The combination of high temperature and Vibrio infection worsens summer mortality in the clam Meretrix petechialis by increasing apoptosis and oxidative stress.

The interaction between environmental factors and Vibrio in bivalves is not well understood, despite the widely held belief that pathogen infection and seawater temperature significantly impact summer mortality. In the present study, we conducted simulated experiments to explore the effects of high temperature and Vibrio infection on the clam Meretrix petechialis. The survival curve analysis revealed that the combined challenge of high temperature and Vibrio infection (31°C-vibrio) led to significantly higher clam mortality compared to the groups exposed solely to Vibrio (27°C-vibrio), high temperature (31°C-control), and the control condition (27°C-control). Furthermore, PCoA analysis of 11 immune genes indicated that Vibrio infection predominated during the incubation period, with a gradual equilibrium between these factors emerging during the course of the infection. Additionally, our investigations into apoptosis and autophagy processes exhibited significant induction of mTOR and Bcl2 of the 31°C-vibrio group in the early challenge stage, followed by inhibition in the later stage. Oxidative stress analysis demonstrated a substantial additive effect on malondialdehyde (MDA) and glutathione (GSH) content in the combined challenge group compared to the control group. Comparative transcriptome analysis revealed a significant increase in differentially expressed genes related to immunity, such as complement C1q-like protein, C-type lectin, big defensin, and lysozyme, in the 31°C-vibrio group, suggesting that the synergistic effect of high temperature and Vibrio infection triggers more robust antibacterial immune responses. These findings provide critical insights for understanding the infection process and uncovering the causes of summer mortality.

Comprehensive Multi-omics Approaches Provide Insights to Summer Mortality in the Clam Meretrix petechialis.

Bivalve mass mortalities have been reported worldwide, which not only can be explained as a result of pathogen infection, but may reflect changes in environments. Although these episodes were often reported, there was limited information concerning the molecular responses to various stressors leading to summer mortality. In the present work, RNA sequencing (RNA-seq), tandem mass tagging (TMT)-based quantitative proteomics, and 16S rRNA sequencing were used to explore the natural outbreak of summer mortality in the clam Meretrix petechialis. We identified a total of 172 differentially expressed genes (DEGs) and 222 differentially expressed proteins (DEPs) in the diseased group compared to the normal group. The inconsistent expression profiles of immune DEGs/DEPs may be due to the immune dysregulation of the diseased clams. Notably, 11 solute carrier family genes were found among the top 20 down-regulated genes in the diseased group, indicating that weakened transmembrane transport ability might occur in the diseased clams. Integration analysis of transcriptomic and proteomic results showed that many metabolic processes such as "arginine and proline metabolism" and "tyrosine metabolism" were inhibited in the diseased group, suggesting metabolic inhibition. Moreover, 16S rRNA sequencing revealed that the microbial composition of clam hepatopancreas was disordered in the diseased group. The comparison of DEGs expression between the natural summer mortality event and an artificial challenge experiment involving both Vibrio infection and heat stress revealed 9/15 genes showing similar expression trends between the two conditions, suggesting that the summer mortality might be caused by a combination of high temperature and Vibrio infection. These results would deepen our understanding of summer mortality and provide candidate resistance markers for clam resistance breeding.

Functional evidence that FGFR regulates MAPK signaling in organizer specification in the gastropod mollusk Lottia peitaihoensis.

The D-quadrant organizer sets up the dorsal-ventral (DV) axis and regulates mesodermal development of spiralians. Studies have revealed an important role of mitogen-activated protein kinase (MAPK) signaling in organizer function, but the related molecules have not been fully revealed. The association between fibroblast growth factor receptor (FGFR) and MAPK signaling in regulating organizer specification has been established in the annelid Owenia fusiformis. Now, comparable studies in other spiralian phyla are required to decipher whether this organizer-inducing function of FGFR is prevalent in Spiralia. Here, we indicate that treatment with the FGFR inhibitor SU5402 resulted in deficiency of organizer specification in the mollusk Lottia peitaihoensis. Subsequently, the bone morphogenetic protein (BMP) signaling gradient and DV patterning were disrupted, suggesting the roles of FGFR in regulating organizer function. Changes in multiple aspects of organizer function (the morphology of vegetal blastomeres, BMP signaling gradient, expression of DV patterning markers, etc.) indicate that these developmental functions have different sensitivities to FGFR/MAPK signaling. Our results reveal a functional role of FGFR in organizer specification as well as DV patterning of Lottia embryos, which expands our knowledge of spiralian organizers. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00194-x.

The gastropod Lottia peitaihoensis as a model to study the body patterning of trochophore larvae.

The body patterning of trochophore larvae is important for understanding spiralian evolution and the origin of the bilateral body plan. However, considerable variations are observed among spiralian lineages, which have adopted varied strategies to develop trochophore larvae or even omit a trochophore stage. Some spiralians, such as patellogastropod mollusks, are suggested to exhibit ancestral traits by producing equal-cleaving fertilized eggs and possessing "typical" trochophore larvae. In recent years, we developed a potential model system using the patellogastropod Lottia peitaihoensis (= Lottia goshimai). Here, we introduce how the species were selected and establish sources and techniques, including gene knockdown, ectopic gene expression, and genome editing. Investigations on this species reveal essential aspects of trochophore body patterning, including organizer signaling, molecular and cellular processes connecting the various developmental functions of the organizer, the specification and behaviors of the endomesoderm and ectomesoderm, and the characteristic dorsoventral decoupling of Hox expression. These findings enrich the knowledge of trochophore body patterning and have important implications regarding the evolution of spiralians as well as bilateral body plans.

Shell field morphogenesis in the polyplacophoran mollusk Acanthochitona rubrolineata.

BACKGROUND: The polyplacophoran mollusks (chitons) possess serially arranged shell plates. This feature is unique among mollusks and believed to be essential to explore the evolution of mollusks as well as their shells. Previous studies revealed several cell populations in the dorsal epithelium (shell field) of polyplacophoran larvae and their roles in the formation of shell plates. Nevertheless, they provide limited molecular information, and shell field morphogenesis remains largely uninvestigated. RESULTS: In the present study, we investigated shell field development in the chiton Acanthochitona rubrolineata based on morphological characteristics and molecular patterns. A total of four types of tissue could be recognized from the shell field of A. rubrolineata. The shell field comprised not only the centrally located, alternatively arranged plate fields and ridges, but also the tissues surrounding them, which were the precursors of the girdle and we termed as the girdle field. The girdle field exhibited a concentric organization composed of two circularly arranged tissues, and spicules were only developed in the outer circle. Dynamic engrailed expression and F-actin (filamentous actin) distributions revealed relatively complicated morphogenesis of the shell field. The repeated units (plate fields and ridges) were gradually established in the shell field, seemingly different from the manners used in the segmentation of Drosophila or vertebrates. The seven repeated ridges also experienced different modes of ontogenesis from each other. In the girdle field, the presumptive spicule-formation cells exhibited different patterns of F-actin aggregations as they differentiate. CONCLUSIONS: These results reveal the details concerning the structure of polyplacophoran shell field as well as its morphogenesis. They would contribute to exploring the mechanisms of polyplacophoran shell development and molluscan shell evolution.

Early mesodermal development in the patellogastropod Lottia goshimai.

Mesodermal development is essential to explore the interlineage variations in the development of spiralians. Compared with model mollusks such as Tritia and Crepidula, knowledge about the mesodermal development of other molluscan lineages is limited. Here, we investigated early mesodermal development in the patellogastropod Lottia goshimai, which shows equal cleavage and has a trochophore larva. The endomesoderm derived from the 4d blastomere, that is, the mesodermal bandlets, was situated dorsally and showed a characteristic morphology. Investigations of the potential mesodermal patterning genes revealed that twist1 and snail1 were expressed in a proportion of these endomesodermal tissues, while all of the five genes we investigated (twist1, twist2, snail1, snail2, and mox) were expressed in ventrally located ectomesodermal tissues. Relatively dynamic snail2 expression suggests additional roles in various internalization processes. By tracing snail2 expression in early gastrulae, the 3a211 and 3b211 blastomeres were suggested to be the precursors of the ectomesoderm, which elongated to become internalized before division. These results help to understand the variations in the mesodermal development of different spiralians and explore the different mechanisms by which ectomesodermal cells are internalized, which has important evolutionary implications.

Nonmuscle Myosin II is Required for Larval Shell Formation in a Patellogastropod.

The molecular mechanisms underlying larval shell development in mollusks remain largely elusive. We previously found evident filamentous actin (F-actin) aggregations in the developing shell field of the patellogastropod Lottia goshimai, indicating roles of actomyosin networks in the process. In the present study, we functionally characterized nonmuscle myosin II (NM II), the key molecule in actomyosin networks, in the larval shell development of L. goshimai. Immunostaining revealed general colocalization of phosphorylated NM II and F-actin in the shell field. When inhibiting the phosphorylation of NM II using the specific inhibitor blebbistatin in one- or 2-h periods during shell field morphogenesis (6-8 h post-fertilization, hpf), the larval shell plate was completely lost in the veliger larva (24 hpf). Scanning electron microscopy revealed that the nascent larval shell plate could not be developed in the manipulated larvae (10 hpf). Further investigations revealed that key events in shell field morphogenesis were inhibited by blebbistatin pulses, including invagination of the shell field and cell shape changes and cell rearrangements during shell field morphogenesis. These factors caused the changed morphology of the shell field, despite the roughly retained "rosette" organization. To explore whether the specification of related cells was affected by blebbistatin treatments, we investigated the expression of four potential shell formation genes (bmp2/4, gata2/3, hox1 and engrailed). The four genes did not show evident changes in expression level, indicating unaffected cell specification in the shell field, while the gene expression patterns showed variations according to the altered morphology of the shell field. Together, our results reveal that NM II contributes to the morphogenesis of the shell field and is crucial for the formation of the larval shell plate in L. goshimai. These results add to the knowledge of the mechanisms of molluskan shell development.

Molluskan Dorsal-Ventral Patterning Relying on BMP2/4 and Chordin Provides Insights into Spiralian Development and Evolution.

Although a conserved mechanism relying on BMP2/4 and Chordin is suggested for animal dorsal-ventral (DV) patterning, this mechanism has not been reported in spiralians, one of the three major clades of bilaterians. Studies on limited spiralian representatives have suggested markedly diverse DV patterning mechanisms, a considerable number of which no longer deploy BMP signaling. Here, we showed that BMP2/4 and Chordin regulate DV patterning in the mollusk Lottia goshimai, which was predicted in spiralians but not previously reported. In the context of the diverse reports in spiralians, it conversely represents a relatively unusual case. We showed that BMP2/4 and Chordin coordinate to mediate signaling from the D-quadrant organizer to induce the DV axis, and Chordin relays the symmetry-breaking information from the organizer. Further investigations on L. goshimai embryos with impaired DV patterning suggested roles of BMP signaling in regulating the behavior of the blastopore and the organization of the nervous system. These findings provide insights into the evolution of animal DV patterning and the unique development mode of spiralians driven by the D-quadrant organizer.

CRISPR/Cas9-mediated mutagenesis reveals the roles of calaxin in gastropod larval cilia.

Obtaining detectable knockout phenotypes in the G0 generation is essential for gene function studies. Although CRISPR/Cas9-mediated gene editing has been employed to knock out molluscan genes, detectable phenotypes in the G0 generation have not been reported in these animals. In this study, we determined the knockout phenotype of a cilium-related gene, calaxin, using CRISPR/Cas9 technology in the gastropod mollusk Lottia goshimai. Injections with the Cas9-sgRNA complex caused approximately 30-80% of the injected larvae to exhibit a short-cilia phenotype characteristic of shortened cilia and decreased motility in the larvae. This phenotype was detectable in the G0 generation and was consistent for two independent sgRNAs. Genotyping of the injected larvae revealed various types of deletions and insertions in the target gene, which occurred in all sequences from the short-cilia larvae. This result indicated that the short-cilia phenotype was indeed caused by calaxin knockout. This possibility was supported by an RNAi assay targeting calaxin, which produced a highly similar short-cilia phenotype. We observed that a single SNP in the target sequences of the sgRNAs could show varied effects on the efficiency of mutagenesis. These results help to establish a foundation for future studies on molluscan gene editing using the CRISPR/Cas9 technique and contribute to the body of knowledge on molluscan ciliary functions.

Early shell field morphogenesis of a patellogastropod mollusk predominantly relies on cell movement and F-actin dynamics.

BACKGROUND: The morphogenesis of the shell field is an essential step of molluscan shell formation, which exhibits both conserved features and interlineage variations. As one major gastropod lineage, the patellogastropods show different characters in its shell field morphogenesis compared to other gastropods (e.g., the pulmonate gastropod Lymnaea stagnalis), likely related to its epibolic gastrulation. The investigation on the shell field morphogenesis of patellogastropods would be useful to reveal the lineage-specific characters in the process and explore the deep conservation among different molluscan lineages. RESULTS: We investigated the early shell field morphogenesis in the patellogastropod Lottia goshimai using multiple techniques. Electron microscopy revealed distinct morphological characters for the central and peripheral cells of the characteristic rosette-like shell field. Gene expression analysis and F-actin staining suggested that the shell field morphogenesis in this species predominantly relied on cell movement and F-actin dynamics, while BrdU assay revealed that cell proliferation contributed little to the process. We found constant contacts between ectodermal and meso/endodermal tissues during the early stages of shell field morphogenesis, which did not support the induction of shell field by endodermal tissues in general, but a potential stage-specific induction was indicated. CONCLUSIONS: Our results emphasize the roles of cell movement and F-actin dynamics during the morphogenesis of the shell field in Lo. goshimai, and suggest potential regulators such as diffusible factors and F-actin modulators. These findings reflect the differences in shell field morphogenesis of different gastropods, and add to the knowledge of molluscan larval shell formation.

Identification of three cell populations from the shell gland of a bivalve mollusc.

The molluscan larval shell formation is a complicated process. There is evidence that the mantle of the primary larva (trochophore) contains functionally different cell populations with distinct gene expression profiles. However, it remains unclear how these cells are specified. In the present study, we identified three cell populations from the shell gland in earlier stages (gastrula) from the bivalve mollusc Crassostrea gigas. These cell populations were determined by analyzing the co-expression relationships among six potential shell formation (pSF) genes using two-color hybridization. The three cell populations, which we designated as SGCPs (shell gland cell populations), formed a concentric-circle pattern from outside to inside of the shell gland. SGCP I was located in the outer edge of the shell gland and the cells expressed pax2/5/8, gata2/3, and bmp2/4. SGCP II was located more internally and the cells expressed two engrailed genes. The last population, SGCP III, was located in the central region of the shell gland and the cells expressed lox4. Determination of the gene expression profiles of SGCPs would help trace their origins and fates and elucidate how these cell populations are specified. Moreover, potential roles of the SGCPs, e.g., development of sensory cells and shell biogenesis, are suggested. Our results reveal the internal organization of the embryonic shell gland at the molecular level and add to the knowledge of larval shell formation.

Dorsoventral decoupling of Hox gene expression underpins the diversification of molluscs.

In contrast to the Hox genes in arthropods and vertebrates, those in molluscs show diverse expression patterns with differences reported among lineages. Here, we investigate 2 phylogenetically distant molluscs, a gastropod and a polyplacophoran, and show that the Hox expression in both species can be divided into 2 categories. The Hox expression in the ventral ectoderm generally shows a canonical staggered pattern comparable to the patterns of other bilaterians and likely contributes to ventral patterning, such as neurogenesis. The other category of Hox expression on the dorsal side is strongly correlated with shell formation and exhibits lineage-specific characteristics in each class of mollusc. This generalized model of decoupled dorsoventral Hox expression is compatible with known Hox expression data from other molluscan lineages and may represent a key characteristic of molluscan Hox expression. These results support the concept of widespread staggered Hox expression in Mollusca and reveal aspects that may be related to the evolutionary diversification of molluscs. We propose that dorsoventral decoupling of Hox expression allowed lineage-specific dorsal and ventral patterning, which may have facilitated the evolution of diverse body plans in different molluscan lineages.

An investigation of oyster TGF-ß receptor genes and their potential roles in early molluscan development.

Though the roles of BMP signaling in development is studied extensively in insects and vertebrates, our knowledge of BMP signaling in molluscan development is limited. In the present study, we performed a genome-based analysis of TGF-ß receptors in the Pacific oyster Crassostrea gigas and revealed that C. gigas possessed all five canonical members of the gene family, including three type I and two type II receptors. Whole mount in situ hybridization revealed that four receptor genes exhibited universal expression at the gastrula stage but cgi-bmprII mRNA was only expressed at the anterior lip of blastopore, indicating the regulation of BMP signaling through restricted expression of TGF-ß receptors. Treatment of oyster embryos using a BMP signaling inhibitor (dorsomorphin) resulted in obvious changes of the expression of developmental regulatory genes. In particular, three dorsally expressed genes were inhibited and two genes expressed at the opposite side showed increased expression, indicating BMP signaling may function in dorsal-ventral (DV) patterning. Some of the influenced genes were potential shell-formation (pSF) genes, including the well-accepted pSF gene engrailed, and thus this indicates the development of shell field was affected by dorsomorphin treatment. Subsequent scanning electronic observation revealed morphological change of the posterior margin of the shell field. Taken together, these results indicate the roles of BMP signaling in both DV patterning and development of shell field in oyster embryo, which expands our knowledge of early molluscan development.

Integrated transcriptomic and proteomic analyses reveal potential mechanisms linking thermal stress and depressed disease resistance in the turbot Scophthalmus maximus.

A worldwide increase in the reports of diseases affecting marine organisms has paralleled the climate warming over the past few decades. In this study, we applied omics to explore the mechanisms underlying thermo-linked epizootics, by comparing both the transcriptome- and proteome-wide response of turbots to a mimic pathogen (poly I:C) between high temperature and low temperature using a time-course approach. Our results showed that myeloperoxidase (MPO) and insulin were differentially expressed transcripts shared by all five time-points post poly I:C-injection between high and low temperature and also had a consistent expression trend as differentially expressed proteins at 24 h post injection. Combined with other data, it was suggested that the elevated temperature enhanced neutrophil-mediated immunity and the resultant MPO-mediated oxidative stress, which lasted for at least 5 days. The contents of malondialdehyde and protein carbonyls, markers of oxidative damage for lipids and proteins, respectively, were compared between different temperature groups, and the results further implied the emergence of oxidative damage under high temperature. It was also suggested that metabolism disorder likely occur considering the sustained expression changes of insulin. Hence, prolonged MPO-mediated oxidative stress and metabolic disorder might be involved in the thermo-linked epizootic.

The sea cucumber genome provides insights into morphological evolution and visceral regeneration.

Apart from sharing common ancestry with chordates, sea cucumbers exhibit a unique morphology and exceptional regenerative capacity. Here we present the complete genome sequence of an economically important sea cucumber, A. japonicus, generated using Illumina and PacBio platforms, to achieve an assembly of approximately 805 Mb (contig N50 of 190 Kb and scaffold N50 of 486 Kb), with 30,350 protein-coding genes and high continuity. We used this resource to explore key genetic mechanisms behind the unique biological characters of sea cucumbers. Phylogenetic and comparative genomic analyses revealed the presence of marker genes associated with notochord and gill slits, suggesting that these chordate features were present in ancestral echinoderms. The unique shape and weak mineralization of the sea cucumber adult body were also preliminarily explained by the contraction of biomineralization genes. Genome, transcriptome, and proteome analyses of organ regrowth after induced evisceration provided insight into the molecular underpinnings of visceral regeneration, including a specific tandem-duplicated prostatic secretory protein of 94 amino acids (PSP94)-like gene family and a significantly expanded fibrinogen-related protein (FREP) gene family. This high-quality genome resource will provide a useful framework for future research into biological processes and evolution in deuterostomes, including remarkable regenerative abilities that could have medical applications. Moreover, the multiomics data will be of prime value for commercial sea cucumber breeding programs.

Scallop genome provides insights into evolution of bilaterian karyotype and development.

Reconstructing the genomes of bilaterian ancestors is central to our understanding of animal evolution, where knowledge from ancient and/or slow-evolving bilaterian lineages is critical. Here we report a high-quality, chromosome-anchored reference genome for the scallop Patinopecten yessoensis, a bivalve mollusc that has a slow-evolving genome with many ancestral features. Chromosome-based macrosynteny analysis reveals a striking correspondence between the 19 scallop chromosomes and the 17 presumed ancestral bilaterian linkage groups at a level of conservation previously unseen, suggesting that the scallop may have a karyotype close to that of the bilaterian ancestor. Scallop Hox gene expression follows a new mode of subcluster temporal co-linearity that is possibly ancestral and may provide great potential in supporting diverse bilaterian body plans. Transcriptome analysis of scallop mantle eyes finds unexpected diversity in phototransduction cascades and a potentially ancient Pax2/5/8-dependent pathway for noncephalic eyes. The outstanding preservation of ancestral karyotype and developmental control makes the scallop genome a valuable resource for understanding early bilaterian evolution and biology.

A SoxC gene related to larval shell development and co-expression analysis of different shell formation genes in early larvae of oyster.

Among the potential larval shell formation genes in mollusks, most are expressed in cells surrounding the shell field during the early phase of shell formation. The only exception (cgi-tyr1) is expressed in the whole larval mantle and thus represents a novel type of expression pattern. This study reports another gene with such an expression pattern. The gene encoded a SoxC homolog of the Pacific oyster Crassostrea gigas and was named cgi-soxc. Whole-mount in situ hybridization revealed that the gene was highly expressed in the whole larval mantle of early larvae. Based on its spatiotemporal expression, cgi-soxc is hypothesized to be involved in periostracum biogenesis, biomineralization, and regulation of cell proliferation. Furthermore, we investigated the interrelationship between cgi-soxc expression and two additional potential shell formation genes, cgi-tyr1 and cgi-gata2/3. The results confirmed co-expression of the three genes in the larval mantle of early D-veliger. Nevertheless, cgi-gata2/3 was only expressed in the mantle edge, and the other two genes were expressed in all mantle cells. Based on the spatial expression patterns of the three genes, two cell groups were identified from the larval mantle (tyr1 +/soxc +/gata2/3 + cells and tyr1 +/soxc +/gata2/3 - cells) and are important to study the differentiation and function of this tissue. The results of this study enrich our knowledge on the structure and function of larval mantle and provide important information to understand the molecular mechanisms of larval shell formation.

Expression patterns indicate that BMP2/4 and Chordin, not BMP5-8 and Gremlin, mediate dorsal-ventral patterning in the mollusk Crassostrea gigas.

Though several bilaterian animals use a conserved BMP2/4-Chordin antagonism to pattern the dorsal-ventral (DV) axis, the only lophotrochozoan species in which early DV patterning has been studied to date, the leech Helobdella robusta, appears to employ BMP5-8 and Gremlin. These findings call into question the conservation of a common DV patterning mechanism among bilaterian animals. To explore whether the unusual DV patterning mechanism in H. robusta is also used in other lophotrochozoan species, we investigated the expression of orthologous genes in the early embryo of a bivalve mollusk, Crassostrea gigas. Searching of the genome and phylogenetic analysis revealed that C. gigas possesses single orthologs of BMP2/4, Chordin, and BMP5-8 and no Gremlin homolog. Whole mount in situ hybridization revealed mRNA localization of BMP2/4 and Chordin on the opposite sides of embryos, suggesting the potential involvement of a BMP2/4-Chordin antagonism in DV patterning in this species. Furthermore, universal BMP5-8 expression and the absence of a Gremlin homolog in the C. gigas genome called into question any major contribution by BMP5-8 and Gremlin to early DV patterning in this species. Additionally, we identified seven genes showing asymmetric expression along the DV axis, providing further insight into DV patterning in C. gigas. We present the first report of a Chordin gene in a lophotrochozoan species and of the opposite expression of BMP2/4 (dorsal) and Chordin (ventral) along the D/V axis of a lophotrochozoan embryo. The findings of this study further the knowledge of axis formation in lophotrochozoan species and provide insight into the evolution of the animal DV patterning mechanism.

Assessment of housekeeping genes as internal references in quantitative expression analysis during early development of oyster.

The early development of mollusks exhibits important characteristics from the developmental and evolutionary perspective. With the increasing number of genome-wide studies, accurate analyses of quantitative gene expression during development are impeded by the lack of validated reference genes. To improve the situation, in this study, we analyzed the expression stability of seven candidate housekeeping genes during early development of the Pacific oyster Crassostrea gigas: actin, glyceraldehyde-3-phosphate dehydrogenase (gapdh), α subunit of elongation factor 1 (elf1α), adp-ribosylation factor 1 (arf1), heterogeneous nuclear ribonucleoprotein q, ubiquitin-conjugating enzyme e2d2 and ribosomal protein s18. We focused on 11 stages from oocyte to D-veliger, which include crucial developmental processes such as axis determination, gastrulation and shell formation. Gene expression stabilities were assessed with the three commonly used programs geNorm, NormFinder and BestKeeper. Although the results obtained with the three programs varied to some extent, in general, arf1, elf1α and gapdh were highly ranked and actin was poorly ranked. This analysis also indicated that multiple genes should be used for normalization, and we concluded that arf1-elf1α-gapdh should be used as internal references. The findings of this study will help researchers to obtain accurate results in future quantitative gene expression analysis of development in bivalve mollusks.

A comparative proteomic analysis reveals important proteins for the fertilization and early embryonic development of the oyster Crassostrea gigas.

Molluscan development involves important features that are important to understanding not only molluscan ontogeny but also animal evolution. To gain insight into the gamete proteome and protein function in fertilization and early development, we analyzed the proteomes of unfertilized oocytes and early embryos (2/4-cell stage) of the Pacific oyster, Crassostrea gigas. An oocyte reference map containing 116 protein spots, of which 69 were identified, revealed a high abundance of vitellogenin-derived protein spots. The differentially regulated protein spots during fertilization were screened using comparative proteomic approaches. In total, 18 differentially regulated protein spots were screened, and 15 of these were identified and divided into three groups. The proteins belonging to the first group function in energy supply and antioxidation and are proposed to ensure successful fertilization by regulating the levels of adenosine triphosphate, resisting oxidative stress, and preventing polyspermy. The proteins of the second group are associated with protein synthesis and modification, reflecting active protein synthesis after fertilization. The three proteins belonging to the final group are hypothesized to function in the regulation of embryonic development through the establishment of cell polarity and modulation of methylation reactions in nuclei. These results will enhance our knowledge of molluscan fertilization and development.