Trochophore Larvae Research Articles

Expression of GATA and POU transcription factors during the development of the planktotrophic trochophore of the polychaete serpulid Hydroides elegans.

The expression of transcription factors with endodermal and mesodermal roles in bilaterians is characterized during the development of Hydroides elegans, a serpulid polychaete with planktotrophic trochophore. GATA 4/5/6 is expressed in endodermal and mesodermal precursors during embryogenesis and in the midgut of trochophore larvae. HeGATA1/2/3a is expressed in animal hemisphere blastomeres 1d121 and 1d122, in dorsal ectoderm and in 4d endomesodermal derivatives that maintain their expression in trochophore larvae. HeGATA1/2/3b is not expressed during embryogenesis, but in several regions of the larva during postembryonic development. During very early gastrulation, Brn1/2/4 is first expressed in cells associated with the prospective oral/foregut side of the blastopore, and during larval development in 4d blastomere descendants. Comparison with orthologs in other metazoans suggests ancestral expression of GATA4/5/6 in the midgut of the last common ancestor of protostomes and deuterostomes. The conserved expression of Brn1/2/4 in the foregut precursors of Hydroides and sea urchins suggests an ancestral role in patterning the tripartite gut of planktotrophic larvae. Broader analysis of these and other regulatory genes reveals variability of developmental gene expression among polychaetes with lecithotrophic larvae, suggesting that they are evolutionarily derived from polychaetes with planktotrophic larvae.

Comparative embryotoxicity and genotoxicity of the herbicide diuron and its metabolites in early life stages of Crassostrea gigas: Implication of reactive oxygen species production

Herbicides are one of the major classes of pollutants contaminating coastal waters over the world. Among them, diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea) is a phenylurea herbicide frequently detected in oyster-producing area, known to be toxic for this important exploited non-target species. With the aim to investigate the mechanisms by which diuron displays its toxicity in oyster, the implication of both biotransformation and oxygen reactive species (ROS) production was studied considering embryotoxicity and genotoxicity as endpoints. Comparative embryotoxicity and genotoxicity of diuron and its main metabolites (DCPMU, DCPU and 3,4-DCA) were thus studied on oyster larvae by the embryo-larval bioassay on D larvae and the comet assay on trochophore larvae, respectively. Exposures were also performed in presence and absence of known ROS scavenger compounds − ascorbic acid and N-acetylcysteine, to evaluate the involvement of oxyradicals in the toxic responses. In the case of diuron, the production of ROS on exposed oyster larvae was also measured using 2′,7′-dichlorodihydrofluorescein diacetate as a probe for flow cytometric analysis.The results we obtained showed the embryotoxicity and genotoxicity of diuron and its metabolites in early life stages of the Pacific oyster. For concentrations ranging from 0.05 to 0.5μgL−1, diuron appeared significantly more embryotoxic than DCPMU and DCPU (p<0.001). Embryotoxicity decreased with diuron metabolism as follows: diuron≥DCPMU=DCPU, highlighting that biotransformation can constitute a true detoxication pathways in oyster larvae by decreasing the toxicity of the parent compound. In the opposite, no difference was observed between diuron and its metabolites concerning larval development when considering a lower and more environmentally realistic range of concentrations (0.002–0.050μgL−1). 3,4-DCA was the only compound that did not show any sign of embryotoxicity, even at concentrations up to 5μgL−1. Concerning genotoxicity, no significant difference was observed between diuron and all of its metabolites including 3, 4 DCA with damages detected from the concentration of 0.05μgL−1. As for diuron, the toxicity of the metabolites seems to be mediated in some part by ROS production as clearly demonstrated by the decrease in genotoxicity and developmental abnormalities in the presence of the oxidant scavenger, ascorbic acid.

Effects of reduced pH on the early larval development of hatchery-reared Donkey's ear abalone, Haliotis asinina (Linnaeus 1758)

Declining pH levels caused by absorption of accumulating atmospheric CO2 in the ocean threaten the normal development of early life stages and particularly impair the ability of calcifying organisms to construct their shells. An experiment was conducted to determine the direct effects of reduced pH on % hatching of fertilized eggs, survival and developmental stages of trochophore larvae of the Donkey's ear abalone, Haliotis asinina. The ambient pH (7.97±0.02) of experimental seawater was reduced by bubbling food-grade CO2 to obtain the desired pH levels of 7.78±0.03, 7.60±0.03 and 7.40±0.02 as treatments. There were increasing negative impacts of reduced pH on the mean % hatched trochophores (97.6 at ambient pH conditions, 83.9 at pH7.78, 24.1 at pH7.60, and 1.4 at pH7.40). Significant impacts of reduced pH were also observed on mean % survival of trochophores (98.3 at ambient pH conditions, 84.9 at pH7.78, 24.1 at pH7.60, and 1.4 at pH7.40). Of the surviving trochophores, 23.2±3.2% (mean±sd) of those exposed to pH7.60 were morphologically deformed but in much lower pH treatment (pH7.40), all the trochophores were deformed. At pH7.78, only 63.3±3.7% of the surviving trochophores developed normally compared to 96.7±1.6% normal trochophores at ambient pH conditions. Normal trochophores have prototrochal cells and girdle fully developed and developing shell tissue. Malformed trochophores showed highly undefined morphological characters and cleavage failed to progress to further developmental stages. In conclusion, the early larval development of H. asinina was found to be highly sensitive to reduced pH levels projected for the end of the present century. Therefore, future rise in CO2 concentration in tropical marine waters will likely pose a significant threat to the natural population densities of this species. Statement of relevance•To understand how lower pH levels can affect this high-value haliotid species is critical because of the importance of this species to fishery and mariculture production;•The deleterious effects of reduced pH on the early larval development of (up to hatch-out stage) of the tropical abalone (H. asinina) were demonstrated in this study;•A significant decrease in hatching rates, increased larval mortality, delayed and abnormal development will most likely have great impact on the population dynamics of this species in their natural habitat;•All the observations in this study suggest that reduced pH can affect early larval development and survival of abalone in the coastal ecosystem; and•these negative effects on larvae may persist into juvenile and adult stages of abalone which would likely contribute to a severe reduction of their natural populations.

Conserved hemopoietic transcription factor Cg-SCL delineates hematopoiesis of Pacific oyster Crassostrea gigas.

Hemocytes are the effective immunocytes in bivalves, which have been reported to be derived from stem-like cells in gill epithelium of oyster. In the present work, a conserved haematopoietic transcription factor Tal-1/Scl (Stem Cell Leukemia) was identified in Pacific oyster (Cg-SCL), and it was evolutionarily close to the orthologs in deuterostomes. Cg-SCL was highly distributed in the hemocytes as well as gill and mantle. The hemocyte specific genes Integrin, EcSOD and haematopoietic transcription factors GATA3, C-Myb, c-kit, were down-regulated when Cg-SCL was interfered by dsRNA. During the larval developmental stages, the mRNA transcripts of Cg-SCL gradually increased after fertilization and peaked at early trochophore larvae stage (10hpf, hours post fertilization), then sharply decreased in late trochophore larvae stage (15hpf) before resuming in umbo larvae (120hpf). Whole-mount immunofluorescence assay further revealed that the immunoreactivity of Cg-SCL appeared in blastula larvae with two approximate symmetric spots, and this expression pattern lasted in gastrula larvae. By trochophore, the immunoreactivity formed a ring around the dorsal region and then separated into two remarkable spots at the dorsal side in D-veliger larvae. After bacterial challenge, the mRNA expression levels of Cg-SCL were significantly up-regulated in the D-veliger and umbo larvae, indicating the available hematopoietic regulation in oyster larvae. These results demonstrated that Cg-SCL could be used as haematopoietic specific marker to trace potential developmental events of hematopoiesis during ontogenesis of oyster, which occurred early in blastula stage and maintained until D-veliger larvae.

Distribution of granuloside in the Antarctic nudibranch Charcotia granulosa (Gastropoda: Heterobranchia: Charcotiidae)

The loss of the shell in nudibranch gastropods has been related to the acquisition of chemical defensive strategies during evolution, such as the use of natural products to deter predation. In the present study, we investigated the origin, location, and putative role of granuloside (1), a homosesterterpene lactone, recently isolated from the Antarctic nudibranch Charcotia granulosa Vayssiere, 1906. Several adults, egg masses, and its bryozoan prey, Beania erecta Waters, 1904, were chemically analyzed by chromatographic and spectroscopic techniques. Light- and transmission electron microscopy of the mantle revealed complex glandular structures, which might be associated with the storage of defensive compounds in analogy to mantle dermal formations described in other nudibranchs. Although preliminary in situ repellence bioassays with live specimens of the nudibranch showed avoidance against the Antarctic generalist sea star predator Odontaster validus, the specific role of the terpene granuloside requires further investigation. The egg masses do not present granuloside, and the glandular structures are absent in the trochophore larvae. Our results suggest that C. granulosa synthesizes granuloside de novo in early stages of its ontogeny, instead of obtaining it from the prey. Considering the wide geographic area inhabited by this slug, this may be advantageous, because natural products produced by the slug will not be affected by fluctuant food availability. Overall, the Antarctic sea slug C. granulosa seems to possess defensive strategies that are similar, in terms of production and storage, to nudibranchs from other regions of the world. This species is one of the few cladobranchs investigated so far that present de novo biosynthesis of a defensive compound.

Standardization of a Patella spp. (Mollusca, Gastropoda) embryo–larval bioassay and advantages of its use in marine ecotoxicology

The use of three limpet species, Patella vulgata Linnaeus, 1758, Patella depressa Pennant, 1777 and Patella ulyssiponensis Gmelin, 1791 as model organisms in marine ecotoxicology has been evaluated. Initial laboratory experiments were aimed to standardize a biological test with embryos and larvae of Patella spp, establishing the percentage of normal trochophore larvae as endpoint. Before conducting in vitro fertilization, oocytes must be maturated artificially by incubation in an alkaline solution; therefore, alkalinizing agent, pH and time of eggs alkalinization were evaluated. Moreover, time of sperm activation, optimum sperm and oocytes concentration during fertilization, gamete contact time, use of stirring during the fertilization, egg concentration and incubation temperature were examined. Minimum sample size per treatment was also estimated. Exposure of oocytes for 10min to FSW alkalinized with NH4OH at pH 9.0, the use of undiluted sperm pre-activated during 45min and a concentration of 200 oocytesmL−1, a gamete-contact time of 180min and egg incubation at 18°C during 24h at a concentration of 80 eggsmL−1 were the conditions allowing maximal embryo-larval development success. With an error of 0.05, a sampling size ≥320 allows a 95% confidence in the estimate. This Patella spp. acute bioassay fulfills a number of important a priori requirements to be used in ecotoxicological studies. Nevertheless, in vitro fertilization requires considerable handling, which may lead to failure in fecundation. Such difficulties are also addressed, in order to facilitate the routine use of this protocol by other laboratories.

Effects of LED light quality on the growth, survival and metamorphosis ofHaliotis discus hannaiIno larvae

Light is a key environmental factor influencing the growth, development and survival of aquatic organisms. We examined the effects of different light qualities (red, orange, white, blue, green or no light) and developmental stage at initial lighting [fertilized egg (FE), trochophore larva (TL), or eye-spot larva (EL)] on the growth, development, and survival of larvae of the Pacific abalone Haliotis discus hannai Ino. Larva-hatching success was significantly higher under blue, green, or no light compared with red, orange or white light (P 0.05). Juvenile survival was significantly higher under blue or green compared with other light qualities (P 0.05). Larval size at completion of the shell was unaffected by stage at initial illumination, but was greater under blue or green light, while size at metamorphosis was greatest following illumination with blue or green light since the TL or EL stage (P 0.05). These results suggest that a blue or green light source applied from the TL stage can increase the hatching and yield of H. discus hannai Ino, with important implications for the development of the aquaculture industry.

Neuromuscular development in Patellogastropoda (Mollusca: Gastropoda) and its importance for reconstructing ancestral gastropod bodyplan features.

Within Gastropoda, limpets (Patellogastropoda) are considered the most basal branching taxon and its representatives are thus crucial for research into evolutionary questions. Here, we describe the development of the neuromuscular system in Lottia cf. kogamogai. In trochophore larvae, first serotonin‐like immunoreactivity (lir) appears in the apical organ and in the prototroch nerve ring. The arrangement and number of serotonin‐lir cells in the apical organ (three flask‐shaped, two round cells) are strikingly similar to those in putatively derived gastropods. First, FMRFamide‐lir appears in veliger larvae in the Anlagen of the future adult nervous system including the cerebral and pedal ganglia. As in other gastropods, the larvae of this limpet show one main and one accessory retractor as well as a pedal retractor and a prototroch muscle ring. Of these, only the pedal retractor persists until after metamorphosis and is part of the adult shell musculature. We found a hitherto undescribed, paired muscle that inserts at the base of the foot and runs towards the base of the tentacles. An apical organ with flask‐shaped cells, one main and one accessory retractor muscle is commonly found among gastropod larvae and thus might have been part of the last common ancestor.

Early development of the blue mussel Mytilus edulis (Linnaeus, 1758) cultured in potassium-fortified inland saline water

The low potassium concentration in inland saline water (ISW) restrains the normal development of cultured marine organisms, and thus, possesses challenges for the development of ISW aquaculture. Therefore, assessing the effects of potassium fortification in ISW on the performance of cultured marine species is an important step to determine the feasibility of their culture in ISW. The aim of this research was to investigate the effects of potassium fortification in ISW on the performance of early life stages of the blue mussel Mytilus edulis including fertilised eggs, trochophore, veliger and pediveliger larvae. These stages were reared in five different levels of potassium-fortified ISW, namely 20, 40, 60, 80 and 100% of potassium levels equivalent to the potassium level in ocean water (OW) and two controls namely, ISW at 27ppt (ISW27) and OW at 25ppt (OW25). The results showed that the higher levels of potassium in ISW, particularly with 100% K+ fortification (ISW100K+), invariably improved the survival and size, and reduced the developmental stage interval and deformities of blue mussel larvae. Deformities, such as faulty cell cleavage, abnormal formation of trochophore larvae, protruding mantle in veliger larvae, and indented shell margin in veliger and in pediveliger, were observed when reared in any ISW. However, rearing in ISW did not result in any deformities in settlement larvae. The number of deformities was reduced at higher K+ fortification levels, and there were no deformities in pediveliger larvae reared in ISW100K+ and in OW. These results showed that K+ fortification in ISW improves the performance of the rearing of the larval stages of the blue mussel. Statement of relevanceThis study is the first attempt to culture larval stages of blue mussel Mytilus edulis in potassium-fortified inland saline water. The results have shown that that the larval development is feasible only if ISW is fortified with K+. By initiating larval culture in inland saline water, we expect better results will be reflected by adult stages.

A “classic” model organism

Wall charts were a universal educational and scientific media until the mid-1900 s, and one of the most prolific of collectors was Karl Georg Friedrich Rudolph Leuckart (1822–1898). The entire collection of Leuckart's wall charts is housed at the University of Pavia, Italy. Shown here is Pavia number 100p, by Berthold Hatschek (1854–1941), a student of Rudolf Leuckart (Leipzig) and Ernst Haeckel (Jena), who received a Ph.D. for his work on the metamorphosis of the Trochophore larvae. A compilation of the Leuckart collection is available in stunning book form: Visual Zoology: The Pavia collection of Leuckart's zoological wall charts (1877), by Carlo Alberto Redi, Silvia Garagna, Maurizio Zuccotti, Ernesto Capanna, and Helmut Zacharias (reprinted with permission from Ibis Publisher © 2000). Shown in this partial reproduction of one wall chart is the anatomy of a Tunicate, a protochordate that is used to study notochord development in the context of vertebrate evolution, fertilization in a self-sterile (plant-like) mechanism, and precise patterning of unequal cleavage.

Identification of β integrin-like- and fibronectin-like proteins in the bivalve mollusk Mytilus trossulus.

Integrins play a key role in the intermediation and coordination between cells and extracellular matrix components. In this study, we first determined the presence of the β integrin-like protein and its presumptive ligand, fibronectin-like protein, during development and in some adult tissues of the bivalve mollusc Mytilus trossulus. We found that β integrin-like protein expression correlated with the development and differentiation of the digestive system in larvae. Besides the presence of β integrin-like protein in the digestive epithelial larval cells, this protein was detected in the hemocytes and some adult tissues of M. trossulus. The fibronectin-like protein was detected firstly at the blastula stage and later, the FN-LP-immunoreactive cells were scattered in the trochophore larvae. The fibronectin-like protein was not expressed in the β integrin-positive cells of either the veliger stage larvae or the adult mussel tissues and the primary hemocyte cell culture. Despite the β integrin- and fibronectin-like proteins being expressed in different cell types of mussel larvae, we do not exclude the possibility of direct interaction between these two proteins during M. trossulus development or in adult tissues.

Larval hatching and development of the wedge shell (Donax trunculus L.) under increased CO2 in southern Portugal

Noticeable changes in global temperatures, climate and ocean carbon chemistry are the result of carbon dioxide increase in the atmosphere. This increase has been mitigated by the oceans capacity to absorb one-fourth of the carbon dioxide in the atmosphere, although this CO2 intake affects oceans carbonate chemistry [i.e., ocean acidification—(OA)]. The detrimental effect of OA in the development and shell formation has been studied in several species of bivalves, although no information is available on the wedge shell Donax trunculus, a gastronomically appreciated species and an important economical resource in several southern European countries. We evaluated the effect of pCO2 increase on hatching and early life stages of D. trunculus, considering two ocean acidification scenarios (ΔpH −0.3 and ΔpH −0.6). Our results showed that elevated pCO2 caused a delay in hatching into D-larvae and reduced larvae survival. In the extreme scenario (ΔpH −0.6), some trochophore larvae persisted to day 9 of the experiment and more abnormal larvae were produced than in the ΔpH −0.3 and control treatments. At day 5, normal veligers under extreme acidification were smaller than in other treatments, but by day 9, these differences were attenuated and the average size of normal D-larvae varied inversely to the pH gradient. Possible underlying mechanisms for these complex response patterns are discussed, including the existence of phenotypic plasticity or genetic pre-adaptive capacity in this D. trunculus population to cope with future environmental changes.

The enkephalinergic nervous system and its immunomodulation on the developing immune system during the ontogenesis of oyster Crassostrea gigas

Enkephalinergic neuroendocrine-immune regulatory system is one of the most important neuroendocrine-immune systems in both vertebrates and invertebrates for its significant role in the immune regulation. In the present study, the early onset of enkephalinergic nervous system and its immunomodulation on the developing immune system during the ontogenesis of oyster Crassostrea gigas were investigated to illustrate the function of neural regulation on the innate immune system in oyster larvae. [Met5]-enkephalin (Met-ENK) was firstly observed on the marginal of the dorsal half of D-hinged larvae. Six immune-related molecules, including four PRRs (CgCTL-1, CgCTL-2, CgCTL-4, CgNatterin-3) and two immune effectors (CgTNF-1 and CgEcSOD) were detected in the early developmental stages of trochophore, D-hinged and umbo larvae of oyster. After incubated with [Met5]-enkephalin, the mRNA expression level of all the PRRs changed significantly (p < 0.05). In trochophore larvae, the expression level of CgNatterin-3 decreased dramatically (p < 0.05) at 6 h, and the expression level of CgCTL-4 was significantly down-regulated at 3 h and 6 h (p < 0.05), respectively. In D-hinged and umbo larvae, only CgCTL-1 was significantly down-regulated and the differences were significant at 3 h and 6 h (p < 0.05), while the expression level of CgCTL-2 and CgCTL-4 increased significantly at 3 h after treatment (p < 0.05). Moreover, the expression levels of immune effectors were up-regulated significantly at 3 h and 6 h in trochophore larvae (p < 0.05). The expression level of CgTNF-1 in both blank and experiment groups was up-regulated but there was no significant difference in D-hinged larvae stage. On the contrary, the expression level of CgEcSOD in D-hinged larvae decreased dramatically at 3 h and 6 h after [Met5]-enkephalin incubation (p < 0.05). In umbo larvae, the expression level of CgTNF-1 and CgEcSOD in the experiment group increased significantly at 6 h after [Met5]-enkephalin treatment (p < 0.05), while no significant difference was found in the blank group. In addition, the anti-bacterial activities of the total protein extract from trochophore, D-hinged and umbo larvae increased significantly (p < 0.05) at both 3 h and 6 h after [Met5]-enkephalin incubation compared to that in the blank group, and PO activities of both D-hinged and umbo larvae total protein extract increased significantly (p < 0.05) while no significant difference was observed in trochophore larvae. The PO activities of the total protein extract in all the experiment groups decreased after the treatment with [Met5]-enkephalin for 6 h, but no significant difference was observed when compared to the blank group. Furthermore, after incubation for 6 h, the concentration of both CgTNF-1 and CgIL17-5 increased dramatically compared to that in the blank group (p < 0.05). These results together indicated that the enkephalinergic nervous system of oyster was firstly appeared in D-hinged larvae, while the primitive immune defense system existed in the region of prototroch in trochophore larvae and developed maturely after D-hinged larvae. The developing immune system could be regulated by the neurotransmitter [Met5]-enkephalin released by the neuroendocrine system in oyster C. gigas.

큰가리비 (Patinopecten yessoensis)의 수온 자극에 의한 조기 성성숙 유도와 발생

본 연구의 결과인 수온조절에 의한 큰가리비의 생식소 발달을 조사한 결과, 암컷 샌식소지수 (GSI) 경우 1월에 실험구 I가 <TEX>$9.12{\pm}2.9$</TEX>, 2월에 <TEX>$14.89{\pm}2.9$</TEX>, 3월에 <TEX>$21.3{\pm}1.4$</TEX>로 지속적으로 상승하였다. 2월에 실험구 I, II, III과는 유의차가 없었으나 (P > 0.05), 실험구와 대조구와는 유의가 있었다 (P < 0.05). 실험구 I은 지속적으로 성숙하여 산란 가능한 범위로 성성숙 및 산란하여 가장 좋은 결과를 보였으며 타 시험구와 유의하게 높았다 (P < 0.05). 조직학적 관찰을 통해 생식소 발달을 조사한 결과, 1월에는 모든 실험구의 큰가리비 난소가 성장기 (growing stage) 단계로 확인되었다. 2월의 대조규 난소는 성장후기 단계였으며, 실험구 I, II 및 III은 모두 성숙기(mature stage)로 확인되었다. 어미의 실내 성성숙 유도를 위하여 수온 <TEX>$12^{\circ}C$</TEX>를 유지한 결과, 11월에 생식소 지수 및 생식소 중량은 각각 <TEX>$4.4{\pm}0.88$</TEX>, 2.8 g 이였으나 1월에 <TEX>$15.1{\pm}2.8$</TEX>, 11.7 g, 2월에 <TEX>$21.7{\pm}5.4$</TEX>, 19.4 g으로 급격히 생식소지수가 증가되어 2월에 방란, 방정이 가능하여 자연 산란군보다 2-3개월의 조기 산란 유도가 가능하였다. 수온 약 <TEX>$16^{\circ}C$</TEX>에서 수정란은 나선상 난할 (spiral cleavage)을 시작하여 낭배기(gastrula stage)를 거쳐 약 1.5일 후에는 담륜자 (trochophore) 유생이 되어 부상하여 유영하였다. 약 10일 후에는 초기원각 (prodissoconch shell)을 형성하여 각정기 유생기 (umbo stage larva)가 시작되며 각정 (umbones)이 형성되었다. 이후 각정기 유생은 성숙하여 수정 후 20-23일 후에는 팜사나 경심망 등의 기질에 부착하여 부착치패가 되어 조기 종묘 생산이 가능하였다. Early sexual maturation through temperature stimulation was induced in female and male of yezo scallop. Gonadosomatic index (GSI) in female showed <TEX>$9.12{\pm}2.9$</TEX> in January, <TEX>$14.89{\pm}2.9$</TEX> in February and <TEX>$21.3{\pm}1.4$</TEX> in March in experiment I. GSI in experiment I showed a significant increase (P < 0.05) and in experiments II and III were not show significant variations (P > 0.05). It also showed significant between the control and the experiments I, II, and III in February (P < 0.05) measurements. Experiment I has showed good results in sexual maturation and spawning when compared with other experiments II and III and the control. Histological observation showed that ovary condition was in a growing stage in all the experiments I, II, and III. In February, ovary condition through histological observation was a late mature stage in all the experiments I, II, and III except the control of a growing stage. GSI and gonad weight were <TEX>$4.4{\pm}0.88$</TEX> and 2.8 g, respectively in November whereas it was <TEX>$15.1{\pm}2.8$</TEX>, and 11.7 g, respectively in January and <TEX>$21.7{\pm}5.4$</TEX>, and 19.4 g, respectively in February after rearing at a water bath of <TEX>$12^{\circ}C$</TEX> depending on the condition of experiment I. It was possible early releasing of eggs and sperms of yezo scallop in February instead of the middle of April to the end of May being spawning period. Fertilized eggs have become a gastrula stage through a spiral cleavage and then become a trochophore larvae after 36 hours. After 10 days, D-shaped larvae have changed into an umbo stage larvae and attached to juveniles in the post larvae after 20-23 days.

Analysis of DNA methylation in tissues and development stages of pearl oyster Pinctada fucata

In this study, we developed a methylation-sensitive amplification polymorphism technique to investigate DNA methylation profiles in different tissues and in the early-development stages of the pearl oyster Pinctada fucata (P. fucata). Methylation levels in adductor muscle, digestive gland, axe foot, heart, and gill ranged from 11.71 to 14.71 %, and significant differences (P < 0.05) between methylation levels in different tissues were observed. The DNA methylation levels of sperm, egg cells, two-cell embryos, morula embryos, trochophore larvae and D-shaped larvae were 13.51, 11.80, 12.14, 12.60, 14.65 and 13.18 %, respectively. Development stages of two-cell embryos, morula embryos, trochophore larvae and D-shaped larvae indicated a higher number of identical DNA methylation status loci in the egg, compared to that in the sperm. It is probable that DNA methylation patterns of the progeny are mainly influenced by the egg, while the sperm may become increasingly important during the process of early embryo development. The observed differences in methylation levels in the tissues and the development stages of P. fucata suggest that DNA methylation may act as an epigenetic regulator during tissue differentiation, individual growth, and development.

Chordate evolution and the three-phylum system.

Traditional metazoan phylogeny classifies the Vertebrata as a subphylum of the phylum Chordata, together with two other subphyla, the Urochordata (Tunicata) and the Cephalochordata. The Chordata, together with the phyla Echinodermata and Hemichordata, comprise a major group, the Deuterostomia. Chordates invariably possess a notochord and a dorsal neural tube. Although the origin and evolution of chordates has been studied for more than a century, few authors have intimately discussed taxonomic ranking of the three chordate groups themselves. Accumulating evidence shows that echinoderms and hemichordates form a clade (the Ambulacraria), and that within the Chordata, cephalochordates diverged first, with tunicates and vertebrates forming a sister group. Chordates share tadpole-type larvae containing a notochord and hollow nerve cord, whereas ambulacrarians have dipleurula-type larvae containing a hydrocoel. We propose that an evolutionary occurrence of tadpole-type larvae is fundamental to understanding mechanisms of chordate origin. Protostomes have now been reclassified into two major taxa, the Ecdysozoa and Lophotrochozoa, whose developmental pathways are characterized by ecdysis and trochophore larvae, respectively. Consistent with this classification, the profound dipleurula versus tadpole larval differences merit a category higher than the phylum. Thus, it is recommended that the Ecdysozoa, Lophotrochozoa, Ambulacraria and Chordata be classified at the superphylum level, with the Chordata further subdivided into three phyla, on the basis of their distinctive characteristics.

Factors relevant to pre‐veliger nutrition ofTridacnidae giant clams

AbstractTridacnid giant clams are of commercial interest for their nutritional, economic, and ecological significance throughout theIndo‐Pacific andOceania. A major impediment to culturing tridacnid giant clams is larval mortality, typically &gt;96%. The effects of suspended and particulate nutrients on tridacnid veliger development have been examined, but factors relevant to the nutritional condition of oocytes and trochophore larvae are unknown. This is significant because the nutritional transition from trochophore to veliger coincides with rises in larval mortality. Our review examines culture techniques known to enhance gamete viability of several marine viable bivalves of commercial interest, and critically assesses whether such methods are applicable to tridacnids in culture conditions. Techniques for optimizing the nutritional requirements of pre‐veliger tridacnids such as broodstock conditioning by supplemental feeding, monitoring the condition of oocytes ready for release, water temperature, egg attributes such as size and biochemical composition, and nutrient loads in culture water chemistry are examined. We include suggestions for optimizing nutritional conditions from gametogenesis through embryonic development, and conclude with a primer of research opportunities based on gaps in our understanding of tridacnid pre‐veliger nutrition compared with other bivalves. A major conclusion is that culture protocols should include pre‐veliger nutritional fitness as a critical factor in enhancing larval survival, growth and development.

A Scallop IGF Binding Protein Gene: Molecular Characterization and Association of Variants with Growth Traits

BackgroundScallops represent economically important aquaculture shellfish. The identification of genes and genetic variants related to scallop growth could benefit high-yielding scallop breeding. The insulin-like growth factor (IGF) system is essential for growth and development, with IGF binding proteins (IGFBPs) serving as the major regulators of IGF actions. Although an effect of IGF on growth was detected in bivalve, IGFBP has not been reported, and members of the IGF system have not been characterized in scallop.ResultsWe cloned and characterized an IGFBP (PyIGFBP) gene from the aquaculture bivalve species, Yesso scallop (Patinopecten yessoensis, Jay, 1857). Its full-length cDNA sequence was 1,445 bp, with an open reading frame of 378 bp, encoding 125 amino acids, and its genomic sequence was 10,193 bp, consisting of three exons and two introns. The amino acid sequence exhibited the characteristics of IGFBPs, including multiple cysteine residues and relatively conserved motifs in the N-terminal and C-terminal domains. Expression analysis indicated that PyIGFBP was expressed in all the tissues and developmental stages examined, with a significantly higher level in the mantle than in other tissues and a significantly higher level in gastrulae and trochophore larvae than in other stages. Furthermore, three single nucleotide polymorphisms (SNPs) were identified in this gene. SNP c.1054A>G was significantly associated with both shell and soft body traits in two populations, with the highest trait values in GG type scallops and lowest in AG type ones.ConclusionWe cloned and characterized an IGFBP gene in a bivalve, and this report also represents the first characterizing an IGF system gene in scallops. A SNP associated with scallop growth for both the shell and soft body was identified in this gene. In addition to providing a candidate marker for scallop breeding, our results also suggest the role of PyIGFBP in scallop growth.

Swimming behavior of the spoon worm Urechis unicinctus (Annelida, Echiura)

Large numbers of swimming and stranding Urechis unicinctus were observed at night during low tide in Sasuhama, Miyagi Prefecture, northeastern Japan, during the periods from January to February in 2012 and 2013. Worms did not drift passively but swam actively, therefore hinting at a certain purpose for such behavior. As trochophore larvae of U. unicinctus were observed to occur simultaneously in the plankton, we infer the possibility that this is an event of reproductive swarming. Anatomical observations of both swimming and stranding U. unicinctus showed that none of the specimens had gametes, which may suggest that these were completely spent after spawning. Urechis unicinctus seemed to begin swimming after dusk and the observed swimming behavior occurred during the evening ebb tide throughout the night low tide during winter time. Stranding U. unicinctus have long been known in Japan and have been attributed to sea storms. The present study shows for the first time the possibility that U. unicinctus swims in order to reproduce at night and that this swimming behavior is closely linked to the stranding of U. unicinctus individuals.

Larval body patterning and apical organs are conserved in animal evolution

BackgroundPlanktonic ciliated larvae are characteristic for the life cycle of marine invertebrates. Their most prominent feature is the apical organ harboring sensory cells and neurons of largely undetermined function. An elucidation of the relationships between various forms of primary larvae and apical organs is key to understanding the evolution of animal life cycles. These relationships have remained enigmatic due to the scarcity of comparative molecular data.ResultsTo compare apical organs and larval body patterning, we have studied regionalization of the episphere, the upper hemisphere of the trochophore larva of the marine annelid Platynereis dumerilii. We examined the spatial distribution of transcription factors and of Wnt signaling components previously implicated in anterior neural development. Pharmacological activation of Wnt signaling with Gsk3β antagonists abolishes expression of apical markers, consistent with a repressive role of Wnt signaling in the specification of apical tissue. We refer to this Wnt-sensitive, six3- and foxq2-expressing part of the episphere as the ‘apical plate’. We also unraveled a molecular signature of the apical organ - devoid of six3 but expressing foxj, irx, nkx3 and hox - that is shared with other marine phyla including cnidarians. Finally, we characterized the cell types that form part of the apical organ by profiling by image registration, which allows parallel expression profiling of multiple cells. Besides the hox-expressing apical tuft cells, this revealed the presence of putative light- and mechanosensory as well as multiple peptidergic cell types that we compared to apical organ cell types of other animal phyla.ConclusionsThe similar formation of a six3+, foxq2+ apical plate, sensitive to Wnt activity and with an apical tuft in its six3-free center, is most parsimoniously explained by evolutionary conservation. We propose that a simple apical organ - comprising an apical tuft and a basal plexus innervated by sensory-neurosecretory apical plate cells - was present in the last common ancestors of cnidarians and bilaterians. One of its ancient functions would have been the control of metamorphosis. Various types of apical plate cells would then have subsequently been added to the apical organ in the divergent bilaterian lineages. Our findings support an ancient and common origin of primary ciliated larvae.