Molecular phylogeny of the Ellobiidae (Gastropoda: Panpulmonata) supports independent terrestrial invasions.

Gastropods of the family Ellobiidae are an interesting group in which to study transitions from intertidal to terrestrial realms. However, the phylogenetic relationships within this family still lack resolution. We present a phylogenetic hypothesis of the Ellobiidae based on Bayesian and maximum likelihood phylograms. We used nuclear (18S, 28S, H3) and mitochondrial (16S, 12S, COI) data, increasing the numbers of markers and data, and making this the most comprehensive phylogenetic study of the family to date. Our results support phylogenetic hypotheses derived from morphological data, and provide a supported framework to evaluate the internal relationships within Ellobiidae. The resulting phylogenetic trees support the previous hypothesis that the Ellobiidae are monophyletic only if the Trimusculinae (Otina, Smeagol and Trimusculus) are considered part of this family. In addition, we found that the Carychiinae, Ellobiinae and Pythiinae are reciprocally monophyletic and closely related, with the Carychiinae as sister group to Ellobiinae. Relationships within Melampodinae and Pedipedinae and their phylogenetic positions remain unresolved. Land invasion by the Ellobiidae occurred independently in Carychiinae and Pythia during different geological times (Mesozoic and Cenozoic, respectively). Diversification in the family does not appear to be related to past climate and biotic changes, neither the Cretaceous-Paleogene boundary nor the lowering of the sea level in the Oligocene.

Evolution of microgastropods (Ellobioidea, Carychiidae): integrating taxonomic, phylogenetic and evolutionary hypotheses.

BACKGROUND: Current biodiversity patterns are considered largely the result of past climatic and tectonic changes. In an integrative approach, we combine taxonomic and phylogenetic hypotheses to analyze temporal and geographic diversification of epigean (Carychium) and subterranean (Zospeum) evolutionary lineages in Carychiidae (Eupulmonata, Ellobioidea). We explicitly test three hypotheses: 1) morphospecies encompass unrecognized evolutionary lineages, 2) limited dispersal results in a close genetic relationship of geographical proximally distributed taxa and 3) major climatic and tectonic events had an impact on lineage diversification within Carychiidae. RESULTS: Initial morphospecies assignments were investigated by different molecular delimitation approaches (threshold, ABGD, GMYC and SP). Despite a conservative delimitation strategy, carychiid morphospecies comprise a great number of unrecognized evolutionary lineages. We attribute this phenomenon to historic underestimation of morphological stasis and phenotypic variability amongst lineages. The first molecular phylogenetic hypothesis for the Carychiidae (based on COI, 16S and H3) reveals Carychium and Zospeum to be reciprocally monophyletic. Geographical proximally distributed lineages are often closely related. The temporal diversification of Carychiidae is best described by a constant rate model of diversification. The evolution of Carychiidae is characterized by relatively few (long distance) colonization events. We find support for an Asian origin of Carychium. Zospeum may have arrived in Europe before extant members of Carychium. Distantly related Carychium clades inhabit a wide spectrum of the available bioclimatic niche and demonstrate considerable niche overlap. CONCLUSIONS: Carychiid taxonomy is in dire need of revision. An inferred wide distribution and variable phenotype suggest underestimated diversity in Zospeum. Several Carychium morphospecies are results of past taxonomic lumping. By collecting populations at their type locality, molecular investigations are able to link historic morphospecies assignments to their respective evolutionary lineage. We propose that rare founder populations initially colonized a continent or cave system. Subsequent passive dispersal into adjacent areas led to in situ pan-continental or mountain range diversifications. Major environmental changes did not influence carychiid diversification. However, certain molecular delimitation methods indicated a recent decrease in diversification rate. We attribute this decrease to protracted speciation.

Use of axonal projection patterns for the homologisation of cerebral nerves in Opisthobranchia, Mollusca and Gastropoda.

INTRODUCTION: Gastropoda are guided by several sensory organs in the head region, referred to as cephalic sensory organs (CSOs). These CSOs are innervated by distinct nerves. This study proposes a unified terminology for the cerebral nerves and the categories of CSOs and then investigates the neuroanatomy and cellular innervation patterns of these cerebral nerves, in order to homologise them. The homologisation of the cerebral nerves in conjunction with other data, e.g. ontogenetic development or functional morphology, may then provide insights into the homology of the CSOs themselves. RESULTS: Nickel-lysine axonal tracing ("backfilling") was used to stain the somata projecting into specific nerves in representatives of opisthobranch Gastropoda. Tracing patterns revealed the occurrence, size and relative position of somata and their axons and enabled these somata to be mapped to specific cell clusters. Assignment of cells to clusters followed a conservative approach based primarily on relative location of the cells. Each of the four investigated cerebral nerves could be uniquely identified due to a characteristic set of soma clusters projecting into the respective nerves via their axonal pathways. CONCLUSIONS: As the described tracing patterns are highly conserved morphological characters, they can be used to homologise nerves within the investigated group of gastropods. The combination of adequate number of replicates and a comparative approach allows us to provide preliminary hypotheses on homologies for the cerebral nerves. Based on the hypotheses regarding cerebral nerve homology together with further data on ultrastructure and immunohistochemistry of CSOs published elsewhere, we can propose preliminary hypotheses regarding homology for the CSOs of the Opisthobranchia themselves.

Transcriptomic evidence that longevity of acquired plastids in the photosynthetic slugs Elysia timida and Plakobranchus ocellatus does not entail lateral transfer of algal nuclear genes.

Sacoglossan sea slugs are unique in the animal kingdom in that they sequester and maintain active plastids that they acquire from the siphonaceous algae upon which they feed, making the animals photosynthetic. Although most sacoglossan species digest their freshly ingested plastids within hours, four species from the family Plakobranchidae retain their stolen plastids (kleptoplasts) in a photosynthetically active state on timescales of weeks to months. The molecular basis of plastid maintenance within the cytosol of digestive gland cells in these photosynthetic metazoans is yet unknown but is widely thought to involve gene transfer from the algal food source to the slugs based upon previous investigations of single genes. Indeed, normal plastid development requires hundreds of nuclear-encoded proteins, with protein turnover in photosystem II in particular known to be rapid under various conditions. Moreover, only algal plastids, not the algal nuclei, are sequestered by the animals during feeding. If algal nuclear genes are transferred to the animal either during feeding or in the germ line, and if they are expressed, then they should be readily detectable with deep-sequencing methods. We have sequenced expressed mRNAs from actively photosynthesizing, starved individuals of two photosynthetic sea slug species, Plakobranchus ocellatus Van Hasselt, 1824 and Elysia timida Risso, 1818. We find that nuclear-encoded, algal-derived genes specific to photosynthetic function are expressed neither in P. ocellatus nor in E. timida. Despite their dramatic plastid longevity, these photosynthetic sacoglossan slugs do not express genes acquired from algal nuclei in order to maintain plastid function.

The taxonomist - an endangered race. A practical proposal for its survival.

BACKGROUND: Taxonomy or biological systematics is the basic scientific discipline of biology, postulating hypotheses of identity and relationships, on which all other natural sciences dealing with organisms relies. However, the scientific contributions of taxonomists have been largely neglected when using species names in scientific publications by not citing the authority on which they are based. DISCUSSION: Consequences of this neglect is reduced recognition of the importance of taxonomy, which in turn results in diminished funding, lower interest from journals in publishing taxonomic research, and a reduced number of young scientists entering the field. This has lead to the so-called taxonomic impediment at a time when biodiversity studies are of critical importance.Here we emphasize a practical and obvious solution to this dilemma. We propose that whenever a species name is used, the author(s) of the species hypothesis be included and the original literature source cited, including taxonomic revisions and identification literature - nothing more than what is done for every other hypothesis or assumption included in a scientific publication. In addition, we postulate that journals primarily publishing taxonomic studies should be indexed in ISISM. SUMMARY: The proposal outlined above would make visible the true contribution of taxonomists within the scientific community, and would provide a more accurate assessment for funding agencies impact and importance of taxonomy, and help in the recruitment of young scientists into the field, thus helping to alleviate the taxonomic impediment. In addition, it would also make much of the biological literature more robust by reducing or alleviating taxonomic uncertainty.

Out of Antarctica?--new insights into the phylogeny and biogeography of the Pleurobranchomorpha (Mollusca, Gastropoda).

The aim of the current study was to gain new insights into the phylogeny, biogeography and evolution of the opisthobranch clade Pleurobranchomorpha. We focused on testing the hypothesis of an Antarctic origin of this clade. The combination of four gene markers (18S rDNA, 28S rDNA, 16S rDNA and CO1) was used to infer a phylogenetic hypothesis of the Pleurobranchomorpha employing Maximum likelihood and Bayesian inference methods. Four methodologically distinct approaches were applied to reconstruct the historical biogeography and dating of the tree was performed via relaxed molecular clock analysis. Phylogenetic analyses supported the monophyly of the Pleurobranchomorpha and their sister group relationship to the Nudibranchia. Monophyly of the main subgroups Pleurobranchaeinae and Pleurobranchinae could not be revealed. Reconstruction of the ancestral area of the Pleurobranchomorpha yielded different possibilities in the diverse analyses. However, the Pleurobranchinae most probably derived from an Antarctic origin. Estimation of divergence times revealed a long credible interval for the Pleurobranchomorpha, whereas the Pleurobranchinae diverged in Early Oligocene and underwent rapid radiation during Oligocene and Early Miocene. Divergence of the Pleurobranchinae into the Antarctic Tomthompsonia and the remaining species in Early Oligocene coincides with two major geological events; namely the onset of glaciation in Antarctica and the opening of the Drake Passage with following formation of the Antarctic Circumpolar Current (ACC). These sudden and dramatic changes in climate probably led to subsequent migration of the last common ancestor of the remaining Pleurobranchinae into warmer regions, while the ACC may have accounted for larval dispersal to the Eastern Atlantic.

The long way to diversity--phylogeny and evolution of the Heterobranchia (Mollusca: Gastropoda).

Heterobranchia are one of the most species rich groups within Gastropoda, with poorly resolved phylogenetic relationships especially in basal taxa. In order to resolve phylogenetic relationships within the Heterobranchia, we pursued a molecular systematic approach by sequencing and analysing a variety of genetic markers (including nuclear 28S rDNA+18S rDNA and mitochondrial 16S rDNA+COI sequences). Maximum likelihood as well as Bayesian inference methods were used for phylogenetic reconstruction. Moreover, data quality was estimated for the purpose of proving the plausibility of the novel phylogenetic hypothesis using a variety of statistical tests as well as network analyses. Finally, a case study was conducted in order to estimate divergence ages using a "relaxed" molecular clock approach with fossils as minimum age constraints. All phylogenetic analyses revealed the Heterobranchia as monophyletic. Within the Heterobranchia, several well supported clades could be resolved. However, the traditional classification based on morphological data could not be confirmed due to paraphyletic Euthyneura as well as paraphyletic Pulmonata and polyphyletic Opisthobranchia. The estimation of data quality yielded a high degree of substitution saturation in many of the nucleotide positions while the Relative-Rate-Test revealed the highest evolution rates within the "Lower Heterobranchia". Although the dataset shows much conflict, many of the proposed hypotheses are supported by splits of the network analysis. The molecular clock approach was able to confirm some evolutionary hypotheses based on fossils such as the late occurrence of Pulmonata and Stylommatophora, respectively, during the Mesozoic. However, large 95% highest posterior density (HPD) intervals at some of the nodes made a precise dating of these nodes difficult. This molecular phylogenetic investigation provides the most comprehensive molecular study of relationships within the Heterobranchia to date. Due to the outstanding taxon sampling of the "Lower Heterobranchia", which have been poorly represented in earlier morphological and molecular studies, substantial insights into the evolution and phylogeny of this enigmatic taxon have been gained.

Neuromuscular development of Aeolidiella stephanieae Valdéz, 2005 (Mollusca, Gastropoda, Nudibranchia).

BACKGROUND: Studies on the development of the nervous system and the musculature of invertebrates have become more sophisticated and numerous within the last decade and have proven to provide new insights into the evolutionary history of organisms. In order to provide new morphogenetic data on opisthobranch gastropods we investigated the neuromuscular development in the nudibranch Aeolidiella stephanieae Valdéz, 2005 using immunocytochemistry as well as F-actin labelling in conjunction with confocal laser scanning microscopy (cLSM). RESULTS: The ontogenetic development of Aeolidiella stephanieae can be subdivided into 8 stages, each recognisable by characteristic morphological and behavioural features as well as specific characters of the nervous system and the muscular system, respectively. The larval nervous system of A. stephanieae includes an apical organ, developing central ganglia, and peripheral neurons associated with the velum, foot and posterior, visceral part of the larva. The first serotonergic and FMRFamidergic neural structures appear in the apical organ that exhibits an array of three sensory, flask-shaped and two non-sensory, round neurons, which altogether disappear prior to metamorphosis. The postmetamorphic central nervous system (CNS) becomes concentrated, and the rhinophoral ganglia develop together with the anlage of the future rhinophores whereas oral tentacle ganglia are not found. The myogenesis in A. stephanieae begins with the larval retractor muscle followed by the accessory larval retractor muscle, the velar or prototroch muscles and the pedal retractors that all together degenerate during metamorphosis, and the adult muscle complex forms de novo. CONCLUSIONS: Aeolidiella stephanieae comprises features of the larval and postmetamorphic nervous as well as muscular system that represent the ground plan of the Mollusca or even the Trochozoa (e. g. presence of the prototrochal or velar muscle ring). On the one hand, A. stephanieae shows some features shared by all nudibranchs like the postmetamorphic condensation of the CNS, the possession of rhinophoral ganglia and the lack of oral tentacle ganglia as well as the de novo formation of the adult muscle complex. On the other hand, the structure and arrangement of the serotonergic apical organ is similar to other caenogastropod and opisthobranch gastropods supporting their sister group relationship.

Phylogenetic support values are not necessarily informative: the case of the Serialia hypothesis (a mollusk phylogeny).

BACKGROUND: Molecular phylogenies are being published increasingly and many biologists rely on the most recent topologies. However, different phylogenetic trees often contain conflicting results and contradict significant background data. Not knowing how reliable traditional knowledge is, a crucial question concerns the quality of newly produced molecular data. The information content of DNA alignments is rarely discussed, as quality statements are mostly restricted to the statistical support of clades. Here we present a case study of a recently published mollusk phylogeny that contains surprising groupings, based on five genes and 108 species, and we apply new or rarely used tools for the analysis of the information content of alignments and for the filtering of noise (masking of random-like alignment regions, split decomposition, phylogenetic networks, quartet mapping). RESULTS: The data are very fragmentary and contain contaminations. We show that that signal-like patterns in the data set are conflicting and partly not distinct and that the reported strong support for a "rather surprising result" (monoplacophorans and chitons form a monophylum Serialia) does not exist at the level of primary homologies. Split-decomposition, quartet mapping and neighbornet analyses reveal conflicting nucleotide patterns and lack of distinct phylogenetic signal for the deeper phylogeny of mollusks. CONCLUSION: Even though currently a majority of molecular phylogenies are being justified with reference to the 'statistical' support of clades in tree topologies, this confidence seems to be unfounded. Contradictions between phylogenies based on different analyses are already a strong indication of unnoticed pitfalls. The use of tree-independent tools for exploratory analyses of data quality is highly recommended. Concerning the new mollusk phylogeny more convincing evidence is needed.

From sea to land and beyond--new insights into the evolution of euthyneuran Gastropoda (Mollusca).

BACKGROUND: The Euthyneura are considered to be the most successful and diverse group of Gastropoda. Phylogenetically, they are riven with controversy. Previous morphology-based phylogenetic studies have been greatly hampered by rampant parallelism in morphological characters or by incomplete taxon sampling. Based on sequences of nuclear 18S rRNA and 28S rRNA as well as mitochondrial 16S rRNA and COI DNA from 56 taxa, we reconstructed the phylogeny of Euthyneura utilising Maximum Likelihood and Bayesian inference methods. The evolution of colonization of freshwater and terrestrial habitats by pulmonate Euthyneura, considered crucial in the evolution of this group of Gastropoda, is reconstructed with Bayesian approaches. RESULTS: We found several well supported clades within Euthyneura, however, we could not confirm the traditional classification, since Pulmonata are paraphyletic and Opistobranchia are either polyphyletic or paraphyletic with several clades clearly distinguishable. Sacoglossa appear separately from the rest of the Opisthobranchia as sister taxon to basal Pulmonata. Within Pulmonata, Basommatophora are paraphyletic and Hygrophila and Eupulmonata form monophyletic clades. Pyramidelloidea are placed within Euthyneura rendering the Euthyneura paraphyletic. CONCLUSION: Based on the current phylogeny, it can be proposed for the first time that invasion of freshwater by Pulmonata is a unique evolutionary event and has taken place directly from the marine environment via an aquatic pathway. The origin of colonisation of terrestrial habitats is seeded in marginal zones and has probably occurred via estuaries or semi-terrestrial habitats such as mangroves.

Opisthobranchia (Mollusca, Gastropoda) - more than just slimy slugs. Shell reduction and its implications on defence and foraging.

BACKGROUND: In general shell-less slugs are considered to be slimy animals with a rather dull appearance and a pest to garden plants. But marine slugs usually are beautifully coloured animals belonging to the less-known Opisthobranchia. They are characterized by a large array of interesting biological phenomena, usually related to foraging and/or defence. In this paper our knowledge of shell reduction, correlated with the evolution of different defensive and foraging strategies is reviewed, and new results on histology of different glandular systems are included. RESULTS: Based on a phylogeny obtained by morphological and histological data, the parallel reduction of the shell within the different groups is outlined. Major food sources are given and glandular structures are described as possible defensive structures in the external epithelia, and as internal glands. CONCLUSION: According to phylogenetic analyses, the reduction of the shell correlates with the evolution of defensive strategies. Many different kinds of defence structures, like cleptocnides, mantle dermal formations (MDFs), and acid glands, are only present in shell-less slugs. In several cases, it is not clear whether the defensive devices were a prerequisite for the reduction of the shell, or reduction occurred before. Reduction of the shell and acquisition of different defensive structures had an implication on exploration of new food sources and therefore likely enhanced adaptive radiation of several groups.

Evolution of leaf warbler songs (Aves: Phylloscopidae).

Songs in passerine birds are important for territory defense and mating. Speciation rates in oscine passerines are so high, due to cultural evolution, that this bird lineage makes up half of the extant bird species. Leaf warblers are a speciose Old-World passerine family of limited morphological differentiation, so that songs are even more important for species delimitation. We took 16 sonographic traits from song recordings of 80 leaf warbler taxa and correlated them with 15 potentially explanatory variables, pairwise, and in linear models. Based on a well-resolved molecular phylogeny of the same taxa, all pairwise correlations were corrected for relatedness with phylogenetically independent contrasts and phylogenetic generalized linear models were used. We found a phylogenetic signal for most song traits, but a strong one only for the duration of the longest and of the shortest element, which are presumably inherited instead of learned. Body size of a leaf warbler species is a constraint on song frequencies independent of phylogeny. At least in this study, habitat density had only marginal impact on song features, which even disappeared through phylogenetic correction. Maybe most leaf warblers avoid the deterioration through sound propagation in dense vegetation by singing from exposed perches. Latitudinal (and longitudinal) extension of the breeding ranges was correlated with most song features, especially verse duration (longer polewards and westwards) and complexity (lower polewards). Climate niche or expansion history might explain these correlations. The number of different element types per verse decreases with elevation, possibly due to fewer resources and congeneric species at higher elevations.

Misperceived invasion: the Lusitanian slug (Arion lusitanicus auct. non-Mabille or Arion vulgaris Moquin-Tandon 1855) is native to Central Europe.

The Lusitanian slug, presumed to be native to south-west Europe, was ranked among the 100 worst invading species in Central Europe. However, from the very beginning of its recognition in the presumed invasion area, there was little evidence that the species was actually anthropogenically introduced. We investigated the invasive status of the species by comparing specific predictions on the population genetic structure in the invasion area with the pattern actually found. In a DNA-taxonomy approach, the species could not be found in its presumed native range. Using statistical phylogeographic techniques on a mitochondrial (COI) and nuclear (ZF) marker and species distribution modelling, we could show that the species is with very high probability not an invasor, but native to Central Europe. The study underlines the value of statistical phylogeography in rigorously testing hypotheses on the dynamics of biological invasions.

Alpine crossroads or origin of genetic diversity? Comparative phylogeography of two sympatric microgastropod species.

The Alpine Region, constituting the Alps and the Dinaric Alps, has played a major role in the formation of current patterns of biodiversity either as a contact zone of postglacial expanding lineages or as the origin of genetic diversity. In our study, we tested these hypotheses for two widespread, sympatric microgastropod taxa--Carychium minimum O.F. Müller, 1774 and Carychium tridentatum (Risso, 1826) (Gastropoda, Eupulmonata, Carychiidae)--by using COI sequence data and species potential distribution models analyzed in a statistical phylogeographical framework. Additionally, we examined disjunct transatlantic populations of those taxa from the Azores and North America. In general, both Carychium taxa demonstrate a genetic structure composed of several differentiated haplotype lineages most likely resulting from allopatric diversification in isolated refugial areas during the Pleistocene glacial periods. However, the genetic structure of Carychium minimum is more pronounced, which can be attributed to ecological constraints relating to habitat proximity to permanent bodies of water. For most of the Carychium lineages, the broader Alpine Region was identified as the likely origin of genetic diversity. Several lineages are endemic to the broader Alpine Region whereas a single lineage per species underwent a postglacial expansion to (re)colonize previously unsuitable habitats, e.g. in Northern Europe. The source populations of those expanding lineages can be traced back to the Eastern and Western Alps. Consequently, we identify the Alpine Region as a significant 'hot-spot' for the formation of genetic diversity within European Carychium lineages. Passive dispersal via anthropogenic means best explains the presence of transatlantic European Carychium populations on the Azores and in North America. We conclude that passive (anthropogenic) transport could mislead the interpretation of observed phylogeographical patterns in general.

A new approach to an old conundrum--DNA barcoding sheds new light on phenotypic plasticity and morphological stasis in microsnails (Gastropoda, Pulmonata, Carychiidae).

The identification of microsnail taxa based on morphological characters is often a time-consuming and inconclusive process. Aspects such as morphological stasis and phenotypic plasticity further complicate their taxonomic designation. In this study, we demonstrate that the application of DNA barcoding can alleviate these problems within the Carychiidae (Gastropoda, Pulmonata). These microsnails are a taxon of the pulmonate lineage and most likely migrated onto land independently of the Stylommatophora clade. Their taxonomical classification is currently based on conchological and anatomical characters only. Despite much confusion about historic species assignments, the Carychiidae can be unambiguously subdivided into two taxa: (i) Zospeum species, which are restricted to karst caves, and (ii) Carychium species, which occur in a broad range of environmental conditions. The implementation of discrete molecular data (COI marker) enabled us to correctly designate 90% of the carychiid microsnails. The remaining cases were probably cryptic Zospeum and Carychium taxa and incipient species, which require further investigation into their species status. Because conventional reliance upon mostly continuous (i.e. nondiscrete) conchological characters is subject to fallibility for many gastropod species assignments, we highly recommend the use of DNA barcoding as a taxonomic, cutting-edge method for delimiting microsnail taxa.

Myogenesis in Aplysia californica (Cooper, 1863) (Mollusca, Gastropoda, Opisthobranchia) with special focus on muscular remodeling during metamorphosis.

To date only few comparative approaches tried to reconstruct the ontogeny of the musculature in invertebrates. This may be due to the difficulties involved in reconstructing three dimensionally arranged muscle systems by means of classical histological techniques combined with light or transmission electron microscopy. Within the scope of the present study we investigated the myogenesis of premetamorphic, metamorphic, and juvenile developmental stages of the anaspidean opisthobranch Aplysia californica using fluorescence F-actin-labeling in conjunction with modern confocal laser scanning microscopy. We categorized muscles with respect to their differentiation and degeneration and found three true larval muscles that differentiate during the embryonic and veliger phase and degenerate during or slightly after metamorphosis. These are the larval retractor, the accessory larval retractor, and the metapodial retractor muscle. While the pedal retractor muscle, some transversal mantle fibers and major portions of the cephalopedal musculature are continued and elaborated during juvenile and adult life, the buccal musculature and the anterior retractor muscle constitute juvenile/adult muscles which differentiate during or after metamorphosis. The metapodial retractor muscle has never been reported for any other gastropod taxon. Our findings indicate that the late veliger larva of A. californica shares some common traits with veligers of other gastropods, such as a larval retractor muscle. However, the postmetamorphic stages exhibit only few congruencies with other gastropod taxa investigated to date, which is probably due to common larval but different adult life styles within gastropods. Accordingly, this study provides further evidence for morphological plasticity in gastropod myogenesis and stresses the importance of ontogenetic approaches to understand adult conditions and life history patterns.

Neurogenesis of cephalic sensory organs of Aplysia californica.

The opisthobranch gastropod Aplysia californica serves as a model organism in experimental neurobiology because of its simple and well-known nervous system. However, its nervous periphery has been less intensely studied. We have reconstructed the ontogeny of the cephalic sensory organs (labial tentacles, rhinophores, and lip) of planktonic, metamorphic, and juvenile developmental stages. FMRFamide and serotonergic expression patterns have been examined by immunocytochemistry in conjunction with epifluorescence and confocal laser scanning microscopy. We have also applied scanning electron microscopy to analyze the ciliary distribution of these sensory epithelia. Labial tentacles and the lip develop during metamorphosis, whereas rhinophores appear significantly later, in stage 10 juveniles. Our study has revealed immunoreactivity against FMRFamides and serotonin in all major nerves. The common labial nerve develops first, followed by the labial tentacle base nerve, oral nerve, and rhinophoral nerve. We have also identified previously undescribed neuronal pathways and other FMRFamide-like-immunoreactive neuronal elements, such as peripheral ganglia and glomerulus-like structures, and two groups of conspicuous transient FMRFamide-like cell somata. We have further found two distinct populations of FMRFamide-positive cell somata located both subepidermally and in the inner regions of the cephalic sensory organs in juveniles. The latter population partly consists of sensory cells, suggesting an involvement of FMRFamide-like peptides in the modulation of peripheral sensory processes. This study is the first concerning the neurogenesis of cephalic sensory organs in A. californica and may serve as a basis for future studies of neuronal elements in gastropod molluscs.