A histochemical and morphological study of the mucus producing pedal gland system in Latia neritoides (Mollusca; Gastropoda; Hygrophila).

The freshwater gastropod Latia neritoides is endemic to the streams of New Zealand's North Island. This species has evolved a unique defence system: it exudes a luminescent mucus thought to deter predators. While the bioluminescence itself has been investigated before, the underlying gland system has remained unstudied and relevant information to understand the defence system has been missing till now. For the release of the glowing mucus of L. neritoides two places of origin were assumed: the lateral foot area or the mantel cavity. In this study the focus was on the first suggestion. To gain insight into the defence system, morphological as well as histochemical analyses were performed involving all secretory gland types in the sub-epithelial foot layer. The results were compared with the foot gland system of Neritina sp., a snail living in a comparable habitat, but using a different survival strategy. The gland types of the two gastropods were compared and their mucus types were investigated. Seven subepithelial gland cell types can be distinguished in the foot region of L. neritoides. Neritina sp., in contrast, has six gland cell types of which three laterally located ones are epithelial. Both species show a pedal gland in the anterior foot region. A striking difference between the species are two prominent subepithelial gland cell types (L1l/L2l) in the lateral foot area of L. neritoides, which are missing in Neritina sp. These gland cells are distributed throughout the entire lateral foot area of L. neritoides and make up about 85% of the mucus gland cells in this area. Defence mucus and trail mucus of L. neritoides show different specificities in lectin staining, but are not equally represented in the gland cell types. Yet, based on the huge size and high density of L1l and L2L, we envision a role for these gland types in the defence system.

Within-family plasticity of nervous system architecture in Syllidae (Annelida, Errantia).

BACKGROUND: The ground pattern underlying the nervous system of the last common ancestor in annelids was long thought to be settled, consisting of a dorsal brain, circumoesophageal connectives and a subepithelial, ladder-like ventral nerve cord with segmental ganglia connected by paired connectives. With the advent of immunocytochemical stainings and confocal laser scanning microscopy, it becomes evident that its architecture is extremely diverse, which makes the reconstruction of a ground pattern in annelida challenging. Whereas the nervous systems of many different families has already been described, only very few studies looked at the diversity of nervous systems within such clades to give a closer estimate on how plastic the annelid nervous system really is. So far, little is known on syllid nervous system architecture, one of the largest and most diverse groups of marine annelids. RESULTS: The position of the brain, the circumoesophageal connectives, the stomatogastric nervous system, the longitudinal nerves that traverse each segment and the innervation of appendages are relatively uniform within the clade. Both the number of connectives within the ventral nerve cord and the number of segmental nerves, which in earlier studies were used to infer phylogenetic relationships and to reconstruct an annelid ground pattern, are highly diverse and differ between genera or even within a given genus. Differences in the distribution of somata of the brain, the nuchal innervation and its associated cell bodies were found between Syllinae and Exogoninae and may be subfamily-specific. CONCLUSIONS: The nervous system morphology of syllids very likely depends on the taxon-specific ecological requirements. Thus, it is not surprising that in a clade, which occupies such diverse niches as the Annelida, we find similar patterns in phylogenetically widely separated species in similar niches and a high degree of modularity within a family. Only standardized protocols and staining methods can lead to comparable results, but so far different approaches have been taken to describe annelid nervous systems, making homologization of certain structures difficult. This study provides the first thorough description of the nervous system in the family Syllidae, allowing more detailed comparisons between annelid families in the future.

Distinct pathways for zinc metabolism in the terrestrial slug Arion vulgaris.

In most organisms, the concentration of free Zn2+ is controlled by metallothioneins (MTs). In contrast, no significant proportions of Zn2+ are bound to MTs in the slug, Arion vulgaris. Instead, this species possesses cytoplasmic low-molecular-weight Zn2+ (LMW Zn) binding compound that divert these metal ions into pathways uncoupled from MT metabolism. Zn2+ is accumulated in the midgut gland calcium cells of Arion vulgaris, where they associate with a low-molecular-weight ligand with an apparent molecular mass of ~ 2,000 Da. Mass spectrometry of the semi-purified LMW Zn binding compound combining an electrospray ion source with a differential mobility analyser coupled to a time-of-flight mass spectrometer revealed the presence of four Zn2+-containing ion signals, which arise from disintegration of one higher MW complex resulting in an ion-mobility diameter of 1.62 nm and a molecular mass of 837 Da. We expect that the novel Zn2+ ion storage pathway may be shared by many other gastropods, and particularly species that possess Cd-selective MT isoforms or variants with only very low affinity to Zn2+.

Complete mitochondrial genomes of two scaphopod molluscs.

Complete mitochondrial genomes were determined for two scaphopod molluscs: the dentaliid Antalis entalis and an unidentified Antarctic gadilid. Both genomes are complete except, in Gadilida sp. indet., a short stretch of nad5 was undetermined and trnR could not be annotated. Organization of the Gadilida sp. genome is nearly identical to that previously reported for the gadilid Siphonodentalium whereas trnK, nad5, trnD, nad4, and nad4l are transposed to the opposite strand in the previously published Graptacme genome relative to that of Antalis. Phylogenetic analysis of the 13 protein-coding and 2 rRNA genes recovered Scaphopoda, Gadilida, and Dentaliida monophyletic with maximal support.

Catalog of taxa introduced by Luitfried Salvini-Plawen (1939-2014).

Luitfried Salvini-Plawen was one of the most distinguished researchers for molluscan phylogenetic systematics of the last decades. In his publications he described a total of 193 species: 134 Solenogastres, 34 Caudofoveata, 14 interstitial Gastropoda, one polyplacophoran and the remaining comprising Cnidaria, Priapulida, Kamptozoa, and Echinodermata. In addition, he introduced 47 genus-group names and 54 names for family-level and higher taxa. This catalog comprises lists of all taxon names published by Luitfried Salvini-Plawen. The catalog entries contain taxonomic information, original citations, type localities and type collections. It aims to facilitate further research on these and related taxa.

Comparison of the SAWNUC model with CLOUD measurements of sulphuric acid-water nucleation.

Binary nucleation of sulphuric acid-water particles is expected to be an important process in the free troposphere at low temperatures. SAWNUC (Sulphuric Acid Water Nucleation) is a model of binary nucleation that is based on laboratory measurements of the binding energies of sulphuric acid and water in charged and neutral clusters. Predictions of SAWNUC are compared for the first time comprehensively with experimental binary nucleation data from the CLOUD chamber at European Organization for Nuclear Research. The experimental measurements span a temperature range of 208-292 K, sulphuric acid concentrations from 1·106 to 1·109 cm-3, and distinguish between ion-induced and neutral nucleation. Good agreement, within a factor of 5, is found between the experimental and modeled formation rates for ion-induced nucleation at 278 K and below and for neutral nucleation at 208 and 223 K. Differences at warm temperatures are attributed to ammonia contamination which was indicated by the presence of ammonia-sulphuric acid clusters, detected by an Atmospheric Pressure Interface Time of Flight (APi-TOF) mass spectrometer. APi-TOF measurements of the sulphuric acid ion cluster distributions ( (H2SO4)i·HSO4- with i = 0, 1, ..., 10) show qualitative agreement with the SAWNUC ion cluster distributions. Remaining differences between the measured and modeled distributions are most likely due to fragmentation in the APi-TOF. The CLOUD results are in good agreement with previously measured cluster binding energies and show the SAWNUC model to be a good representation of ion-induced and neutral binary nucleation of sulphuric acid-water clusters in the middle and upper troposphere.

Neutral molecular cluster formation of sulfuric acid-dimethylamine observed in real time under atmospheric conditions.

For atmospheric sulfuric acid (SA) concentrations the presence of dimethylamine (DMA) at mixing ratios of several parts per trillion by volume can explain observed boundary layer new particle formation rates. However, the concentration and molecular composition of the neutral (uncharged) clusters have not been reported so far due to the lack of suitable instrumentation. Here we report on experiments from the Cosmics Leaving Outdoor Droplets chamber at the European Organization for Nuclear Research revealing the formation of neutral particles containing up to 14 SA and 16 DMA molecules, corresponding to a mobility diameter of about 2 nm, under atmospherically relevant conditions. These measurements bridge the gap between the molecular and particle perspectives of nucleation, revealing the fundamental processes involved in particle formation and growth. The neutral clusters are found to form at or close to the kinetic limit where particle formation is limited only by the collision rate of SA molecules. Even though the neutral particles are stable against evaporation from the SA dimer onward, the formation rates of particles at 1.7-nm size, which contain about 10 SA molecules, are up to 4 orders of magnitude smaller compared with those of the dimer due to coagulation and wall loss of particles before they reach 1.7 nm in diameter. This demonstrates that neither the atmospheric particle formation rate nor its dependence on SA can simply be interpreted in terms of cluster evaporation or the molecular composition of a critical nucleus.

Characterisation and localisation of the opsin protein repertoire in the brain and retinas of a spider and an onychophoran.

BACKGROUND: Opsins have been found in the majority of animals and their most apparent functions are related to vision and light-guided behaviour. As an increasing number of sequences have become available it has become clear that many opsin-like transcripts are expressed in tissues other than the eyes. Opsins can be divided into three main groups: rhabdomeric opsins (r-opsins), ciliary opsins (c-opsins) and group 4 opsins. In arthropods, the main focus has been on the r-opsins involved in vision. However, with increased sequencing it is becoming clear that arthropods also possess opsins of the c-type, group 4 opsins and the newly discovered arthropsins but the functions of these opsins are unknown in arthropods and data on their localisation is limited or absent. RESULTS: We identified opsins from the spider Cupiennius salei and the onychophoran Euperipatoides kanangrensis and characterised the phylogeny and localisation of these transcripts. We recovered all known visual opsins in C. salei, and in addition found a peropsin, a c-opsin and an opsin resembling Daphnia pulex arthropsin. The peropsin was expressed in all eye types except the anterior median eyes. The arthropsin and the c-opsin were expressed in the central nervous system but not the eyes. In E. kanangrensis we found: a c-opsin; an opsin resembling D. pulex arthropsins; and an r-opsin with high sequence similarity to previously published onychophoran onychopsins. The E. kanangrensis c-opsin and onychopsin were expressed in both the eyes and the brain but the arthropsin only in the brain. CONCLUSION: Our novel finding that opsins of both the ciliary and rhabdomeric type are present in the onychophoran and a spider suggests that these two types of opsins were present in the last common ancestor of the Onychophora and Euarthropoda. The expression of the c-opsin in the eye of an onychophoran indicates that c-opsins may originally have been involved in vision in the arthropod clade. The lack of c-opsin expression in the spider retina suggests that the role for c-opsin in vision was lost in the euarthropods. Our discovery of arthropsin in onychophorans and spiders dates the emergence of arthropsin to the common ancestor of Onychophora and Euarthropoda and their expression in the brain suggests a non-visual function.

The effect of carrier gas contaminants on the charging probability of aerosols under bipolar charging conditions.

This work concentrates on the experimental determination of the properties of ionic molecular clusters that are produced in the bipolar ionic atmosphere of a radioactivity based (241)Am charger. The main scope of this study was to investigate the dependency of the ions' properties on carrier gas contaminants caused by the evaporation of trace gases from different kinds of frequently encountered tubing materials. A recently developed high resolution mobility spectrometer allows the precise determination of the ions' electrical mobility; an empirical mass-mobility relationship was used to approximate the corresponding ion masses. It was found that impurities in the carrier gas dramatically change the pattern of the ion mobility/size distribution, resulting in very different ion properties that strongly depend on the carrier gas composition. Since the ion properties control the charging process of aerosols, it was further investigated how the different ion properties affect the calculation of the charging probabilities of aerosols. The results show that despite large variations of the ions' properties, only a minor effect on the calculated charging probabilities can be found.

Quantitative characterization of critical nanoclusters nucleated on large single molecules.

First order phase transitions involve nucleation, formation of nanoscale regions of a new phase within a metastable parent phase. Using the heterogeneous nucleation theorem we show how clusters formed by nucleation on single molecules evolve from the gas phase and determine the critical size beyond which condensation starts to form aerosol particles. Our experiments reveal the activation of molecules into droplets to happen via formation of critical clusters substantially larger than the seed molecule. The nanosized critical clusters were found to be well predicted by the Kelvin-Thomson relation pointing directly to the key step in the phase transition.

Expression of Hox genes during the larval development of the snail, Gibbula varia (L.)-further evidence of non-colinearity in molluscs.

Hox transcription factors, a subfamily of homeobox genes, are expressed in distinct, often overlapping domains along the anterior-posterior body axis of animal embryos. Here, we report the sequence and expression pattern of Hox2, Hox3, Hox4, Hox5, Lox5, Hox7, Lox4, and Lox2 in different larval stages during the encapsulated development of the marine gastropod Gibbula varia. Our results show that all Gva-Hox genes are expressed in ectoderm-derived cells. Hox2, Hox3, Hox4, Hox5, and Hox7 are expressed in overlapping patterns in the pedal, pleural, oesophageal, and visceral ganglia, supporting the ancestral role of Hox genes in the neurogenesis processes in bilaterians. Gva-Hox1, Gva-Post2, and Gva-Post1 genes are involved in shell morphogenesis and have apparently lost their role in neurogangliogenesis. Lox5, Lox4, and Lox2 are expressed in different cells of the apical organ during the earlier larval stage (trochophore) and the cerebral ganglia during later larval stages (veliger). These results support the hypothesis that apical organ neurosensory cells contribute to the formation of cerebral ganglia commissures during metamorphosis. Gva-Hox7 and Gva-Lox4 are additionally expressed in the prototroch of the trochophore and in the velar area of the veliger larvae. This contradicts with the expression of these genes in the annelids, where most of Hox genes are expressed in the posttrochal area and are involved in segmental determination. Therefore, expression of Hox genes may serve as an example of co-option and plasticity of gene function during evolution of gastropods.

Conservation of ParaHox genes' function in patterning of the digestive tract of the marine gastropod Gibbula varia.

BACKGROUND: Presence of all three ParaHox genes has been described in deuterostomes and lophotrochozoans, but to date one of these three genes, Xlox has not been reported from any ecdysozoan taxa and both Xlox and Gsx are absent in nematodes. There is evidence that the ParaHox genes were ancestrally a single chromosomal cluster. Colinear expression of the ParaHox genes in anterior, middle, and posterior tissues of several species studied so far suggest that these genes may be responsible for axial patterning of the digestive tract. So far, there are no data on expression of these genes in molluscs. RESULTS: We isolated the complete coding sequences of the three Gibbula varia ParaHox genes, and then tested their expression in larval and postlarval development. In Gibbula varia, the ParaHox genes participate in patterning of the digestive tract and are expressed in some cells of the neuroectoderm. The expression of these genes coincides with the gradual formation of the gut in the larva. Gva-Gsx patterns potential neural precursors of cerebral ganglia as well as of the apical sensory organ. During larval development this gene is involved in the formation of the mouth and during postlarval development it is expressed in the precursor cells involved in secretion of the radula, the odontoblasts. Gva-Xolx and Gva-Cdx are involved in gut patterning in the middle and posterior parts of digestive tract, respectively. Both genes are expressed in some ventral neuroectodermal cells; however the expression of Gva-Cdx fades in later larval stages while the expression of Gva-Xolx in these cells persists. CONCLUSIONS: In Gibbula varia the ParaHox genes are expressed during anterior-posterior patterning of the digestive system. This colinearity is not easy to spot during early larval stages because the differentiated endothelial cells within the yolk permanently migrate to their destinations in the gut. After torsion, Gsx patterns the mouth and foregut, Xlox the midgut gland or digestive gland, and Cdx the hindgut. ParaHox genes of Gibbula are also expressed during specification of cerebral and ventral neuroectodermal cells. Our results provide additional support for the ancestral complexity of Gsx expression and its ancestral role in mouth patterning in protostomes, which was secondarily lost or simplified in some species.

Involvement of Hox genes in shell morphogenesis in the encapsulated development of a top shell gastropod (Gibbula varia L.).

Regulatory gene expression during the patterning of molluscan shells has only recently drawn the attention of scientists. We show that several Hox genes are expressed in association with the shell gland and the mantle in the marine vetigastropod Gibbula varia (L.). The expression of Gva-Hox1, Gva-Post2, and Gva-Post1 is initially detected in the trochophore larval stage in the area of the shell field during formation of embryonic shell. Later, during development, these genes are expressed in the mantle demonstrating their continuous role in larval shell formation and differentiation of mantle edge that secretes the adult shell. Gva-Hox4 is expressed only late during the development of the veliger-like larva and may also be involved in the adult shell morphogenesis. Additionally, this gene also seems to be associated with secretion of another extracellular structure, the operculum. Our data provide further support for association of Hox genes with shell formation which suggest that the molecular mechanisms underlying shell synthesis may consist of numerous conserved pattern-formation genes. In cephalopods, the only other molluscan class in which Hox gene expression has been studied, no involvement of Hox genes in shell formation has been reported. Thus, our results suggest that Hox genes are coopted to various functions in molluscs.

Pyrolysis GC/MS and IR spectroscopy in chitin analysis of molluscan shells.

Chitin is an insoluble component in the shells of several molluscan species. It is thought to play important roles, in biomineralization and shell structure. To date, however, reports are scarce and sometimes contradictory, and suffer from methodological problems. Only in a single cephalopod species has the chitin been identified as beta-chitin. We present data on chitin occurrence in 22 species of shell-bearing Mollusca (Conchifera) and Polyplacophora, including the first evidence for scaphopods, based on pyrolysis gas chromatography, mass spectrometry (GC-MS), and infrared spectroscopy (IR). Pyrolysis GC-MS detected chitin in every tested member of the Conchifera. IR spectroscopy before and after chitinase treatment revealed at least three distinct patterns of peak changes. The contents of the insoluble shell organics included not only chitin and proteins, but also insoluble polysaccharides, e.g., glucan. We conclude that chitin was present in the last common ancestor of the Conchifera and that its abundance in the shell matrix depends on the differentiation of the shell.

Heterogeneous nucleation experiments bridging the scale from molecular ion clusters to nanoparticles.

Generation, investigation, and manipulation of nanostructured materials are of fundamental and practical importance for several disciplines, including materials science and medicine. Recently, atmospheric new particle formation in the nanometer-size range has been found to be a global phenomenon. Still, its detailed mechanisms are mostly unknown, largely depending on the incapability to generate and measure nanoparticles in a controlled way. In our experiments, an organic vapor (n-propanol) condenses on molecular ions, as well as on charged and uncharged inorganic nanoparticles, via initial activation by heterogeneous nucleation. We found a smooth transition in activation behavior as a function of size and activation to occur well before the onset of homogeneous nucleation. Furthermore, nucleation enhancement for charged particles and a substantial negative sign preference were quantitatively detected.

The complete sequences and gene organisation of the mitochondrial genomes of the heterodont bivalves Acanthocardia tuberculata and Hiatella arctica--and the first record for a putative Atpase subunit 8 gene in marine bivalves.

BACKGROUND: Mitochondrial (mt) gene arrangement is highly variable among molluscs and especially among bivalves. Of the 30 complete molluscan mt-genomes published to date, only one is of a heterodont bivalve, although this is the most diverse taxon in terms of species numbers. We determined the complete sequence of the mitochondrial genomes of Acanthocardia tuberculata and Hiatella arctica, (Mollusca, Bivalvia, Heterodonta) and describe their gene contents and genome organisations to assess the variability of these features among the Bivalvia and their value for phylogenetic inference. RESULTS: The size of the mt-genome in Acanthocardia tuberculata is 16.104 basepairs (bp), and in Hiatella arctica 18.244 bp. The Acanthocardia mt-genome contains 12 of the typical protein coding genes, lacking the Atpase subunit 8 (atp8) gene, as all published marine bivalves. In contrast, a complete atp8 gene is present in Hiatella arctica. In addition, we found a putative truncated atp8 gene when re-annotating the mt-genome of Venerupis philippinarum. Both mt-genomes reported here encode all genes on the same strand and have an additional trnM. In Acanthocardia several large non-coding regions are present. One of these contains 3.5 nearly identical copies of a 167 bp motive. In Hiatella, the 3' end of the NADH dehydrogenase subunit (nad)6 gene is duplicated together with the adjacent non-coding region. The gene arrangement of Hiatella is markedly different from all other known molluscan mt-genomes, that of Acanthocardia shows few identities with the Venerupis philippinarum. Phylogenetic analyses on amino acid and nucleotide levels robustly support the Heterodonta and the sister group relationship of Acanthocardia and Venerupis. Monophyletic Bivalvia are resolved only by a Bayesian inference of the nucleotide data set. In all other analyses the two unionid species, being to only ones with genes located on both strands, do not group with the remaining bivalves. CONCLUSION: The two mt-genomes reported here add to and underline the high variability of gene order and presence of duplications in bivalve and molluscan taxa. Some genomic traits like the loss of the atp8 gene or the encoding of all genes on the same strand are homoplastic among the Bivalvia. These characters, gene order, and the nucleotide sequence data show considerable potential of resolving phylogenetic patterns at lower taxonomic levels.

The complete sequence and gene organization of the mitochondrial genome of the gadilid scaphopod Siphonondentalium lobatum (Mollusca).

Comparisons of mitochondrial gene sequences and gene arrangements can be informative for reconstructing high-level phylogenetic relationships. We determined the complete sequence of the mitochondrial genome of Siphonodentalium lobatum, (Mollusca, Scaphopoda). With only 13,932 bases, it is the shortest molluscan mitochondrial genome reported so far. The genome contains the usual 13 protein-coding genes, two rRNA and 22 tRNA genes. The ATPase subunit 8 gene is exceptionally short. Several transfer RNAs show truncated TpsiC arms or DHU arms. The gene arrangement of S. lobatum is markedly different from all other known molluscan mitochondrial genomes and shows low similarity even to an unpublished gene order of a dentaliid scaphopod. Phylogenetic analyses of all available complete molluscan mitochondrial genomes based on amino acid sequences of 11 protein-coding genes yield trees with low support for the basal branches. None of the traditionally accepted molluscan taxa and phylogenies are recovered in all analyses, except for the euthyneuran Gastropoda. S. lobatum appears as the sister taxon to two of the three bivalve species. We conclude that the deep molluscan phylogeny is probably beyond the resolution of mitochondrial protein sequences. Moreover, assessing the phylogenetic signal in gene order data requires a much larger taxon sample than is currently available, given the exceptional diversity of this character set in the Mollusca.