Evolutionary regularities of development of somatic polyploidy in salivary glands of gastropod mollusks: V. Subclasses Opisthobranchia and Pulmonata

Salivary glands of 25 species of Opisthobranchia and Pulmonata gastropod mollusks were studied with the use of histochemical methods and cytophotometry of DNA in cellular nuclei. In the secretory epithelium, cells of three main types are identified: granular cells (with granular glycoprotein inclusions), mucocytes-I (containing sulphatized acid mucopolysaccharides), and mucocytes-II (containing neutral and acid nonsulphatized polysaccharides and protein), as well as epithelial ciliated cells and cells of ducts. It is shown that the salivary gland secretory cells of all studied mollusk species are polyploidized, but to different degree. In most species, the maximal degree of polyploidy estimated by the DNA content amounts to 64–128c. Giant polyploidy—up to 4096c—is revealed in salivary gland cells of Tritonia diomedea. The functional properties due to the peculiarities of nutrition of different molluscan species and phylogenetic tendencies of development of somatic polyploidy in the class of Gastropoda are discussed. The high degree of obligatory polyploidization revealed in salivary gland cells of the opisthobranchian and pulmonate mollusks is considered as a peculiar cytological arogenesis as compared with allogenic, facultative, and slight manifestations of polyploidy in prosobranchian gastropod mollusks. The probable causes of such differences are due to the euthyneural type of organization of the central nervous system and to the giant neuronal polyploidy in opisthobranchian and pulmonate mollusks. The causes, mechanisms, and significance of such correlations are so far unclear.     

Evolutionary conservation of mechanisms for neural regionalization, proliferation and interconnection in brain development

Comparative studies of brain development in vertebrate and invertebrate model systems demonstrate remarkable similarities in expression and action of developmental control genes during embryonic patterning, neural proliferation and circuit formation in the brain. Thus, comparable sets of developmental control genes are involved in specifying the early brain primordium as well as in regionalized patterning along its anteroposterior and dorsoventral axes. Furthermore, similar cellular and molecular mechanisms underlie the formation and proliferation of neural stem cell-like progenitors that generate the neurons in the central nervous systems. Finally, neural identity and some complex circuit interconnections in specific brain domains appear to be comparable in vertebrates and invertebrates and may depend on similar developmental control genes.     

Evolutionary ecology of intraspecific brain size variation: a review

The brain is a trait of central importance for organismal performance and fitness. To date, evolutionary studies of brain size variation have mainly utilized comparative methods applied at the level of species or higher taxa. However, these studies suffer from the difficulty of separating causality from correlation. In the other extreme, studies of brain plasticity have focused mainly on within‐population patterns. Between these extremes lie interpopulational studies, focusing on brain size variation among populations of the same species that occupy different habitats or selective regimes. These studies form a rapidly growing field of investigations which can help us to understand brain evolution by providing a test bed for ideas born out of interspecific studies, as well as aid in uncovering the relative importance of genetic and environmental factors shaping variation in brain size and architecture. Aside from providing the first in depth review of published intraspecific studies of brain size variation, we discuss the prospects embedded with interpopulational studies of brain size variation. In particular, the following topics are identified as deserving further attention: (i) studies focusing on disentangling the contributions of genes, environment, and their interactions on brain variation within and among populations, (ii) studies applying quantitative genetic tools to evaluate the relative importance of genetic and environmental factors on brain features at different ontogenetic stages, (iii) apart from utilizing simple gross estimates of brain size, future studies could benefit from use of neuroanatomical, neurohistological, and/or molecular methods in characterizing variation in brain size and architecture.     

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.     

Microtubule-associated protein 1B function during normal development, regeneration, and pathological conditions in the nervous system

Microtubule-associated protein 1B is the first MAP to be expressed during the development of the nervous system. Several different approaches have revealed that MAP1B function is associated with microtubule and actin microfilament polymerization and dynamics. In recent years, the generation of molecular models to inactivate MAP1B function in invertebrates and mammals has sparked some controversy about the real role of MAP1B. Despite discrepancies between some studies, it is clear that MAP1B plays a principal role in the development of the nervous system. In this article, we summarize the evidence for MAP1B function in a wide variety of cellular processes implicated in the proper construction of the nervous system. We also discuss the role of MAP1B in pathological processes.     

The neurobiology and evolution of cannabinoid signalling

The plant Cannabis sativa has been used by humans for thousands of years because of its psychoactivity. The major psychoactive ingredient of cannabis is Delta(9)-tetrahydrocannabinol, which exerts effects in the brain by binding to a G-protein-coupled receptor known as the CB1 cannabinoid receptor. The discovery of this receptor indicated that endogenous cannabinoids may occur in the brain, which act as physiological ligands for CB1. Two putative endocannabinoid ligands, arachidonylethanolamide ('anandamide') and 2-arachidonylglycerol, have been identified, giving rise to the concept of a cannabinoid signalling system. Little is known about how or where these compounds are synthesized in the brain and how this relates to CB1 expression. However, detailed neuroanatomical and electrophysiological analysis of mammalian nervous systems has revealed that the CB1 receptor is targeted to the presynaptic terminals of neurons where it acts to inhibit release of 'classical' neurotransmitters. Moreover, an enzyme that inactivates endocannabinoids, fatty acid amide hydrolase, appears to be preferentially targeted to the somatodendritic compartment of neurons that are postsynaptic to CB1-expressing axon terminals. Based on these findings, we present here a model of cannabinoid signalling in which anandamide is synthesized by postsynaptic cells and acts as a retrograde messenger molecule to modulate neurotransmitter release from presynaptic terminals. Using this model as a framework, we discuss the role of cannabinoid signalling in different regions of the nervous system in relation to the characteristic physiological actions of cannabinoids in mammals, which include effects on movement, memory, pain and smooth muscle contractility. The discovery of the cannabinoid signalling system in mammals has prompted investigation of the occurrence of this pathway in non-mammalian animals. Here we review the evidence for the existence of cannabinoid receptors in non-mammalian vertebrates and invertebrates and discuss the evolution of the cannabinoid signalling system. Genes encoding orthologues of the mammalian CB1 receptor have been identified in a fish, an amphibian and a bird, indicating that CB1 receptors may occur throughout the vertebrates. Pharmacological actions of cannabinoids and specific binding sites for cannabinoids have been reported in several invertebrate species, but the molecular basis for these effects is not known. Importantly, however, the genomes of the protostomian invertebrates Drosophila melanogaster and Caenorhabditis elegans do not contain CB1 orthologues, indicating that CB1-like cannabinoid receptors may have evolved after the divergence of deuterostomes (e.g. vertebrates and echinoderms) and protostomes. Phylogenetic analysis of the relationship of vertebrate CB1 receptors with other G-protein-coupled receptors reveals that the paralogues that appear to share the most recent common evolutionary origin with CB1 are lysophospholipid receptors, melanocortin receptors and adenosine receptors. Interestingly, as with CB1, each of these receptor types does not appear to have Drosophila orthologues, indicating that this group of receptors may not occur in protostomian invertebrates. We conclude that the cannabinoid signalling system may be quite restricted in its phylogenetic distribution, probably occurring only in the deuterostomian clade of the animal kingdom and possibly only in vertebrates.     

Nature’s Palette: Characterization of Shared Pigments in Colorful Avian and Mollusk Shells

Pigment-based coloration is a common trait found in a variety of organisms across the tree of life. For example, calcareous avian eggs are natural structures that vary greatly in color, yet just a handful of tetrapyrrole pigment compounds are responsible for generating this myriad of colors. To fully understand the diversity and constraints shaping nature’s palette, it is imperative to characterize the similarities and differences in the types of compounds involved in color production across diverse lineages. Pigment composition was investigated in eggshells of eleven paleognath bird taxa, covering several extinct and extant lineages, and shells of four extant species of mollusks. Birds and mollusks are two distantly related, calcareous shell-building groups, thus characterization of pigments in their calcareous structures would provide insights to whether similar compounds are found in different phyla (Chordata and Mollusca). An ethylenediaminetetraacetic acid (EDTA) extraction protocol was used to analyze the presence and concentration of biliverdin and protoporphyrin, two known and ubiquitous tetrapyrrole avian eggshell pigments, in all avian and molluscan samples. Biliverdin was solely detected in birds, including the colorful eggshells of four tinamou species. In contrast, protoporphyrin was detected in both the eggshells of several avian species and in the shells of all mollusks. These findings support previous hypotheses about the ubiquitous deposition of tetrapyrroles in the eggshells of various bird lineages and provide evidence for its presence also across distantly related animal taxa.     

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.     

Brain development in the yellow fever mosquito Aedes aegypti: a comparative immunocytochemical analysis using cross-reacting antibodies from Drosophila melanogaster

Considerable effort has been directed towards understanding the organization and function of peripheral and central nervous system of disease vector mosquitoes such as Aedes aegypti. To date, all of these investigations have been carried out on adults but none of the studies addressed the development of the nervous system during the larval and pupal stages in mosquitoes. Here, we first screen a set of 30 antibodies, which have been used to study brain development in Drosophila, and identify 13 of them cross-reacting and labeling epitopes in the developing brain of Aedes. We then use the identified antibodies in immunolabeling studies to characterize general neuroanatomical features of the developing brain and compare them with the well-studied model system, Drosophila melanogaster, in larval, pupal, and adult stages. Furthermore, we use immunolabeling to document the development of specific components of the Aedes brain, namely the optic lobes, the subesophageal neuropil, and serotonergic system of the subesophageal neuropil in more detail. Our study reveals prominent differences in the developing brain in the larval stage as compared to the pupal (and adult) stage of Aedes. The results also uncover interesting similarities and marked differences in brain development of Aedes as compared to Drosophila. Taken together, this investigation forms the basis for future cellular and molecular investigations of brain development in this important disease vector.     

From variable to constant cell numbers: cellular characteristics of the arthropod nervous system argue against a sister-group relationship of Chelicerata and “Myriapoda” but favour the Mandibulata concept

In the new debate on arthropod phylogeny, structure and development of the nervous system provide important arguments. The architecture of the brain of Hexapoda, Crustacea and Chelicerata in recent years has been thoroughly compared against an evolutionary background. However, comparative aspects of the nervous systems in these taxa at the cellular level have been examined in only a few studies. This review sets out to summarize these aspects and to analyse the existing data with respect to the concept of individually identifiable neurons. In particular, mechanisms of neurogenesis, the morphology of serotonergic interneurons, the number of motoneurons, and cellular features and development of the lateral eyes are discussed. We conclude that in comparison to the Mandibulata, in Chelicerata the numbers of neurons in the different classes examined are much higher and in many cases are not fixed but variable. The cell numbers in Mandibulata are lower and the majority of neurons are individually identifiable. The characters explored in this review are mapped onto an existing phylogram, as derived from brain architecture in which the Hexapoda are an in-group of the Crustacea, and there is not any conflict of the current data with such a phylogenetic position of the Hexapoda. Nevertheless, these characters argue against a sister-group relationship of Myriapoda and Chelicerata as has been recently suggested in several molecular studies, but instead provide strong evidence in favour of the Mandibulata concept.Edited by D. Tautz     

Antibodies to FMRF amide, and the related pentapeptide LPLRF amide, reveal two groups of immunoreactive peptides in chicken brain

A radioimmunoassay has been developed for the chicken brain peptide, Leu-Pro-Leu-Arg-Phe-amide (LPLRF amide); this peptide was originally discovered because it reacts with antibodies to the molluscan neuropeptide FMRF amide. The present antibody to LPLRF amide reacts about twenty times less well with FMRF amide compared with LPLRF amide. Using radioimmunoassays employing antibodies raised against LPLRF amide and FMRF amide we have separated by gel filtration and HPLC several different immunoreactive peptides in acid alcohol extracts of chicken brain. When LPLRF amide was used as the assay standard one group of peptides reacted similarly with the two types of antibody; the other group, which was represented by a single major component, reacted at least 50 times better with FMRF amide antibodies compared with LPLRF amide antibodies. It seems, therefore, that in the avian central nervous system, and probably other vertebrates, there are several different groups of peptides immunochemically related to FMRF amide.     

Chromatographic characterisation and biological activity of neuropeptides immunoreactive to antisera against Met5-enkephalin-Arg6-Phe7 (YGGFMRF) extracted from the blowfly Calliphora vomitoria (Diptera).

Neuropeptides identified with a radioimmunoassay specific for the C-terminus of Met5-enkephalin-Arg6-Phe7 (YGGFMRF) have been extracted from nervous tissues of the blowfly Calliphora vomitoria and also from whole flies. Chromatographic characterisation, based on criteria of molecular weight, charge and hydrophobicity, reveals a complex multiplicity of immunoreactive peptides. Variations in the amounts and types of peptides found within different nervous tissues is evidence that the cellular precursor processing is selective. Physiological studies on the isolated blowfly salivary gland show that synthetic YGGFMRF is a potent secretagogue with a maximal rate of fluid secretion induced at a concentration of between 10(-13) and 10(-12) M. The tetrapeptide comprising the last four residues of the C-terminus of YGGFMRF, Phe-Met-Arg-Phe, is equally potent. However, the carboxyamidated variants, YGGFMRF-NH2 and the molluscan cardioacceleratory peptide FMRF-NH2, as well as the opioid peptides Met5- and Leu5-enkephalin, have no activity. Partially purified YGGFMRF-immunoreactive peptides from the blowfly have ED50 values in the bioassay approximating to 0.3 thoracic ganglion, 2.1 hypocerebral ganglion and 3.0 brain equivalents.     

Taxonomic and regional patterns in benthic macroinvertebrate elemental composition in streams

1. Ecological stoichiometry has been used to better understand dynamics in consumer growth and the role of consumer‐recycled nutrients because it focuses on more than one element. Most research has focused on pelagic rather than benthic consumers. Variation in elemental composition among benthic consumer taxa would suggest that taxa differ in their susceptibility to nutrient limitation or in their role in recycling nutrients. 2. We collected benthic macroinvertebrates from streams in two regions (Indiana–Michigan and Wisconsin, U.S.A.) to examine taxonomic and regional variation in benthic macroinvertebrate body carbon (C), nitrogen (N), and phosphorus (P) concentrations and ratios. 3. Elemental composition varied little within taxa common to both regions. In contrast, elemental composition differed greatly among taxa and appeared to be related to phylogeny. The elemental composition of macroinvertebrates clustered into three distinct groups: insects, mollusks, and crustaceans. To a lesser extent, insects and mollusks also differed in elemental composition among genera. 4. Functional feeding groups (FFGs) differed in elemental composition, with predators having a higher N content than other groups. Substantial elemental imbalances between C and N were found between most primary consumers and their likely food sources, and the magnitude of the imbalance depended in part on the FFG. 5. Our results support an assumption of most ecological stoichiometry models that, within a species, the elemental composition of aquatic invertebrates is relatively constant. Variation in elemental composition among taxa at various higher taxonomic levels suggests that susceptibility of stream invertebrates to nutrient limitation and their role in nutrient cycling will strongly depend on phylogeny.     

Nociceptive behavior and physiology of molluscs: animal welfare implications

Molluscs have proven to be invaluable models for basic neuroscience research, yielding fundamental insights into a range of biological processes involved in action potential generation, synaptic transmission, learning, memory, and, more recently, nociceptive biology. Evidence suggests that nociceptive processes in primary nociceptors are highly conserved across diverse taxa, making molluscs attractive models for biomedical studies of mechanisms that may contribute to pain in humans but also exposing them to procedures that might produce painlike sensations. We review the physiology of nociceptors and behavioral responses to noxious stimulation in several molluscan taxa, and discuss the possibility that nociception may result in painlike states in at least some molluscs that possess more complex nervous systems. Few studies have directly addressed possible emotionlike concomitants of nociceptive responses in molluscs. Because the definition of pain includes a subjective component that may be impossible to gauge in animals quite different from humans, firm conclusions about the possible existence of pain in molluscs may be unattainable. Evolutionary divergence and differences in lifestyle, physiology, and neuroanatomy suggest that painlike experiences in molluscs, if they exist, should differ from those in mammals. But reports indicate that some molluscs exhibit motivational states and cognitive capabilities that may be consistent with a capacity for states with functional parallels to pain. We therefore recommend that investigators attempt to minimize the potential for nociceptor activation and painlike sensations in experimental invertebrates by reducing the number of animals subjected to stressful manipulations and by administering appropriate anesthetic agents whenever practicable, welfare practices similar to those for vertebrate subjects.     

GnRH and GnRH receptors: distribution,function and evolution

Gonadotropin‐releasing hormone (GnRH) was originally identified because of its essential role in regulating reproduction in all vertebrates. Since then, three phylogenetically related GnRH decapeptides have been characterized in vertebrates and invertebrates. Almost all tetrapods investigated have at least two GnRH forms (GnRH1 and GnRH2) in the central nervous system. From distributional and functional studies in vertebrates, GnRH1 in the hypothalamus projects predominantly to the pituitary and regulates reproduction via gonadotropin release. GnRH2, which is located in the midbrain, projects to the whole brain and is thought to be involved in sexual behaviour and food intake. GnRH3, located in the forebrain, has only been found in teleost fish and appears to be involved in sexual behaviour, as well as, in some fish species, gonadotropin release. Multiple GnRH receptors (GnRH‐Rs), G‐protein‐coupled receptors regulate endocrine functions and neural transmissions in vertebrates. Phylogenetic and structural analyses of coding sequences show that all vertebrate GnRH‐Rs cluster into two main receptor types comprised of four subfamilies. This suggests that at least two rounds of GnRH receptor gene duplications may have occurred in different groups within each lineage. Functional studies suggest that two particular subfamilies of GnRH receptors have independently evolved to act as species‐specific endocrine modulators in the pituitary, and these show the greatest variety in regulating neuron networks in the brain. Given the long evolutionary history of the GnRH system, it seems likely that much more remains to be understood about its roles in behaviour and function of vertebrates.     

Development and differentiation of neuronal subsets in asexually reproducing Microstomum lineare. Immunocytochemistry of 5-HT, RF-amide and SCPB

The development of immunoreactivity (IR) in the nervous system of asexually reproducing Microstomum lineare has been studied by a combination of simultaneous and double immunostaining with antisera to 5-HT and RF-amide, as well as with monoclonal antibodies to SCPB (molluscan small cardioactive peptide). Immunoreactivity appears in a distinct sequential order. 5-HT antigenicity in the postpharyngeal commissure indicates the initiation of the development of a new zooid. The development of a new brain and pharyngeal plexus always starts in connection to the parental nerve cords. Significantly different developmental patterns are observed for the IR to 5-HT and RF-amide, whereas IR to SCPB has the same localization as that to RF-amide, but appears both weaker and later during the development. Influences of the immunoreactive substances on the asexual reproduction and the feeding behaviour are discussed.     

Molluscan biological and chemical diversity: secondary metabolites and medicinal resources produced by marine molluscs

The phylum Mollusca represents an enormous diversity of species with eight distinct classes. This review provides a taxonomic breakdown of the published research on marine molluscan natural products and the medicinal products currently derived from molluscs, in order to identify priority targets and strategies for future research. Some marine gastropods and bivalves have been of great interest to natural products chemists, yielding a diversity of chemical classes and several drug leads currently in clinical trials. Molluscs also feature prominently in a broad range of traditional natural medicines, although the active ingredients in the taxa involved are typically unknown. Overall secondary metabolites have only been investigated from a tiny proportion (<1%) of molluscan species. At the class level, the number of species subject to chemical studies mirrors species richness and our relative knowledge of the biology of different taxa. The majority of molluscan natural products research is focused within one of the major groups of gastropods, the opisthobranchs (a subgroup of Heterobranchia), which are primarily comprised of soft‐bodied marine molluscs. Conversely, most molluscan medicines are derived from shelled gastropods and bivalves. The complete disregard for several minor classes of molluscs is unjustified based on their evolutionary history and unique life styles, which may have led to novel pathways for secondary metabolism. The Polyplacophora, in particular, have been identified as worthy of future investigation given their use in traditional South African medicines and their abundance in littoral ecosystems. As bioactive compounds are not always constitutively expressed in molluscs, future research should be targeted towards biosynthetic organs and inducible defence reactions for specific medicinal applications. Given the lack of an acquired immune system, the use of bioactive secondary metabolites is likely to be ubiquitous throughout the Mollusca and broadening the search field may uncover interesting novel chemistry.