Trochophore Stage Research Articles

The Increased Expression of an Engrailed to Sustain Shell Formation in Response to Ocean Acidification.

Engrailed is a transcription factor required in numerous species for important developmental steps such as neurogenesis, segment formation, preblastoderm organization, and compartment formation. Recent study has proved that engrailed is also a key gene related to shell formation in marine bivalves. In the present study, the expression pattern of an engrailed gene (Cgengrailed-1) in Pacific oyster Crassostrea gigas under CO2-driven acidification was investigated to understand its possible role in the regulation of shell formation and adaptation to ocean acidification (OA). The open reading frame (ORF) of Cgengrailed-1 was obtained, which was of 690 bp encoding a polypeptide of 229 amino acids with a HOX domain. Phylogenetic analysis indicated that the deduced amino acid sequence of Cgengrailed-1 shared high homology with other engraileds from Drosophila melanogaster, Mizuhopecten yessoensi, and Crassostrea virginica. The mRNA transcripts of Cgengrailed-1 were constitutively expressed in various tissues with the highest expression levels detected in labial palp and mantle, which were 86.83-fold (p < 0.05) and 75.87-fold (p < 0.05) higher than that in hepatopancreas. The mRNA expression of Cgengrailed-1 in mantle decreased dramatically after moderate (pH 7.8) and severe (pH 7.4) acidification treatment (0.75- and 0.15-fold of that in control group, p < 0.05). The results of immunofluorescence assay demonstrated that the expression level of Cgengrailed-1 in the middle fold of mantle increased significantly upon moderate and severe acidification treatment. Moreover, after the oyster larvae received acidification treatment at trochophore stage, the mRNA expression levels of Cgengrailed-1 increased significantly in D-shape larvae stages, which was 3.11- (pH 7.8) and 4.39-fold (pH 7.4) of that in control group (p < 0.05). The whole-mount immunofluorescence assay showed that Cgengrailed-1 was mainly expressed on the margin of shell gland, and the periostracum in trochophore, early D-shape larvae and D-shape larvae in both control and acidification treatment groups, and the intensity of positive signals in early D-shape larvae and D-shape larvae increased dramatically under acidification treatment. These results collectively suggested that the expression of Cgengrailed-1 could be triggered by CO2-driven acidification treatment, which might contribute to induce the initial shell formation in oyster larvae and the formation of periostracum in adult oyster to adapt to the acidifying marine environment.

Evaluation of using veliger stage larvae for the preparation of metaphase spreads from the pacific abalone (Haliotis discus hannai)

Karyotype analysis is a major work in the process of triploid abalone production for the purpose of productivity and quality improvement. However, the metaphase spreads for karyotype analysis have been prepared just from the larvae at trochophore stage, which has restricted the spectrum of sample correction inhibiting more efficient analysis. Here, we investigated the feasibility of preparing metaphase spreads from the larvae at veliger stage that is the next developmental stage of trochophore. For this, diploid and triploid larvae at trochophore and veliger stages from Pacific abalone (Haliotis discus hannai ) were subjected to metaphase spread preparation and its efficiencies were measured and compared each other. As the results, although the efficiencies of metaphase spread preparation were significantly lower in the larvae at veliger stage compared to the ones at trochophore stage regardless of ploidy status, we found that the preparation of metaphase spreads, which showed the clear chromosomal images containing the normal number of chromosomes, was possible from the veliger stage larvae. On the other hands, all larvae used in this study regardless of developmental stage and ploidy did not show colchicine sensitivity. Moreover, no significant difference was observed in cell cycle distribution of the cells comprising larvae between two developmental stages regardless of ploidy status. These suggested that the details of protocol to prepare metaphase spreads from abalone larvae should be optimized depending on its developmental stages. Taken together, we demonstrated the feasibility of preparing metaphase spreads from H. discus hannai veliger stage larvae for karyotype analysis.

Abnormal Karyotype of Pen Shell (Atrina pectinata) During Its Early Embryonic Development in Late Breeding Season

The pen shell, Atrina pectinata, distributes globally. It is one of the most important edible bivalves in east Asian countries. However, there are multiple difficulties in rearing pen shell larvae and juveniles because of their high mortality. To understand the mechanism underlining such high mortality at the early embryonic development stage, we obtained approximately 100 million larvae during later breeding season in June, 2017, and checked a large amount of mitotic chromosomal plates of the early embryos and post-spawning gonads tissue slices of their parents. The results showed that most diploid A. pectinata embryos have 17 pairs of chromosomes (2n = 34) as their parents do. The first pair of particularly large chromosomes are heterotypic in some diploid embryos while they are homomorphic in others. The primary sex-determination chromosome type is XX/XY. A lot of triploid, pentaploid and aneuploid embryos with different numbers of the largest homomorphic or heteromorphic chromosomes were found due to the degeneration of overmatured parent gonads which hold normal karyotype. These larvae will die even though most of them may develop into the trochophore stage with 34 chromosomes. Genetic deficiency of chromosomes will cause a high rate of mortality in early embryos in late breeding season. These findings should enrich the current knowledge of juvenile pen shell aquaculture.

Breeding and Larval Rearing of Asian Moon Scallop Amusium pleuronectes in Eastern Samar, Philippines

Asian moon scallop, Amusium pleuronectes, is among the most common commercially harvested scallop in the Philippines. This study investigated suitable conditioning methods, induced spawning, and documented scallop larval development at the Marine Fisheries Research and Development Center (MFRDC) Guiuan, Eastern Samar. Scallops held in tanks with flow-through water system without substrate yielded high survival rate of 48.06 ± 5.95%. Optimum water temperature was 28-29°C for maintaining scallops. Isochrysis galbana and Chaetoceros calcitrans were suitable microalgal feed for A. pleuronectes consumed at 7,388,888 cells min-1 per scallop. These significant results were applied in maintaining scallops at the hatchery. As a result, scallops with an average 19.8 mm shell length (SL) reached 37.5 mm SL within four months with a survival rate of 96.57 ± 2.04%, average daily growth rate (ADGR) of 0.13 ± 0.04 mm.day-1, and specific growth rate (SGR) of 3.92 ± 1.31%.d-1. Natural spawning was successful under controlled conditions while induced spawning trial through thermal stimulation, food shock, sexual stimulation, and serotonin injection resulted unsuccessful release of sperm and eggs. The estimated number of fertilized eggs per spawning ranged from 0.22-1.4 million. Fertilized eggs appeared spherical and dark in color with 54.2-62.57 µm in diameter. After nine hours, the larvae developed into trochophore stage with 59.08-84.4 µm in length. D-veliger with 120.37-157.07µm shell length developed after 24 hours. Development of the early umbone stage was reached on day 5; and on day 7, the umbo larvae become well-developed with shell length of 135.45-173.36 µm. On day 9, pedi-veligers were observed in the culture. Spat grew 312.41-509.48 µm on day 16 and survived until four months with final shell length of 4-10 mm. For the larval rearing, stocking density of A. pleuronectes larvae observed highest survival rate of 0.04 ± 0.03% at 200 larvae/L, while no larvae survived at 800 larvae/L due to contamination of protozoans in the culture medium.

Состав жирных кислот в трохофорах мидий Mytilus galloprovincialis, выращенных в условиях загрязнённости полихлорбифенилами

Состояние черноморских популяций Mytilus galloprovincialis в естественной среде обитания напрямую зависит от развития мидии на всех стадиях, в том числе на начальных стадиях личиночных форм, наиболее чувствительных к загрязнению окружающей среды. Поллютанты органического происхождения оказывают негативное влияние на личинки моллюска, проявляющееся в торможении их роста и развития. Закономерности размножения мидии хорошо изучены, что даёт возможность получать в контролируемых лабораторных условиях личинки из искусственно оплодотворённых яйцеклеток этого вида моллюсков. В работе впервые исследован жирнокислотный состав общих липидов, выделенных из биомассы тканей личинок M. galloprovincialis на стадии трохофоры в контроле и после их трёхдневной экспозиции в среде с добавлением различных концентраций полихлорбифенилов. Жирнокислотный состав суммарных липидов в биомассе личинок, полученных на третьи сутки эксперимента, исследовали методом хромато-масс-спектрометрии. Всего идентифицировано 14 жирных кислот: 59 % из них относились к насыщенным, 24 % — к моноеновым, 17 % — к полиеновым. Для статистического анализа использовали программу MATLAB (версия 8.2). В условиях проведённого эксперимента в липидах трохофор M. galloprovincialis достоверно отличались значения суммы мононенасыщенных и полиненасыщенных жирных кислот. Сумма насыщенных жирных кислот статистически значимо не изменялась. Основными насыщенными жирными кислотами во всех исследуемых трохофорах мидий являлись пальмитиновая (C16:0) и стеариновая (C18:0). Их концентрации значительно не изменялись под действием полихлорбифенилов. Наиболее значимые мононенасыщенные жирные кислоты — олеиновая (C18:1ω9), пальмитолеиновая (C16:1ω7) и вакценовая (C18:1ω7). Содержание мононенасыщенных жирных кислот понижалось вдвое при действии полихлорбифенилов с концентрациями 0,1 и 1 мкг·л−1; при концентрации полихлорбифенилов 10 мкг·л−1 суммарное содержание этих кислот было равно таковому в контроле. Среди полиненасыщенных жирных кислот, обладающих положительной биологической активностью, были идентифицированы арахидоновая (C20:4ω6), эйкозапентаеновая (C20:5ω3) и докозагексаеновая (C22:6ω3). Суммарное содержание Омега-3 и Омега-6 кислот в личинках мидий в контрольном опыте не превышало 12,8 %. С увеличением концентрации полихлорбифенилов в среде выращивания трохофор с 0,1 до 1 мкг·л−1 концентрация полиненасыщенных жирных кислот повышалась в 2,5 раза. При концентрации полихлорбифенилов 10 мкг·л−1 и в пробе с ацетоном суммарное содержание полиненасыщенных жирных кислот было сопоставимо с таковым в контрольном опыте. Результаты исследования свидетельствуют о том, что жирнокислотный отклик трохофор мидий M. galloprovincialis максимален при воздействии концентраций полихлорбифенилов от 0,1 до 1 мкг·л−1. При концентрации загрязнителей 10 мкг·л−1 и выше биохимические процессы в личинках, по-видимому, замедляются. Результаты данного исследования могут способствовать лучшему пониманию перестроек, позволяющих моллюскам на личиночных стадиях развития адаптироваться к условиям загрязнения среды обитания органическими поллютантами.

Practical implementation of in vitro culture of Lanice conchilega (Polychaeta) in a coastal defence context

ABSTRACT Current coastal protection measures become increasingly insufficient under circumstances of climate change and increasing anthropogenic pressures. Aggregations of the ecosystem engineer Lanice conchilega (Pallas, 1766) stabilize the sediment bed of sandy shorelines. Therefore, this polychaete is considered an interesting target species in the search for nature-inspired designs in coastal zone management. This study investigated the potential of L. conchilega as a resilient coastal builder by trying to cultivate them and enhancing the larval settling process by using artificial substrata. A closed Recirculating aquaculture system (RAS) combining spawning induction and larval rearing was designed and tested. The tanks with adults were exposed to different photoperiods prior to water temperature rise. A spawning and fertilisation event was recorded in the 0:24 Light:Dark (LD) tank up to the trochophore stage. Additionally, the temperature-shock spawning induction method was experimentally proved effective. All practical experience on the in vitro spawning induction and larval rearing of L. conchilega is discussed and summarized as a guideline for future culturing trials. In vitro substratum settlement experiments with aulophore larvae sampled with plankton hauling revealed the potential of artificial substrata to trap larvae. Nonetheless, a preference in substratum type is not to be excluded yet.

SEM observation of early shell formation and expression of biomineralization-related genes during larval development in the pearl oyster Pinctada fucata

Shell formation of Pinctada fucata in larval development stages plays a crucial role in their survival. Scanning electron microscopy (SEM) was used to observe the morphological changes during larval development. We found that the early shell forms soon after enlargement of the blastopore at the anterior end of the trochophore stage and the complete shell forms in the spats stage, required for metamorphosis of P. fucata. Based on our transcriptome data of trochophore, D-shaped, umbonal, eyespots and spats stages, including the whole process of shell formation, 93 differentially expressed biomineralization-related genes were identified, of which 25 genes were unique to P. fucata, 30 were identical to genes in pacific oyster, and the remaining genes were annotated to other species. Two-dimensional and three-dimensional principal components analysis (PCA) showed that different developmental stages were significantly different, with the early two stages exhibiting a larger difference compared with the next stages. The 93 genes were sorted into 20 trends with three trends being significantly enriched: an initial increase and then a decrease, a monotonic decrease, and a monotonic increase. Gene expression patterns changed with regulatory function during shell formation. Almost all the biomineralization-related genes were up-regulated in the D-shaped stage, but only five genes were up-regulated in that stage but down-regulated in the remaining stages. There were also 11 genes up-regulated in the last three stages, and a total of 24 genes showed high expression level during the last four stages. The 55 genes selected for shell incision experiment sorted into five trends and most genes presented differences in expression between 24 h and other time points. Considering all these results, there is a correlation with the morphological change and the expression of biomineralization-related genes during larval developmental stages, especially of differently expressed genes.

Cloning and functional analysis of BMP3 in the pearl oyster (Pinctada fucata)

ABSTRACT Bone morphogenetic proteins (BMPs) are multifunctional secreted cytokines that play important roles in bone formation. In the present study, a homologous BMP3 gene was identified from Pinctada fucata (PfBMP3). PfBMP3 was 2710 bp length, contained 1470 bp of ORFs. PfBMP3 gene was expressed at all stages tested, with the highest expression during the trochophore and metamorphosis stages. PfBMP3 gene was expressed in all tissues tested, with high levels detected in the gill and mantle. In situ hybridization of the mantle revealed that PfBMP3 was highly expressed in the inner epidermis of the inner fold, the outer epidermis of the middle fold, and outer epidermis of the mantle. In the shell notching experiments, the expression of PfBMP3 was up-regulated after treatment. These results indicated that PfBMP3 might play a role in P. fucata shell formation. The expression level of PfBMP3 in a low calcium aquacultural environment was higher than that in a high calcium aquacultural environment, suggesting that PfBMP3 might participate in calcium ion metabolism regulation. RNA interference (RNAi) showed that the expression level of the calmodulin gene PfCaM was down-regulated after RNAi, implying that the involvement of PfBMP3 in calcium ion metabolism might be through action with PfCaM.

Transcriptome-wide identification, functional characterization, and expression analysis of two novel invertebrate-type Toll-like receptors from disk abalone (Haliotis discus discus)

Toll-like receptors (TLRs) are well-known pattern recognition receptors that play key immunological roles in a diverse range of organisms. In this study, two novel invertebrate TLRs from disk abalone (designated as AbTLR-A and AbTLR-B) were identified and functionally characterized for the first time. AbTLR-A and AbTLR-B comprised the typical TLR domain architecture containing an extracellular leucine-rich repeat domain, transmembrane domain, and Toll/interleukin-1 receptor domain. Expressional analysis revealed that both TLRs were constitutively expressed at all the early embryonic stages of disk abalone analyzed, with the highest level of AbTLR-A found at the 16-cell stage and AbTLR-B at the trochophore stage. According to tissue distribution analysis, prominent mRNA expression of AbTLR-A and AbTLR-B was detected in the hemocytes and gills, respectively. AbTLR-A and AbTLR-B mRNAs were significantly up-regulated in response to Gram-negative Vibrio parahemolyticus, Gram-positive Listeria monocytogenes, and viral hemorrhagic septicemia virus injections in abalone hemocytes and gills. Overexpression of AbTLR-A and AbTLR-B in HEK293T cells directly activated nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) responsive reporters. Neither TLRs showed a high response to pathogen-associated molecular patterns in vitro. Co-expression of AbTLR-A and AbTLR-B with AbMyD88-2 and AbMyD88-X activated NF-κB-responsive reporters in a synergetic manner. These findings demonstrate the involvement of AbTLR-A and AbTLR-B in abalone innate immunity.

Developmental dynamics of myogenesis in Pacific oyster Crassostrea gigas.

Pacific oyster (Crassostrea gigas) is a sessile bivalve living in the intertidal zone. It has become an attractive model for developmental studies due to its metamorphic transition from a mobile planktonic larvae to a sessile adults. To determine the effect of metamorphosis on muscle development in oyster larvae, we characterized myogenesis during larval development and metamorphosis by phalloidin staining which labels filamentous actin filaments. Our data revealed a dynamic pattern of myogenesis during embryonic and larval development. It appears that simple "U-shaped" muscle ring first developed at the trochophore stage. This was followed by a more complex musculature including an anterior adductor, velum ventral retractors at the veliger stage, and the addition of posterior adductors and foot retractors at the veliger and pediveliger stages. During metamorphosis, muscle structures in the anterior adductor, velum retractors and ventral retractors were degenerated. At the same time, mantle and gill musculature appeared and became the primary muscle system in juveniles together with the posterior adductor. In addition, indirect immunofluorescence with the monoclonal antibody against C. gigas muscle proteins (myosin heavy chains (MYHC) and α-actinin) were used to monitor changes in the developing musculature at different larval stages. The immunofluorescence staining results of muscle proteins were consistent with phalloidin staining. The expression locations of two muscle proteins were similar and mainly located in larval velum retractor and adductor muscle. The α-actinin expression positions were located in Z-lined of velum striated retractors. Data from these studies provide a comprehensive description of myogenesis in C. gigas embryos and larvae. Moreover, our data showed that metamorphosis has a significant impact on remolding the musculature after transition from a mobile planktonic larvae to a sessile mollusk, associated with certain muscle group degradation.

Cloning and molecular characterization of two ferritins from red abalone Haliotis rufescens and their expressions in response to bacterial challenge at juvenile and adult life stages

Ferritins are ubiquitous proteins with a pivotal role in iron storage and homeostasis, and in host defense responses during infection by pathogens in several organisms, including mollusks. In this study, we characterized two ferritin homologues in the red abalone Haliotis rufescens, a species of economic importance for Chile, USA and Mexico. Two ferritin subunits (Hrfer1 and Hrfer2) were cloned. Hrfer1 cDNA is an 807 bp clone containing a 516 bp open reading frame (ORF) that corresponds to a novel ferritin subunit in H. rufescens. Hrfer2 cDNA is an 868 bp clone containing a 516 bp ORF that corresponds to a previously reported ferritin subunit, but in this study 5′- and 3′-UTR sequences were additionally found. We detected a putative Iron Responsive Element (IRE) in the 5′-UTR sequence, suggesting a posttranscriptional regulation of Hrfer2 translation by iron. The deduced protein sequences of both cDNAs possessed the motifs and domains required in functional ferritin subunits. Expression patterns of both ferritins in different tissues, during different developmental stages, and in response to bacterial (Vibrio splendidus) exposure were examined. Both Hrfer1 and Hrfer2 are most expressed in digestive gland and gonad. Hrfer1 mRNA levels increased about 34-fold along with larval developmental process, attaining the highest level in the creeping post-larvae. Exogenous feeding is initiated at the creeping larva stage; thus, the increase of Hrfer1 may suggest and immunity-related role upon exposure to bacteria. Highest Hrfer2 expression levels were detected at trochophore stage; which may be related with early shell formation. Upon challenge with, the bacteria an early mild induction of Hrfer2 (2 h post-challenge), followed by a stronger induction of Hrfer1 at 15 h post-challenge, was observed in haemocytes from adult abalones. While maximal upregulation of both genes in the whole individual occurred at 24 h post-challenge, in juveniles. A significant increase in ferritin protein levels from 6 h to 24 h post-challenge was also detected. Our results suggest an involvement of Hrfer1 and Hrfer2, and of ferritin proteins in the immune response of H. rufescens to bacterial infection.

Copper and ocean acidification interact to lower maternal investment, but have little effect on adult physiology of the Sydney rock oyster Saccostrea glomerata

It remains unknown how molluscs will respond to oceans which are increasingly predicted to be warmer, more acidic, and heavily polluted. Ocean acidification and trace metals will likely interact to increase the energy demands of marine organisms, especially oysters. This study tested the interactive effect of exposure to elevated pCO2 and copper on the energetic demands of the Sydney rock oyster (Saccostrea glomerata) during reproductive conditioning and determined whether there were any positive or negative effects on their offspring. Oysters were exposed to elevated pCO2 (1000 μatm) and elevated copper (Cu 50 μg L−1 [0.787 μM]) in an orthogonal design for eight weeks during reproductive conditioning. After eight weeks, energetic demands on oysters were measured including standard metabolic rate (SMR), nitrogen excretion, molar oxygen to nitrogen (O:N) ratio, and pHe of adult oysters as well as the size and total lipid content of their eggs. To determine egg viability, the gametes were collected and fertilised from adult oysters, the percentage of embryos that had reached the trochophore stage after 24 h was recorded. Elevated pCO2 caused a lower extracellular pH and there was a greater O:N ratio in adult oysters exposed to copper. While the two stressors did not interact to cause significant effects on adult physiology, they did interact to reduce the size and lipid content of eggs indicating that energy demand on adult oysters was greater when both elevated pCO2 and copper were combined. Despite the lower energy, there were no negative effects on early embryonic development. In conclusion, elevated pCO2 can interact with metals and cause greater energetic demands on oysters; in response oysters may lower maternal investment to offspring.

The ultrastructural features of embryonic and early larval development in Yesso scallop, Mizuhopecten yessoensis.

Fine structural features of Mizuhopecten yessoensis in different embryonic and larval developmental stages from oocyte to early veliger have been studied. Spermatozoa have a conical head, consisting of a short acrosome, nucleus, and 4 mitochondria in the middle piece. The flagellum is approximately 40 μm in length. Oocyte cytoplasm contains numerous yolk granules, which are gradually expended during the larval development and disappear at the early veliger stage. Gastrula has two invaginations: shell gland anlage on the dorsal side and ventral side archenteron. Prototroch of a trochophore consists of two ciliary rings. Telotroch is absent. Myoblasts containing bundles of myofilaments are found in the blastocoel of trochophore. Velum and shell begin forming at the late trochophore stage. Cells of gut epithelium form numerous microvilli, organized into a brush border structure on the surface of enterocytes. A suggestion is made that larvae at the late trochophore stage are capable of digesting food and feeding. The structure of a M. yessoensis veliger is typical for pectinids. The velar retractor muscles have cross striation with a period of 1 μm. The digestive system of an 8-day-old veliger consists of the esophagus, stomach (with subdivided gastric shield and style sac), and short intestine. The digestive gland (with no discernible loops at this stage) is a part of the stomach epithelium with two types of digestive enzyme-producing cells.

Neuronal Development in the Larvae of the Invasive Biofouler Dreissena polymorpha (Mollusca: Bivalvia), with Special Attention to Sensory Elements and Swimming Behavior.

Although understanding of the neuronal development of Trochozoa has progressed recently, little attention has been paid to freshwater bivalves, including species with a strong ecological impact, such as the zebra mussel (Dreissena polymorpha). Therefore, an important question might concern how the developing nervous system is involved in the formation of the rapid and successful invasive behavior of this species. Our aim was to reveal the neuronal development of trochophore and veliger larvae of Dreissena, with special attention to the organization of sensory structures and their possible involvement in detecting environmental cues. After applying serotonin and FMRFamide immunocytochemistry, the first serotonin immunoreactive sensory elements appeared 16-18 hours after fertilization, whereas the first FMRFamide immunoreactive sensory cell was seen only at 32 hours of development (trochophore stage). Later, sensory elements were found in three parts of the larval body, including the apical organ, the posterior region, and the stomach. Although differences in the timing of appearance and the morphology of cells were observed, the two signaling systems showed basic similarity in their organization pattern until the end of the veliger stage. Pharmacological, physiological, and quantitative immunocytochemical investigations were also performed, suggesting the involvement of both the serotoninergic system and the FMRFamidergic system in sensomotor processes. Manipulation of the serotonin synthesis by para-chloroplenylalanine and 5-hydroxytryptophane, as well as application of increased salinity, influenced larval swimming activity, both accompanied by changes in immunofluorescence intensity. We concluded that these two early sensory systems may play an important role in the development of settlement competency of this biofouling invasive bivalve, Dreissena.

Nervous system development in the Pacific oyster, Crassostrea gigas (Mollusca: Bivalvia)

BackgroundBivalves comprise a large, highly diverse taxon of invertebrate species. Developmental studies of neurogenesis among species of Bivalvia are limited. Due to a lack of neurogenesis information, it is difficult to infer a ground pattern for Bivalvia. To provide more comprehensive morphogenetic data on bivalve molluscs and relationships among molluscan clades, we investigated neurogenesis in the Pacific oyster, Crassostrea gigas, from the appearance of the first sensory cells to the formation of the larval ganglionic nervous system by co-immunocytochemistry of the neuronal markers FMRFamide or 5-HT and vesicular acetylcholine transporter (VAChT).ResultsNeurogenesis begins with the emergence of the apical serotonin-immunoreactive (5-HT-ir) sensory cells and paired sensory posttrochal dorsal and ventral FMRFamide-immunoreactive (FMRFamide-ir) cells at the early trochophore stage. Later, at the early veliger stage, the apical organ (AO) includes 5-HT-ir, FMRFamide-ir, and VAChT-ir cells. At the same stage, VAChT-ir cells appear in the posterior region of larvae and send axons towards the AO. Thus, FMRFamide-ir neurites and VAChT-ir processes form scaffolds for longitudinal neurite bundles develop into the paired ventral nerve cords (VNC). Later-appearing axons from the AO/CG neurons join the neurite bundles comprising the VNC. All larval ganglia appear along the VNC as paired or fused (epiathroid) clusters in late veliger and pediveliger larvae. We observed the transformation of the AO into the cerebral ganglia, which abundantly innervated the velum, and the transformation of ventral neurons into the pedal ganglia, innervating the foot, gills, and anterior adductor muscle. The visceral ganglia appear last in the pediveliger oyster and innervate the visceral mass and posterior adductor of premetamorphic larvae. In addition, a local FMRFamide-ir network was detected in the digestive system of pediveliger larvae. We identified VAChT-ir nervous elements in oyster larvae, which have not been observed previously in molluscs. Finally, we performed a morphology-based comparative analysis of neuronal structures among bivalve, conchiferan, and aculiferan species.ConclusionsWe described the development of the nervous system during the larval development in Crassostrea gigas. These data greatly advance the currently limited understanding of neurodevelopment in bivalves and mollusks, which has hampered the generation of a ground pattern reconstruction of the last common ancestor of Mollusca. Our morphological data support phylogenomic data indicating a closer Bivalvia-Gastropoda sister group relationship than the Bivalvia-Scaphopoda (Diasoma) group relationship.

Towards a ground pattern reconstruction of bivalve nervous systems: neurogenesis in the zebra mussel Dreissena polymorpha

Bivalvia is a taxon of aquatic mollusks that includes clams, oysters, mussels, and scallops. Within heterodont bivalves, Dreissena polymorpha is a small, mytiliform, freshwater mussel that develops indirectly via a planktotrophic veliger larva. Currently, only a few studies on bivalve neurogenesis are available, impeding the reconstruction of a ground pattern in Bivalvia. In order to inject novel data into this discussion, we describe herein the development of the serotonin-like and α-tubulin-like immunoreactive (lir) neuronal components of D. polymorpha from the early trochophore to the late veliger stage. Neurogenesis starts in the early trochophore stage at the apical pole with the appearance of one flask-shaped serotonin-lir cell. When larvae reach the veliger stage, four flask-shaped serotonin-lir cells are present in the apical organ. At the same time, the anlagen of the cerebral ganglia start to form at the base of the apical organ. From the apical organ, one pair of cerebro-visceral connectives projects posteriorly and connects to a posterior larval sensory organ that contains serotonin- and α-tubulin-like flask-shaped cells. Additional, paired serotonin-lir neurites originate from the apical organ and project into the velum. One unpaired stomatogastric serotonin-lir cell develops ventrally to the stomach at the veliger stage. The low number of serotonin-lir cells in the apical organ of bivalve veligers is shared with larvae of basally branching gastropods and scaphopods and is thus considered a feature of the last common ancestor of Conchifera, while the overall simplicity of the larval neural architecture appears to be a specific trait of Bivalvia.

Molecular identification of disk abalone (Haliotis discus discus) tetraspanin 33 and CD63: Insights into potent players in the disk abalone host defense system

Tetraspanins are a superfamily of transmembrane proteins involved in a diverse range of physiological processes including differentiation, adhesion, signal transduction, cell motility, and immune responses. In the present study, two tetraspanins, CD63 and tetraspanin 33 (TSPAN33) from disk abalone (AbCD63 and AbTSPAN33), were identified and characterized at the molecular level. The coding sequences for AbCD63 and AbTSPAN33 encoded polypeptides of 234 and 290 amino acids (aa) with predicted molecular mass of 25.3 and 32.5 kDa, respectively. The deduced AbCD63 and AbTSPAN33 protein sequences were also predicted to have a typical tetraspanin domain architecture, including four transmembrane domains (TM), short N- and C- terminal regions, a short intracellular loop, as well as a large and small extracellular loop. A characteristic CCG motif and cysteine residues, which are highly conserved across CD63 and TSPAN33 proteins of different species, were present in the large extracellular loop of both abalone tetraspanins. Phylogenetic analysis revealed that the AbCD63 and AbTSPAN33 clustered in the invertebrate subclade of tetraspanins, thus exhibiting a close relationship with tetraspanins of other mollusks. The AbCD63 and AbTSPAN33 mRNA transcripts were detected at early embryonic development stages of disk abalone with significantly higher amounts at the trochophore stage, suggesting the involvement of these proteins in embryonic development. Both AbCD63 and AbTSPAN33 were ubiquitously expressed in all the tissues of unchallenged abalones analyzed, with the highest expression levels found in hemocytes. Moreover, significant induction of AbCD63 and AbTSPAN33 mRNA expression was observed in immunologically important tissues, such as hemocytes and gills, upon stimulation with live bacteria (Vibrio parahaemolyticus and Listeria monocytogenes), virus (viral hemorrhagic septicemia virus), and two potent immune stimulators [polyinosinic:polycytidylic acid (poly I:C) and lipopolysaccharide (LPS)]. Collectively, these findings suggest that AbCD63 and AbTSPAN33 are involved in innate immune responses in disk abalone during pathogenic stress.

Rate of oxygen consumption during embryonic development of great ramshorn Planorbarius corneus (Gastropoda)

Energy metabolism is studied in great ramshorn Planorbarius corneus during embryonic development. It is shown that the rate of oxygen consumption is constantly increasing in the process of embryogenesis. The respiration intensity (rate of the oxygen consumption per unit of the embryo volume) initially increases and then slowly decreases until eclosion. At the early stages of development until the early trochophore stage, the embryo is not growing, and, thus, the change in the rate of oxygen consumption during this period is not associated with the change of the embryo volume. Reduction in the intensity of respiration begins simultaneously with the beginning of the growth of the embryo at the stage of the middle trochophore. Starting from the middle trochophore and until eclosion, an association between oxygen consumption rate and volume of the embryo can be described with an allometric equation with exponential coefficient equal to approximately 0.23.

Larval development and post‐larval growth of Branchiomma bairdi (Annelida: Sabellidae) from a Mediterranean population

AbstractBranchiomma bairdi is a Caribbean fan worm introduced in several localities worldwide, including the Mediterranean Sea, where the species’ range has rapidly expanded. Reproduction in B. bairdi was previously investigated in both extra‐Mediterranean and Mediterranean areas, but no information is available on larval development and post‐larval growth. In the present article, we examined these features for a population from the Mar Grande of Taranto (Ionian Sea). The species is hermaphrodite, and fertilization occurs in situ. Mucus seems to play an important role in fertilization, and also in preserving eggs before fertilization. The trochophore stage develops within the mucus and after hatching, larvae swim for about 3 d before settlement. The trochophore showed a distinct prototroch and two red dorsolateral larval eyes. The pelagic stage takes only 96 h even though prototroch is maintained after settlement, disappearing at 5 d, when larvae showed three chaetigers and branchial crown consisted of four radioles. Some interesting observations concerning changes in the morphology of chaetae and in the number of uncini during growth are also reported, together with discussion of the development of stylodes, an important diagnostic feature in Branchiomma species identification.

Embryology, larval development, settlement and metamorphosis in the New Zealand Serpulid Polychaete Galeolaria hystrix

Galeolaria hystrix are a large, long-lived serpulid worm, abundant in the intertidal and subtidal of New Zealand, where they can form important biogenic reefs. The developmental biology, settlement and metamorphosis of the species was examined in laboratory cultures at 13 °C, over a 27-day period, providing new insight into the reproductive biology of this ecologically important species. Gametes were obtained by dissection, and fertilised eggs (≈65 μm diameter) developed to an 80-μm free-swimming trochophore stage by 24 h. The trochophore increased in body length to 100 μm by day 9, and by day 12 commenced differentiation into a three-setiger stage larva that reached competency and a size of ≈320 μm by day 16. We detail the anatomical changes that occur in the larvae over this period, and compared these with previous descriptions of serpulid development. Competent demersal larvae commenced settlement through a searching behaviour that resulted in metamorphosis within 24 h after permanent attachment. The formation of a neck constriction in the larvae, a reduction of the larval head and the formation of tentacle buds of the rudimentary branchial crown delineated metamorphosis. A calcified tube large enough to encase the juvenile was secreted by five-days post-metamorphosis.