receptors (SKR). These receptors, Cragi-CCKR1 and Cragi-CCKR2, were characterised functionally using a cell-based assay. We identified di- and mono-sulphated forms of oyster Cragi-CCK1 (pEGAWDY(SO3H)DY(SO3H)GLGGGRF-NH2) as the potent endogenous agonists for these receptors. The Cragi-CCK genes were expressed in the visceral ganglia of the nervous system. The Cragi-CCKR1 gene was expressed in a variety of tissues, while Cragi-CCKR2 gene expression was more restricted to nervous tissues. An in vitro bioassay revealed that different forms of Cragi-CCK1 decreased the frequency of the spontaneous contractions of oyster hindgut. Expression analyses in oysters with contrasted nutritional statuses or in the course of their reproductive cycle highlighted the plausible role of Cragi-CCK signalling in the regulation of feeding and its possible involvement in the coordination of nutrition and energy storage in the gonad. This study confirms the early origin of the CCK/SK signalling system from the common bilaterian ancestor and delivers new insights into its structural and functional evolution in the lophotrochozoan lineage.

A Framework for Automatic Recognition of Cell Damage on Microscopic Images using Artificial Neural Networks.

Despite several technological advances in the past years, the vast majority of microscopy examinations continue to be performed in a very laborious, time-consuming manner, requiring highly experienced personnel to spend several hours to visually examine each microscope slide. Due to recent improvements in modern Digital Image Processing, professionals that work on microscopic exams could benefit from new tools that can apply image processing possibilities to their specific field. We propose a framework consisting of an image segmentation stage, feature extraction, and then a Shallow Neural Network related to human perception. The framework is used to classify among 5 types of animal cell damage analyzed in a case study. The case study used applies the Single Cell Gel Electrophoresis assay (SCGE, also known as comet assay) to the cells of land mollusk Helix aspersa in order to measure the DNA damage caused by mutagenic agents. To train and analyze the performance of our approach, we used a dataset manually segmented by a biologist and comprised of 130 slide samples with labeled cells. Our framework proved to be robust, achieving an average accuracy of 88.3%.

[Early Peripheral Sensory Neurons in the Development of Trochozoan Animals].

Neuronal development of the majority of trochozoan animals with biphasic pelago-bentic life cycle starts from transient peripheral neurons, which do not belong to the central nervous system and are mainly located in the apical sensory organ and in the hyposphere. Some of these neurons are pioneer and send neurites that form a scaffold upon which the adult central nervous system later develops. In representative species of molluscs and polychaetes, immunolabelling with the antibodies against neurotransmitters serotonin and FMRFamide, and acetylated α-tubulin revealed that the structure of almost all early peripheral neurons is typical for sensory, most probably chemosensory cells: flask shape, and cilia at the end of the apical dendrite or inside the distal ampoule. Morphology, transmitter specificity, location and projections of the early sensory cells differ in trochophores of different species thus suggesting different origin of these cells. In polychaete larvae, pharmacological inhibition of serotonin synthesis in early peripheral neurons did not affect the development, whereas its increase resulted in developmental arrest and neural malformations, suggesting that early peripheral sensory neurons are involved in developmental regulation.

Cell Proliferation Pattern and Twist Expression in an Aplacophoran Mollusk Argue Against Segmented Ancestry of Mollusca.

The study of aplacophoran mollusks (i.e., Solenogastres or Neomeniomorpha and Caudofoveata or Chaetodermomorpha) has traditionally been regarded as crucial for reconstructing the morphology of the last common ancestor of the Mollusca. Since their proposed close relatives, the Polyplacophora, show a distinct seriality in certain organ systems, the aplacophorans are also in the focus of attention with regard to the question of a potential segmented ancestry of mollusks. To contribute to this question, we investigated cell proliferation patterns and the expression of the twist ortholog during larval development in solenogasters. In advanced to late larvae, during the outgrowth of the trunk, a pair of longitudinal bands of proliferating cells is found subepithelially in a lateral to ventrolateral position. These bands elongate during subsequent development as the trunk grows longer. Likewise, expression of twist occurs in two laterally positioned, subepithelial longitudinal stripes in advanced larvae. Both, the pattern of proliferating cells and the expression domain of twist demonstrate the existence of extensive and long-lived mesodermal bands in a worm-shaped aculiferan, a situation which is similar to annelids but in stark contrast to conchiferans, where the mesodermal bands are usually rudimentary and ephemeral. Yet, in contrast to annelids, neither the bands of proliferating cells nor the twist expression domain show a separation into distinct serial subunits, which clearly argues against a segmented ancestry of mollusks. Furthermore, the lack of twist expression during the development of the ventromedian muscle argues against homology of a ventromedian longitudinal muscle in protostomes with the notochord of chordates.

Allocation of cytoplasm to macromeres in embryos of annelids and molluscs is positively correlated with egg size.

Evolutionary transitions between feeding and nonfeeding larval development have occurred many times in marine invertebrates, but the developmental changes underlying these frequent and ecologically important transitions are poorly known, especially in spiralians. We use phylogenetic comparative methods to test the hypothesis that evolutionary changes in egg size and larval nutritional mode are associated with parallel changes in allocation of cytoplasm to macromere cell lineages in diverse annelids and molluscs. Our analyses show that embryos of species with large eggs and nonfeeding larvae tend to allocate relatively more embryonic cytoplasm to macromeres at 3rd cleavage than do embryos of species with small eggs and feeding larvae. The association between egg size and allocation to macromeres in these spiralians may be driven by constraints associated with mitotic spindle positioning and size, or may be a result of "adaptation in cleavage" to maintain rapid cell cycles in micromeres, position yolk in cell lineages where it can be most efficiently used, or adjust allocation to ectoderm to accommodate changes in embryonic surface area/volume ratio.

Lubinska Phenomenon: Simultaneous Bidirectional Axoplasmic Flow in Nerve Fibers.

Experiments on live mollusk neurons isolated with a neurite fragmentsat its various levels demonstrated that axoplasm is characterized by mechanical strain realized in the form of retraction up to complete invagination of the axoplasm into the soma. Changes in axon geometry were attributed to neuroplasm movement. It was found that the direction of axoplasm movement depends on the location of adhesion points. It was always simultaneous and oppositely directed, as is the case with contractile myofibrils. The formation of distant paired adhesion sites can promote moving away of the axoplasm mass and organelles carried by it. The velocity and activity of axoplasm movement depend on the quantity and intensity of adhesion points along the axon.

[REACTION OF HAEMOCYTES OF THE MOLLUSK PLANORBARIUS CORNEUS TO A XENOTRANSPLANT].

Tissue reaction of the mollusk Planorbarius corneus to the introduction of a transplant (cat vibrissa) was examined. The transplant was introduced into mollusk tissues with the use of an injection needle. After a day, flattened haemocytes were found on the surface of the transplant. The wound channel formed by the needle was arrested by a capsule formed of 5-15 layers of flattened cells. The cavity of the wound channel and the core of the vibrissa were also filled with haemocytes. During incubation of the vibrissa in vitro, adhesion and sedimentation of haemocytes on its surface was observed.

[Probable structural functional evolution of sensory surface of osphradia of aquatic prosobranchian molluscs].

At present, in ecotoxicological studies, as biomarkers there are used physiological reactions of invertebrates, based on diverse reflex. The primary chain of the reflex is chemo-, mechano-, and osmoreceptors. The structures are exposed on the surface of body and mantle cavity. Earlier, a hypothesis was put forward, which suggested that the polymodal osphradial organ of the pond snail might participate in adaptive reactions of aquatic molluscs to toxicants. The known homology of osphradial structures allows spreading this suggestion on marine representatives of various subclasses of Mollusca, although diversity of structure, of ways of nutrition, and multiplicity of aquatic molluscs can impede interpretation of future ecotoxicological studies. To elucidate this issue, we carried out the comparative electron microscopy study of osphradial organs in representatives of various families of Prosobranchia (Mollusca, Gastropoda). By ultrastructural parameters in the osphradial organs, five sensory cellular complexes (SCC) have been revealed. A probable connection is demonstrated of these cellular complexes with the known chemo-, mechano-, and osmoreceptor modalities. Structure of the complexes become more complicated in the process of evolution of gastropods and depends on the way of nutrition of molluscs. Thus, the primitively built osphradium of the herbivorous mollusc Viviparus sp. is a polymodal receptor and initial prototype for further morphophysiological constructions. Osphradium of littorine is the next chain of evolitionary transformations and combines in itself the osmo- and chemosensory SCC. In osphradia of the new, ctenidial type the total receptor surface increases and the ultrastructural specialization of cellular complexes occurs, which promotes the appearance in them of the sarcophagous way of nutrition. For predator marine molluscs actively searching for their preys by odor, there is identified an additional superficial sensory cellular complex. It is located on lateral surfaces and on ventral edge of petals of osphradia, near zones of cilium supportive cells. These seem to be mechanoreceptor structures tracing direction and rate of flow of liquid along osphradium. A connection of SCC with the certain modality of outer nutrition and with the way of nutrition is suggested.

A generalized approach to the modeling and analysis of 3D surface morphology in organisms.

The surface geometry of an organism represents the boundary of its three-dimensional (3D) form and can be used as a proxy for the phenotype. A mathematical approach is presented that describes surface morphology using parametric 3D equations with variables expressed as x, y, z in terms of parameters u, v. Partial differentiation of variables with respect to parameters yields elements of the Jacobian representing tangent lines and planes of every point on the surface. Jacobian elements provide a compact size-free summary of the entire surface, and can be used as variables in principal components analysis to produce a morphospace. Mollusk and echinoid models are generated to demonstrate that whole organisms can be represented in a common morphospace, regardless of differences in size, geometry, and taxonomic affinity. Models can be used to simulate theoretical forms, novel morphologies, and patterns of phenotypic variation, and can also be empirically-based by designing them with reference to actual forms using reverse engineering principles. Although this study uses the Jacobian to summarize models, they can also be analyzed with 3D methods such as eigensurface, spherical harmonics, wavelet analysis, and geometric morphometrics. This general approach should prove useful for exploring broad questions regarding morphological evolution and variation.

Structure and formation of the unusual sperm of Patelloida latistrigata (Mollusca : Patellogastropoda): implications for fertilization biology.

The structure of the spermatozoa and spermatogenesis of the lottiid limpet Patelloida latistrigata is described by transmission electron microscopy. Although the lengths of the spermatozoa (about 60 µm) and their head region (about 12 µm) are similar to those of other patellogastropods, the structure of the sperm head and midpiece are very different. The head consists of an unusually large acrosome (about 11-µm long) with a broad posterior invagination that houses the relatively small nucleus. The midpiece mitochondria, which are rather elongate with large folded tubular cristae, are housed in a cytoplasmic sheath posterior to the nucleus. The proximal centriole is unusually elongate (about 2-µm long). The axoneme that emerges from the distal centriole is surrounded anteriorly by the cytoplasmic sheath in which the cytoplasmic side of the plasma membrane has electron-dense material. The flagellum is enlarged at its terminal end. Spermatogenesis is similar to that described for other patellogastropods. Patelloida latistrigata, therefore, has spermatozoa that seem to meet the morphological criteria of ent-aquasperm, which raises the question of whether fertilization is truly external in this limpet. However, it is also possible that the modifications to the sperm are linked to unknown specializations of the egg or egg envelope.

Molluscan neurons in culture: shedding light on synapse formation and plasticity.

From genes to behaviour, the simple model system approach has played many pivotal roles in deciphering nervous system function in both invertebrates and vertebrates. However, with the advent of sophisticated imaging and recording techniques enabling the direct investigation of single vertebrate neurons, the utility of simple invertebrate organisms as model systems has been put to question. To address this subject meaningfully and comprehensively, we first review the contributions made by invertebrates in the field of neuroscience over the years, paving the way for similar breakthroughs in higher animals. In particular, we focus on molluscan (Lymnaea, Aplysia, and Helisoma) and leech (Hirudo) models and the pivotal roles they have played in elucidating mechanisms of synapse formation and plasticity. While the ultimate goal in neuroscience is to understand the workings of the human brain in both its normal and diseased states, the sheer complexity of most vertebrate models still makes it difficult to define the underlying principles of nervous system function. Investigators have thus turned to invertebrate models, which are unique with respect to their simple nervous systems that are endowed with a finite number of large, individually identifiable neurons of known function. We start off by discussing in vivo and semi-intact preparations, regarding their amenability to simple circuit analysis. Despite the 'simplicity' of invertebrate nervous systems however, it is still difficult to study individual synaptic connections in detail. We therefore emphasize in the next section, the utility of studying identified invertebrate neurons in vitro, to directly examine the development, specificity, and plasticity of synaptic connections in a well-defined environment, at a resolution that it is still unapproachable in the intact brain. We conclude with a discussion of the future of invertebrates in neuroscience in elucidating mechanisms of neurological disease and developing neuron-silicon interfaces.

Morphological features of the inflammatory response in molluscs.

Over the last few years, there has been a large increase in studying the biology and pathology of molluscs, predominantly in addressing the molecular patterns involved in their immune-mediated and inflammatory responses. Conversely, the literature-based diagnostic criteria concerning the morphology of the above phenomena still involves pathogenetic confusion and conflicting terminology. A comparison of bibliographic resources, such as the Abridged Glossary of Terms Used in Invertebrate Pathology and the National Status manual for molluscan histopathological examination and analysis from the NOAA, have revealed variability in the definitions of superimposable lesions, emphasising the need for further efforts in establishing standard terminology and methodologies in this field of study. This review suggests some possible solutions for overcoming the use of parallel terminologies in diagnosing inflammation in molluscs and also highlights conflicting features requiring further discussion.

[The new glutamic acid derivative RGPU-135 (glutaron, neuroglutamin) modulates ion currents in mollusk neurons].

The new glutamic acid derivative RGPU-135 (3-phenylglutamic acid hydrochloride, glutaron, neuroglutamin) produces dose-dependent and reversible modulation of transmembrane sodium, potassium and, to a greater extent, calcium ion currents in neurons of Lymnaea stagnalis and Planorbarius corneus mollusks at concentrations of 1, 10, 100, and 1000 microM. At concentrations within 1 - 10 microM micromole, Ca and K currents are activated rather insignificantly; at 100 pmole, the amplitude of calcium currents is increased by 5 - 10%; and at 1000 microM, the Na and K ion currents are suppressed by 5 - 12%. RGPU-135 does not influence the membrane surface charge potential and the gating of ion channels. The effects of RGPU-135 were quickly reversible, which indicated the relatively weak drug binding to the membrane structures and ion channels.

Autofluorescence imaging, an excellent tool for comparative morphology.

Here we present a set of methods for documenting (exo-)morphology by applying autofluorescence imaging. For arthropods, but also for other taxa, autofluorescence imaging combined with composite imaging is a fast documentation method with high-resolution capacities. Compared to conventional micro- and macrophotography, the illumination is much more homogenous, and structures are often better contrasted. Applying different wavelengths to the same object can additionally be used to enhance distinct structures. Autofluorescence imaging can be applied to dried and embedded specimens, but also directly on specimens within their storage liquid. This has an enormous potential for the documentation of rare specimens and especially type specimens without the need of preparation. Also for various fossils, autofluorescence can be used to enhance the contrast between the fossil and the matrix significantly, making even smallest details visible. 'Life-colour' fluorescence especially is identified as a technique with great potential. It provides additional information for which otherwise more complex methods would have to be applied. The complete range of differences and variations between fluorescence macrophotography and different types of fluorescence microscopy techniques are here explored and evaluated in detail. Also future improvements are suggested. In summary, autofluorescence imaging is a powerful, easy and fast-to-apply tool for morphological studies.

Acute effect of exposure of mollusk single neuron to 900-MHz mobile phone radiation.

The goal of the present work was to explore the influence of commercially available cell phone irradiation on the single neuron excitability and memory processes. A Transverse Electromagnetic Cell (TEM Cell) was used to expose single neurons of mollusk to the electromagnetic field. Finite-Difference Time-Domain (FDTD) method was used for modeling the TEM Cell and the electromagnetic field interactions with living nerve ganglion and neurons. Neuron electrophysiology was investigated using standard microelectrode technique. The specific absorption rate (SAR) deposited into the single neuron was calculated to be 0.63 W/kg with a temperature increment of 0.1°C. After acute exposure, average firing threshold of the action potentials was not changed. However, the average latent period was significantly decreased. This indicates that together with latent period the threshold and the time of habituation might be altered during exposure. However, these alterations are transient and only latent period remains on the changed level.

Sperm chemotaxis, fluid shear, and the evolution of sexual reproduction.

Chemical communication is fundamental to sexual reproduction, but how sperm search for and find an egg remains enigmatic. For red abalone (Haliotis rufescens), a large marine snail, the relationship between chemical signaling and fluid motion largely determines fertilization success. Egg-derived attractant plumes are dynamic, changing their size and shape in response to unique combinations of physical and chemical environmental features. Attractant plumes that promote sexual reproduction, however, are limited to a precise set of hydrodynamic conditions. Performance-maximizing shears are those that most closely match flows in native spawning habitats. Under conditions in which reproductive success is chronically limited by sperm availability, gametes are under selection for mechanisms that increase sperm-egg encounter. Here, chemoattraction is found to provide a cheap evolutionary alternative for enhancing egg target size without enlarging cytoplasmic and/or cell volume. Because egg signaling and sperm response may be tuned to meet specific fluid-dynamic constraints, shear could act as a critical selective pressure that drives gamete evolution and determines fitness.

[The golden age of comparative morphology: laser scanning microscopy and neurogenesis of trochophore animals].

Immunochemical labeling of neuronal elements and laser confocal microscopy have considerably expanded the capacity of comparative morphology and allowed us to monitor the neurogenesis of various trochophore animals at the level of individual identified neurons and their projections. It has been demonstrated that many generally accepted concepts of the larval nervous system and the phylogenetic theories constructed on this basis are incorrect. Comparative analysis has demonstrated that the orthogonal brain is absent at all developmental stages in the representative Lophotrochozoa members. Fundamental differences in the structure and development of the nervous system have been found in the trochophores belonging to different taxonomic groups within Lophotrochozoa; these differences demonstrate that the trochophore larva in these groups are not homologous, while their similarity is most likely a result of convergence. Our results challenge the concept of trochophore as the ancestral form common for all trochophore animals. It is necessary to exclude from phylogenetic discussions the orthogon as a basic plan for the structure of the nervous system and the trochophore as an ancestral form for all Lophtrochozoa.

Histochemical and ultrastructural characterization of the posterior esophagus of Bulla striata (Mollusca, Opisthobranchia).

The posterior esophagus of Bulla striata, running from the gizzard to the stomach, was investigated with light and electron microscopy to obtain new data for a comparative analysis of the digestive system in cephalaspidean opisthobranchs. In this species, the posterior esophagus can be divided into two regions. In the first, the epithelium is formed by columnar cells with apical microvilli embedded in a cuticle. Many epithelial and subepithelial secretory cells are present in this region. In both, electron-lucent secretory vesicles containing filaments and a peripheral round mass of secretory material fill the cytoplasm. These acid mucus-secreting cells may also contain a few dense secretory vesicles. In the second part of the posterior esophagus, the cuticle is absent and the epithelium is ciliated. In this region, epithelial cells may contain larger lipid droplets and glycogen reserves. Subepithelial secretory cells are not present, and in epithelial secretory cells the number of dense vesicles increases, but most secretory cells still contain some electron-lucent vesicles. These cells secrete a mixture of proteins and acid polysaccharides and should be considered seromucous. The secretory cells of the posterior esophagus are significantly different from those previously reported in the anterior esophagus of this herbivorous species.