Discovery and functional understanding of MiRNAs in molluscs: a genome-wide profiling approach.

Small non-coding RNAs play a pivotal role in gene regulation, repression of transposable element and viral activity in various organisms. Among the various categories of these small non-coding RNAs, microRNAs (miRNAs) guide post-translational gene regulation in cellular development, proliferation, apoptosis, oncogenesis, and differentiation. Here, we performed a genome-wide computational prediction of miRNAs to improve the understanding of miRNA observation and function in molluscs. As an initial step, hundreds of conserved miRNAs were predicted in 35 species of molluscs through genome scanning. Afterwards, the miRNAs' population, isoforms, organization, and function were characterized in detail. Furthermore, the key miRNA biogenesis factors, including AGO2, DGCR8, DICER, DROSHA, TRABP2, RAN, and XPO5, were elucidated based on homologue sequence searching. We also summarized the miRNAs' function in biomineralization, immune and stress response, as well as growth and development in molluscs. Because miRNAs play a vital role in various lifeforms, this study will provide insight into miRNA biogenesis and function in molluscs, as well as other invertebrates.

Microhabitat selection, and seasonal and daily activity of the snail Tikoconus costarricanus (Stylommatophora: Euconulidae) in tropical montane wet forest understory / Selección de microhábitat, y actividad diaria y estacional del caracol Tikoconus costarricanus (Stylommatophora: Euconulidae) en el sotobosque de bosques tropicales húmedos de montaña

Introduction: The integument of terrestrial mollusks is highly susceptible to dehydration; therefore, microhabitat selection, seasonality and behavior around the day are crucial to their survival. However, they are still poorly understood, especially for tropical montane wet forest species. Objetive: To analyze activity patterns and microhabitat selection on shrubs of the land snail Tikoconus costarricanus, according to season, daytime and weather. Methods: I conducted the study near "El llano" water dam in a tropical montane wet forest in Costa Rica. I observed daily activity of T. costarricanus, during the rainy and the dry season on a 2 km long trail. I observed 167 specimens and made 781 observations in total. I took note of: season, time, activity, weather, temperature and relative humidity inside the forest, and part of the leaf and height where snails were. Active snails had optical tentacles extended. I analysed daily rainfall data from a nearby meteorological station. Results: This species can be found mainly on understory leaves between 1.1 and 2 m above the floor. They are active the day around during the whole year. Only 24 % of the snails were on the upper side of leaves, but 92 % of them were active. The following behaviors were season dependent: activity peaks, leaf side selection according to daytime and weather, quantity of snails aestivating and vertical distribution. During the dry season, I found more active snails in rainy and cloudy days. During the rainy season I found more active snails in sunny days. During rainy season light hours, active snail percentage on the upper side of leaves decreased with rainfall increase, while in the dry season decreased with temperature increase and relative humidity decrease. During night hours of both seasons, the number of snails on the upper side of leaves decreased considerably in relation to active snails, especially in the rainy season. This snail aestivated on the underside of leaves during periods shorter than 21 h, mainly around noon and afternoons during the dry season. High humidity in this forest allowed snail activity around the day at any season. However, this species aestivated when moisture reached the lowest values, mainly, around noon and in the afternoons in the dry season. The rainfall pattern during the rainy season light hours explains the specimen decrease on the upper side of leaves, but in the dry season it is better explained by the temperature increase and the decrease in relative humidity. During dark hours, the almost complete snail absence on the upper side of leaves during the rainy season is explained if the effect of canopy drip produced by rain and dew condensed on leaves is added to the rain pattern. The situation is slightly different in dark hours of the dry season, in this case, snail decrease on the upper side of leaves is explained by canopy drip from dew and fog precipitation. Seasonal difference in vertical distribution may be a way to avoid rain and rain splash out on the soil. Conclusions: Moisture, temperature, rain, and canopy drip from dew, fog and rain, affect behavior and substrate selection of small terrestrial mollusks that inhabit shrubs in wet tropical rainforests.

Introducción: El integumento de los moluscos terrestres es susceptible a desecación, por eso la selección de microhábitats, la estacionalidad y el comportamiento a lo largo del día son cruciales para su sobrevivencia. No obstante, es poco lo que se ha investigado al respecto, en especial para moluscos tropicales de arbustos del sotobosque en bosques húmedos de montaña. Objetivo: Analizar los patrones de actividad y de la selección de microhábitats del caracol terrestre Tikoconus costarricanus según la estación del año, la hora del día y el clima. Métodos: El trabajo lo realicé en un bosque tropical húmedo de montaña cerca de la represa hidrológica "El Llano", Costa Rica. Analicé la actividad diaria de especímenes de T.costarricanus en un trayecto de 2 km: observé 167 especímenes e hice 781 observaciones. Tomé nota de: estación del año, hora, actividad, parte de la hoja donde estaba el caracol, altura en la planta, clima y humedad relativa y temperatura dentro del bosque. Consideré que un espécimen estaba activo cuando los tentáculos ópticos estaban extendidos. Analicé la precipitación pluvial por horas con datos de una estación meteorológica cercana. Resultados: Encontré caracoles principalmente en las hojas de arbustos entre 1.1 y 2 m sobre el suelo; encontré individuos activos a todas horas y en cualquier época del año. Solo el 24 % de los individuos estaban en el haz, de esos el 92 % estaban activos. Encontré variación estacional en el patrón de actividad, la selección del lado de la hoja de acuerdo a la hora del día y el clima, la cantidad de especímenes estivando, y la distribución vertical. En la época seca encontré más caracoles activos con clima húmedo, por el contrario, en la estación lluviosa encontré más caracoles activos con clima seco. Durante las horas de luz de la estación lluviosa, la cantidad de caracoles activos sobre el haz de las hojas disminuyó con el aumento de precipitación pluvial, en tanto que en la estación seca disminuyó con el aumento de la temperatura y la disminución de la humedad relativa. Durante las horas nocturnas de ambas estaciones, la cantidad de caracoles sobre el haz de las hojas disminuyó considerablemente en relación a los caracoles activos, especialmente en la época lluviosa. Este caracol estivó principalmente a mediodía y en las tardes de la época seca en el envés de las hojas por periodos inferiores a 21 h seguidas. La elevada humedad del bosque tropical húmedo de montaña permite que estos caracoles estén activos en cualquier momento. Sin embargo, la estivación les permite protegerse de la desecación durante los periodos más secos. El patrón de lluvias durante las horas de luz de la época lluviosa explica la disminución de especímenes presentes en el haz de las hojas, pero en la época seca lo explica mejor el aumento de la temperatura y la disminución de la humedad relativa. Durante las horas oscuras, la casi ausencia de caracoles en el haz de las hojas durante la estación lluviosa se explica si al patrón de lluvia se le agrega el efecto del goteo foliar producido por la lluvia y el rocío que se condensa sobre las hojas. La situación es un poco diferente en las horas oscuras de la estación seca, en este caso, la disminución de caracoles en el haz, se explica por el goteo foliar derivado del rocío y de la precipitación de neblina. Es posible que la diferencia en la preferencia vertical se deba a que evitan la lluvia y la salpicadura de la lluvia al caer en el suelo. Conclusiones: La humedad, la temperatura, la lluvia y el goteo foliar derivado de rocío, neblina y lluvia, afectan el comportamiento y la selección de sustrato de moluscos terrestres pequeños que habitan en arbustos de bosques tropicales húmedos de montaña.

Staggered Hox expression is more widespread among molluscs than previously appreciated.

Hox genes are expressed along the anterior-posterior body axis in a colinear fashion in the majority of bilaterians. Contrary to polyplacophorans, a group of aculiferan molluscs with conserved ancestral molluscan features, gastropods and cephalopods deviate from this pattern by expressing Hox genes in distinct morphological structures and not in a staggered fashion. Among conchiferans, scaphopods exhibit many similarities with gastropods, cephalopods and bivalves, however, the molecular developmental underpinnings of these similar traits remain unknown. We investigated Hox gene expression in developmental stages of the scaphopod Antalis entalis to elucidate whether these genes are involved in patterning morphological traits shared by their kin conchiferans. Scaphopod Hox genes are predominantly expressed in the foot and mantle but also in the central nervous system. Surprisingly, the scaphopod mid-stage trochophore exhibits a near-to staggered expression of all nine Hox genes identified. Temporal colinearity was not found and early-stage and late-stage trochophores, as well as postmetamorphic individuals, do not show any apparent traces of staggered expression. In these stages, Hox genes are expressed in distinct morphological structures such as the cerebral and pedal ganglia and in the shell field of early-stage trochophores. Interestingly, a re-evaluation of previously published data on early-stage cephalopod embryos and of the gastropod pre-torsional veliger shows that these developmental stages exhibit traces of staggered Hox expression. Considering our results and all gene expression and genomic data available for molluscs as well as other bilaterians, we suggest a last common molluscan ancestor with colinear Hox expression in predominantly ectodermal tissues along the anterior-posterior axis. Subsequently, certain Hox genes have been co-opted into the patterning process of distinct structures (apical organ or prototroch) in conchiferans.

Brain regionalization genes are co-opted into shell field patterning in Mollusca.

The 'brain regionalization genes' Six3/6, Otx, Pax2/5/8, Gbx, and Hox1 are expressed in a similar fashion in the deuterostome, ecdysozoan, and the cephalopod brain, questioning whether this holds also true for the remaining Mollusca. We investigated developmental Gbx-expression in representatives of both molluscan sister groups, the Aculifera and Conchifera. Gbx is expressed in the posterior central nervous system of an aculiferan polyplacophoran and solenogaster but not in a conchiferan bivalve suggesting that Gbx, together with Six3/6, Otx, Pax2/5/8, and Hox1, is involved in central nervous system regionalization as reported for other bilaterians. Gbx is, however, also expressed in the anterior central nervous system, i.e. the anlagen of the cerebral ganglia, in the solenogaster, a condition not reported for any other bilaterian so far. Strikingly, all Gbx-orthologs and the other 'posterior brain regionalization genes' such as Pax2/5/8 and Hox1 are expressed in the mantle that secretes shell(s) and spicules of mollusks (except cephalopods). In bivalves, the ancestral condition has even been lost, with Gbx and Pax2/5/8 not being expressed in the developing central nervous system anymore. This suggests an additional role in the formation of the molluscan shell field(s) and spicule-bearing cells, key features of mollusks.

Ancestral and novel roles of Pax family genes in mollusks.

BACKGROUND: Pax genes are transcription factors with significant roles in cell fate specification and tissue differentiation during animal ontogeny. Most information on their tempo-spatial mode of expression is available from well-studied model organisms where the Pax-subfamilies Pax2/5/8, Pax6, and Paxα/ß are mainly involved in the development of the central nervous system (CNS), the eyes, and other sensory organs. In certain taxa, Pax2/5/8 seems to be additionally involved in the development of excretion organs. Data on expression patterns in lophotrochozoans, and in particular in mollusks, are very scarce for all the above-mentioned Pax-subfamilies, which hampers reconstruction of their putative ancestral roles in bilaterian animals. Thus, we studied the developmental expression of Pax2/5/8, Pax6, and the lophotrochozoan-specific Paxß in the worm-shaped mollusk Wirenia argentea, a member of Aplacophora that together with Polyplacophora forms the Aculifera, the proposed sister taxon to all primarily single-shelled mollusks (Conchifera). RESULTS: All investigated Pax genes are expressed in the developing cerebral ganglia and in the ventral nerve cords, but not in the lateral nerve cords of the tetraneural nervous system. Additionally, Pax2/5/8 is expressed in epidermal spicule-secreting or associated cells of the larval trunk and in the region of the developing protonephridia. We found no indication for an involvement of the investigated Pax genes in the development of larval or adult sensory organs of Wirenia argentea. CONCLUSIONS: Pax2/5/8 seems to have a conserved role in the development of the CNS, whereas expression in the spicule-secreting tissues of aplacophorans and polyplacophorans suggests co-option in aculiferan skeletogenesis. The Pax6 expression pattern in Aculifera largely resembles the common bilaterian expression during CNS development. All data available on Paxß expression argue for a common role in lophotrochozoan neurogenesis.

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.

Identification of a female spawn-associated Kazal-type inhibitor from the tropical abalone Haliotis asinina.

Abalone (Haliotis) undergoes a period of reproductive maturation, followed by the synchronous release of gametes, called broadcast spawning. Field and laboratory studies have shown that the tropical species Haliotis asinina undergoes a two-week spawning cycle, thus providing an excellent opportunity to investigate the presence of endogenous spawning-associated peptides. In female H. asinina, we have isolated a peptide (5145 Da) whose relative abundance in hemolymph increases substantially just prior to spawning and is still detected using reverse-phase high-performance liquid chromatography chromatograms up to 1-day post-spawn. We have isolated this peptide from female hemolymph as well as samples prepared from the gravid female gonad, and demonstrated through comparative sequence analysis that it contains features characteristic of Kazal-type proteinase inhibitors (KPIs). Has-KPI is expressed specifically within the gonad of adult females. A recombinant Has-KPI was generated using a yeast expression system. The recombinant Has-KPI does not induce premature spawning of female H. asinina when administered intramuscularly. However it displays homomeric aggregations and interaction with at least one mollusc-type neuropeptide (LRDFVamide), suggesting a role for it in regulating neuropeptide endocrine communication. This research provides new understanding of a peptide that can regulate reproductive processes in female abalone, which has the potential to lead to the development of greater control over abalone spawning. The findings also highlight the need to further explore abalone reproduction to clearly define a role for novel spawning-associated peptide in sexual maturation and spawning. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Hox and ParaHox gene expression in early body plan patterning of polyplacophoran mollusks.

Molecular developmental studies of various bilaterians have shown that the identity of the anteroposterior body axis is controlled by Hox and ParaHox genes. Detailed Hox and ParaHox gene expression data are available for conchiferan mollusks, such as gastropods (snails and slugs) and cephalopods (squids and octopuses), whereas information on the putative conchiferan sister group, Aculifera, is still scarce (but see Fritsch et al., 2015 on Hox gene expression in the polyplacophoran Acanthochitona crinita). In contrast to gastropods and cephalopods, the Hox genes in polyplacophorans are expressed in an anteroposterior sequence similar to the condition in annelids and other bilaterians. Here, we present the expression patterns of the Hox genes Lox5, Lox4, and Lox2, together with the ParaHox gene caudal (Cdx) in the polyplacophoran A. crinita. To localize Hox and ParaHox gene transcription products, we also investigated the expression patterns of the genes FMRF and Elav, and the development of the nervous system. Similar to the other Hox genes, all three Acr-Lox genes are expressed in an anteroposterior sequence. Transcripts of Acr-Cdx are seemingly present in the forming hindgut at the posterior end. The expression patterns of both the central class Acr-Lox genes and the Acr-Cdx gene are strikingly similar to those in annelids and nemerteans. In Polyplacophora, the expression patterns of the Hox and ParaHox genes seem to be evolutionarily highly conserved, while in conchiferan mollusks these genes are co-opted into novel functions that might have led to evolutionary novelties, at least in gastropods and cephalopods.

Ancestral role of Pax2/5/8 in molluscan brain and multimodal sensory system development.

BACKGROUND: Mollusks represent the largest lophotrochozoan phylum and exhibit highly diverse body plans. Previous studies have demonstrated that transcription factors such as Pax genes play important roles during their development. Accordingly, in ecdysozoan and vertebrate model organisms, orthologs of Pax2/5/8 are among others involved in the formation of the midbrain/hindbrain boundary, the auditory/geosensory organ systems, and the excretory system. METHODS: Pax2/5/8 expression was investigated by in situ hybridization during the development of representatives of the two major molluscan subclades, Aculifera and Conchifera. RESULTS: Compared to the investigated polyplacophoran and bivalve species that lack larval statocysts as geosensory organs and elaborate central nervous systems (CNS), cephalopods possess highly centralized brains and statocysts. Pax2/5/8 is expressed in regions where sensory cells develop subsequently during ontogenesis. Expression domains include esthetes and the ampullary system in polyplacophorans as well as the eyes of cephalopods. No Pax2/5/8 expression was observed in the less centralized CNS of bivalve, polyplacophoran, and gastropod embryos, thus arguing for a loss of Pax2/5/8 involvement in CNS development in these lineages. In contrast, Pax2/5/8 is expressed among others in brain lobes along the trajectory of the esophagus that divides the cephalopod brain. CONCLUSIONS: Our results, along with those on Otx- and Hox-gene expression, demonstrate that the cephalopod condition is similar to that in mouse and fruit fly, with Otx being expressed in the anterior-most brain region (except for the vertical lobe) and a Pax2/5/8 expression domain separating the Otx-domain from a Hox-gene expressing posterior brain region. Thus, Pax2/5/8 appears to have been recruited independently into regionalization of non-homologous complex brains of organisms as different as squid, fruit fly, and mouse. In addition, Pax2/5/8 is expressed in multimodal sensory systems in mollusks such as the esthetes and the ampullary system of polyplacophorans as well as the eyes of cephalopods. Pax2/5/8-expressing cells are present in regions where the future sensory cells such as the polyplacophoran esthetes are situated and hence Pax2/5/8 expression probably predates sensory cell development during ontogeny. In mollusks, Pax2/5/8 is only expressed in derivatives of the ectoderm and hence an ancestral role in molluscan ectoderm differentiation is inferred.

From complex to simple: myogenesis in an aplacophoran mollusk reveals key traits in aculiferan evolution.

BACKGROUND: Recent studies suggest a bifurcation at the base of Mollusca, resulting in the primarily single-shelled Conchifera (Bivalvia, Gastropoda, Scaphopoda, Monoplacophora, Cephalopoda) and the spicule-bearing Aculifera (Polyplacophora, Neomeniomorpha, Chaetodermomorpha). A recent study revealed a complex larval musculature exclusively shared by Neomeniomorpha and Polyplacophora, supporting a close relationship of both taxa. However, the ontogenetic transition from the complex larval to the simple adult neomeniomorph musculature, which mainly consists of a three-layered body-wall musculature and serially iterated dorsoventral muscles, remains unknown. To close this gap in knowledge, we studied remodeling of the larval musculature during metamorphosis in the neomeniomorph Wirenia argentea. A comparative analysis with a novel data set of a polyplacophoran, Leptochiton asellus, allows us to infer the morphology of the last common ancestor of Aculifera and the evolution of its subclades therefrom. RESULTS: The complex larval musculature of Wirenia argentea persists through metamorphosis and becomes modified to form two of the three muscle layers of the adult body wall. The innermost longitudinal layer of the three-layered body wall musculature is generated by transformation and expansion of distinct larval longitudinal muscle bundles. The larval ventrolateral muscle strands are remodeled and eventually become the most ventral part of the adult longitudinal layer of the body wall musculature. The paired larval enrolling muscle forms the lateral parts and the former rectus muscle is destined to become the most dorsal part of the longitudinal layer of the body wall musculature. The transient ventromedian muscle is lost during postmetamorphic development. CONCLUSIONS: Postmetamorphic remodeling in W. argentea supports the hypothesis of a complex myoanatomy rather than a three-layered body wall musculature at the base of Aculifera, and thus argues against homology of the body wall musculature of adult Neomeniomorpha and other potential molluscan sister groups. Our data show that the neomeniomorph body wall musculature is a derived condition and not an aculiferan or molluscan plesiomorphy.

Histological validation of morphochromatically-defined gonadal maturation stages of green abalone (Haliotis fulgens) Philippi,1845 and pink abalone (Haliotis corrugata) Wood, 1828 / Validación histológica de las fases morfocromáticas de maduración gonádica del abulón azul (Haliotis fulgens) Philippi, 1845 y del abulón amarillo (Haliotis corrugata) Wood, 1828

Identifying the maturity stage of specimens is essential for the determination of the spawning season, which in turn facilitates the formulation of adequate fisheries management measures for the sustainable use of sea resources, such as abalone. This study aims to validate, based on histological evidence, a gonad maturation scale that encompasses four morphochromatically-defined stages (developing, maturity, spawning and resting) for females and males, plus an additional stage for reproductively inactive (undifferentiated) specimens, to be used as an identification key. Given the similarity between Haliotis fulgens and Haliotis corrugata as to their morphochromatic features, the gonadal maturation scale can be used for both species.

La determinación de la fase de madurez es importante para identificar la temporada de desove y permitir establecer medidas de manejo pesquero para el aprovechamiento responsable de los recursos como el abulón. El presente trabajo valida histológicamente una escala de maduración gonádica a manera de guía de identificación, que consiste de cuatro fases morfocromáticas para hembras y machos (desarrollo, madurez, desove y reposo), y un estadio para los organismos reproductivamente inactivos (indiferenciado). Dada la similitud entre las características morfocromáticas de Haliotis fulgens y Haliotis corrugata, la escala de maduración gonádica puede ser utilizada indistintamente.

Unexpected co-linearity of Hox gene expression in an aculiferan mollusk.

BACKGROUND: Mollusca is an extremely diverse animal phylum that includes the aculiferans (worm-like aplacophorans and eight-shelled polyplacophorans) and their sister group, the conchiferans, comprising monoplacophorans, bivalves (clams, mussels), gastropods (snails, slugs), scaphopods (tusk shells) and cephalopods (squids, octopuses). Studies on mollusks have revealed an overall number of 11 Hox genes in seven out of eight molluscan "class"-level taxa, but expression data of key developmental regulators such as homeotic genes are only available for three gastropod and two cephalopod species. These show that Hox genes are involved in the formation of specific features including shell, foot, funnel or tentacles and not in antero-posterior body plan patterning as in most other bilaterian animals. The role of Hox genes in non-conchiferan (i.e., aculiferan) mollusks remains entirely unknown. RESULTS: Here we present the first data on the expression of seven Hox genes in apolyplacophoran mollusk, Acanthochitona crinita. In A. crinita the Hox genes Acr-Hox1-5, Hox7 and Post2 are expressed in a co-linear pattern along the antero-posterior axis, but not in molluscan-specific features such as the shell or the foot. The expression pattern is restricted to the post-trochal region and the transcripts are present in ecto-, endo- and mesodermal cell layers. Contrary to the situation in gastropods and cephalopods, we did neither find Hox gene expression in distinct neural subsets of A. crinita, nor in its developing shell plates. CONCLUSIONS: Our analysis and comparison with other lophotrochozoans indicate that the basal role of Hox genes is in antero-posterior axis patterning in mollusks, similar to the vast majority of bilaterian animals, and that this role has been conserved in polyplacophorans, while co-option into patterning of evolutionary novelties emerged either at the base of Conchifera or independently in gastropods and cephalopods. These morphological innovations most likely contributed to the evolutionary success of its representatives, as exemplified by, e.g., the wide ecological range and species richness of gastropods.

Aplacophoran mollusks evolved from ancestors with polyplacophoran-like features.

Mollusca is an animal phylum with vast morphological diversity and includes worm-shaped aplacophorans, snails, bivalves, and the complex cephalopods. The interrelationships of these class-level taxa are still contentious, but recent phylogenomic analyses suggest a dichotomy at the base of Mollusca, resulting in a monophyletic Aculifera (comprising the shell-less, sclerite-bearing aplacophorans and the eight-shelled polyplacophorans) and Conchifera (all other, primarily univalved groups). The Aculifera concept has recently gained support via description of the fossil Kulindroplax, which shows both aplacophoran- and polyplacophoran-like features and suggests that the aplacophorans originated from a shelled ancestor, but the overall morphology of the last common aculiferan ancestor remains obscure. Here we show that larvae of the aplacophoran Wirenia argentea have several sets of muscles previously known only from polyplacophoran mollusks. Most of these are lost during metamorphosis, and we interpret them as ontogenetic remnants of an ancestor with a complex, polyplacophoran-like musculature. Moreover, we find that the first seven pairs of dorsoventral muscles develop synchronously in Wirenia, similar to juvenile polyplacophorans, which supports the conclusions based on the seven-shelled Kulindroplax. Accordingly, we argue that the simple body plan of recent aplacophorans is the result of simplification and does not represent a basal molluscan condition.

[Macrobenthos community structure and its relationships with environmental factors in Weihe River basin, Northwest China].

In October 2011, an investigation was conducted on the macrobenthos at 45 sampling sites in the Weihe River basin of Northwest China. A total of 116 macrobenthos species were collected, of which, 91 species (78.4%) were aquatic insects, 12 species (10.3%) were mollusks, 9 species (7.8%) were annelids, and 4 species (3.4%) were crustaceans. According to the species composition and relative abundance of macrobenthos, the 45 sampling sites could be divided into three groups by using two-way indicator species analysis (TWINSPAN) and detrended correspondence analysis (DCA). In the first group, Serratella sp., Hydropsyche sp., and Antocha sp. were the indicator species. In the second group, Tabanus sp., Alotanypus venustus, one species of Pelecorhynchidae, Liodessus sp., and Limnodrilus hoffmeistteri were the indicator species. In the third group, Ephemera nigroptera and Polypylis hemisphaerula were the indicator species. The canonical correspondence analysis (CCA) indicated that 'cobble + boulder' type substrate, current velocity, water conductivity, water depth, and water total nitrogen content had significant effects on the spatial distribution of macrobenthos in Weihe River basin.

On the function of chitin synthase extracellular domains in biomineralization.

Molluscs with various shell architectures evolved around 542-525 million years ago, as part of a larger phenomenon related to the diversification of metazoan phyla. Molluscs deposit minerals in a chitin matrix. The mollusc chitin is synthesized by transmembrane enzymes that contain several unique extracellular domains. Here we investigate the assembly mechanism of the chitin synthase Ar-CS1 via its extracellular domain ArCS1_E22. The corresponding transmembrane protein ArCS1_E22TM accumulates in membrane fractions of the expression host Dictyostelium discoideum. Soluble recombinant ArCS1_E22 proteins can be purified as monomers only at basic pH. According to confocal fluorescence microscopy experiments, immunolabeled ArCS1_E22 proteins adsorb preferably to aragonitic nacre platelets at pH 7.75. At pH 8.2 or pH 9.0 the fluorescence signal is less intense, indicating that protein-mineral interaction is reduced with increasing pH. Furthermore, ArCS1_E22 forms regular nanostructures on cationic substrates as revealed by atomic force microscopy (AFM) experiments on modified mica cleavage planes. These experiments suggest that the extracellular domain ArCS1_E22 is involved in regulating the multiple enzyme activities of Ar-CS1 such as chitin synthesis and myosin movements by interaction with mineral surfaces and eventually by protein assembly. The protein complexes could locally probe the status of mineralization according to pH unless ions and pCO2 are balanced with suitable buffer substances. Taking into account that the intact enzyme could act as a force sensor, the results presented here provide further evidence that shell formation is coordinated physiologically with precise adjustment of cellular activities to the structure, topography and stiffness at the mineralizing interface.

The growth performance of Jade Tiger cultured abalone fed diets supplemented with fish oil and vegetable oils.

BACKGROUND: The effects of fish oil (FO) supplementation and the dietary replacement of FO with flaxseed oil (FlaxO) and canola oil (CO) on the growth of cultured abalone was investigated. The study involved three growth experiments: (E1) diets containing 0.5, 1.0, 1.5, 2.0 and 2.5% of FO, respectively; (E2) diets in which FO was serially replaced by 25, 50, 75 and 100% FlaxO, respectively; and (E3) diets in which FO was serially replaced by 25, 50, 75 and 100% CO, respectively. RESULTS: In Experiment 1, abalone fed a diet supplemented with 1.5% FO showed a significantly higher (121.2 ± 1.1 mg day(-1)) daily growth rate of weight (DGRw ) compared to control (70.1 ± 1.71 mg day(-1)). In Experiment 2, abalone fed 1.5% FO diet and diets containing 25-75% FlaxO showed no significant differences in DGRw. The diet containing 100% FlaxO showed significantly lower (63.3 ± 6.7 mg day(-1)) DGRw. In Experiment 3, abalone fed diets containing 25% and 50% CO showed similar DGRw as those fed a 1.5% FO diet. The diet containing 75% and 100% CO showed significantly lower (63.7 ± 5.0 to 95.4 ± 5.1 mg day(-1)) DGRw. CONCLUSION: Supplementation with 1.5% of dietary FO can improve growth performance in cultured abalone. It is feasible to replace 75% of dietary FO with FlaxO and 50% of dietary FO with CO, without negative effect on growth performance.

Medicinal benefits of marine invertebrates: sources for discovering natural drug candidates.

Marine invertebrates are one of the major groups of organisms, which could be diversified under the major taxonomic groups of Porifera, Cnidaria, Mollusca, Arthropoda, Echinodermata, and many other minor phyla. To date, range of medicinal benefits and a significant number of marine natural products (MNPs) have been discovered from marine invertebrates. Seafood diet from edible marine invertebrates such as mollusks and crustaceans has been linked with various medicinal benefits to improve human health. Among marine invertebrates, spongers from phylum Porifera is the most dominant group responsible for discovering large number of MNPs, which have been used as template to develop therapeutic drugs. MNPs isolated from invertebrates have shown wide range of therapeutic properties including antimicrobial, antioxidant, antihypertensive, anticoagulant, anticancer, anti-inflammatory, wound healing and immune modulator, and other medicinal effects. Therefore, marine invertebrates are rich sources of chemical diversity and health benefits for developing drug candidates, cosmetics, nutritional supplements, and molecular probes that can be supported to increase the healthy life span of human.

Development of the excretory system in the polyplacophoran mollusc, Lepidochitona corrugata: the protonephridium.

A single pair of protonephridia is the typical larval excretory organ of molluscs. Their presence in postlarval developmental stages was discovered only recently. We found that the protonephridia of the polyplacophoran mollusc, Lepidochitona corrugata, achieve their most elaborate differentiation and become largest during the postlarval period. This study describes the protonephridia of L. corrugata using light and electron microscopy and interactive three-dimensional visualization. We focus on the postlarval developmental period, in which the protonephridia consist of three parts: the terminal part with the ultrafiltration sites at the distal end, the voluminous protonephridial kidney, and the efferent nephroduct leading to the nephropore. The ultrafiltration sites show filtration slits between regularly arranged thin pedicles. The ciliary flame originates from both the terminal cell and the duct cells of the terminal portion. The efferent duct also shows ciliation. The most conspicuous structures, the protonephridial kidneys, are voluminous swellings composed of reabsorptive cells ("nephrocytes"). These cells exhibit strong vacuolization and an infolding system increasing the basal surface. The protonephridial kidneys, previously not reported at such a level of organization in molluscs, strikingly resemble (metanephridial) kidneys of adult molluscan excretory systems.