A dynein-associated photoreceptor protein prevents ciliary acclimation to blue light.

Light-responsive regulation of ciliary motility is known to be conducted through modulation of dyneins, but the mechanism is not fully understood. Here, we report a novel subunit of the two-headed f/I1 inner arm dynein, named DYBLUP, in animal spermatozoa and a unicellular green alga. This subunit contains a BLUF (sensors of blue light using FAD) domain that appears to directly modulate dynein activity in response to light. DYBLUP (dynein-associated BLUF protein) mediates the connection between the f/I1 motor domain and the tether complex that links the motor to the doublet microtubule. Chlamydomonas lacking the DYBLUP ortholog shows both positive and negative phototaxis but becomes acclimated and attracted to high-intensity blue light. These results suggest a mechanism to avoid toxic strong light via direct photoregulation of dyneins.

Phylogenetic comparisons reveal mosaic histories of larval and adult shell matrix protein deployment in pteriomorph bivalves.

Molluscan shells are organo-mineral composites, in which the dominant calcium carbonate is intimately associated with an organic matrix comprised mainly of proteins and polysaccharides. However, whether the various shell matrix proteins (SMPs) date to the origin of hard skeletons in the Cambrian, or whether they represent later deployment through adaptive evolution, is still debated. In order to address this issue and to better understand the origins and evolution of biomineralization, phylogenetic analyses have been performed on the three SMP families, Von Willebrand factor type A (VWA) and chitin-binding domain-containing protein (VWA-CB dcp), chitobiase, and carbonic anhydrase (CA), which exist in both larval and adult shell proteomes in the bivalves, Crassostrea gigas and Pinctada fucata. In VWA-CB dcp and chitobiase, paralogs for larval and adult SMPs evolved before the divergence of these species. CA-SMPs have been taken as evidence for ancient origins of SMPs by their presumed indispensable function in biomineralization and ubiquitous distribution in molluscs. However, our results indicate gene duplications that gave rise to separate deployments as larval and adult CA-SMPs occurred independently in each lineage after their divergence, which is considerably more recent than hitherto assumed, supporting the "recent heritage and fast evolution" scenario for SMP evolution.

Three multi-allelic gene pairs are responsible for self-sterility in the ascidian Ciona intestinalis.

Many hermaphroditic organisms possess a self-incompatibility system to avoid inbreeding. Although the mechanisms of self-incompatibility in flowering plants are well known, little is known about the mechanisms of self-sterility in hermaphroditic marine invertebrates. Ascidians are hermaphroditic sessile marine invertebrates that release sperm and eggs into the surrounding seawater. Several species, including Ciona intestinalis type A (Ciona robusta), exhibit strict self-sterility. In a previous study, we found that the candidate genes responsible for self-sterility in Ciona reside in chromosome 2q (locus A) and chromosome 7q (locus B). Two pairs of multi-allelic genes, named s(sperm)-Themis-A and v(vitelline-coat)-Themis-A in locus A and s-Themis-B and v-Themis-B in locus B, are responsible for self-sterility. In this study, we identified a third multi-allelic gene pair, s-Themis-B2 and v-Themis-B2, within locus B that is also involved in this system. Genetic analysis revealed that the haplotypes of s/v-Themis-A, s/v-Themis-B and s/v-Themis-B2 play essential roles in self-sterility. When three haplotypes were matched between s-Themis and v-Themis, fertilization never occurred even in nonself crossing. Interestingly, gene targeting of either s/v-Themis-B/B2 or s/v-Themis-A by genome editing enabled self-fertilization. These results indicate that s/v-Themis-A, -B and -B2 are S-determinant genes responsible for self-sterility in the ascidian C. intestinalis type A.

The male gamete membrane protein DMP9/DAU2 is required for double fertilization in flowering plants.

All flowering plants exhibit a unique type of sexual reproduction called 'double fertilization' in which each pollen tube-delivered sperm cell fuses with an egg and a central cell. Proteins that localize to the plasma membrane of gametes regulate one-to-one gamete pairing and fusion between male and female gametes for successful double fertilization. Here, we have identified a membrane protein from Lilium longiflorum generative cells using proteomic analysis and have found that the protein is an ortholog of Arabidopsis DUF679 DOMAIN MEMBRANE PROTEIN 9 (DMP9)/DUO1-ACTIVATED UNKNOWN 2 (DAU2). The flowering plant DMP9 proteins analyzed in this study were predicted to have four transmembrane domains and be specifically expressed in both generative and sperm cells. Knockdown of DMP9 resulted in aborted seeds due to single fertilization of the central cell. Detailed imaging of DMP9-knockdown sperm cells during in vivo and semi-in vitro double fertilization revealed that DMP9 is involved in gamete interaction that leads to correct double fertilization.

Dual Gene Repertoires for Larval and Adult Shells Reveal Molecules Essential for Molluscan Shell Formation.

Molluscan shells, mainly composed of calcium carbonate, also contain organic components such as proteins and polysaccharides. Shell organic matrices construct frameworks of shell structures and regulate crystallization processes during shell formation. To date, a number of shell matrix proteins (SMPs) have been identified, and their functions in shell formation have been studied. However, previous studies focused only on SMPs extracted from adult shells, secreted after metamorphosis. Using proteomic analyses combined with genomic and transcriptomic analyses, we have identified 31 SMPs from larval shells of the pearl oyster, Pinctada fucata, and 111 from the Pacific oyster, Crassostrea gigas. Larval SMPs are almost entirely different from those of adults in both species. RNA-seq data also confirm that gene expression profiles for larval and adult shell formation are nearly completely different. Therefore, bivalves have two repertoires of SMP genes to construct larval and adult shells. Despite considerable differences in larval and adult SMPs, some functional domains are shared by both SMP repertoires. Conserved domains include von Willebrand factor type A (VWA), chitin-binding (CB), carbonic anhydrase (CA), and acidic domains. These conserved domains are thought to play crucial roles in shell formation. Furthermore, a comprehensive survey of animal genomes revealed that the CA and VWA-CB domain-containing protein families expanded in molluscs after their separation from other Lophotrochozoan linages such as the Brachiopoda. After gene expansion, some family members were co-opted for molluscan SMPs that may have triggered to develop mineralized shells from ancestral, nonmineralized chitinous exoskeletons.

Chitin-based barrier immunity and its loss predated mucus-colonization by indigenous gut microbiota.

Mammalian gut microbiota are integral to host health. However, how this association began remains unclear. We show that in basal chordates the gut space is radially compartmentalized into a luminal part where food microbes pass and an almost axenic peripheral part, defined by membranous delamination of the gut epithelium. While this membrane, framed with chitin nanofibers, structurally resembles invertebrate peritrophic membranes, proteome supports its affinity to mammalian mucus layers, where gut microbiota colonize. In ray-finned fish, intestines harbor indigenous microbes, but chitinous membranes segregate these luminal microbes from the surrounding mucus layer. These data suggest that chitin-based barrier immunity is an ancient system, the loss of which, at least in mammals, provided mucus layers as a novel niche for microbial colonization. These findings provide a missing link for intestinal immune systems in animals, revealing disparate mucosal environment in model organisms and highlighting the loss of a proven system as innovation.

Isolation of GFP-tagged plasma membrane protein from Arabidopsis egg cells.

Angiosperms possess a double fertilization system for sexual reproduction. Double fertilization is regulated by interactions among proteins localized in the plasma membrane of each sex gamete. A few plasma membrane resident proteins regulating double fertilization have been identified in male gametes. In contrast, no fertilization regulators in female gamete plasma membrane have been identified, largely due to difficulties in the isolation and collection of female gametes. We had produced Arabidopsis transgenic plant pDD45::GFP-AtPIP2;1 where the egg cell plasma membrane was specifically labeled with GFP (Igawa et al. 2013). The protein extract derived from approximately 200 pistils, which contained unfertilized and mature egg cells, was subjected to immunoprecipitation using anti-GFP antibody. As a result, both GFP and AtPIP2;1 were specifically detected in immunoprecipitated proteins from pistil tissues of pDD45::GFP-AtPIP2;1 transgenic plant, but not in those of wild type pistils. It was revealed that specific proteins expressed in the egg cells were successfully isolated from pistil cell population. The method described here showed the feasibility of isolating specific egg cell plasma membrane protein without gamete isolation and collection procedures.

Stepwise Evolution of Coral Biomineralization Revealed with Genome-Wide Proteomics and Transcriptomics.

Despite the importance of stony corals in many research fields related to global issues, such as marine ecology, climate change, paleoclimatogy, and metazoan evolution, very little is known about the evolutionary origin of coral skeleton formation. In order to investigate the evolution of coral biomineralization, we have identified skeletal organic matrix proteins (SOMPs) in the skeletal proteome of the scleractinian coral, Acropora digitifera, for which large genomic and transcriptomic datasets are available. Scrupulous gene annotation was conducted based on comparisons of functional domain structures among metazoans. We found that SOMPs include not only coral-specific proteins, but also protein families that are widely conserved among cnidarians and other metazoans. We also identified several conserved transmembrane proteins in the skeletal proteome. Gene expression analysis revealed that expression of these conserved genes continues throughout development. Therefore, these genes are involved not only skeleton formation, but also in basic cellular functions, such as cell-cell interaction and signaling. On the other hand, genes encoding coral-specific proteins, including extracellular matrix domain-containing proteins, galaxins, and acidic proteins, were prominently expressed in post-settlement stages, indicating their role in skeleton formation. Taken together, the process of coral skeleton formation is hypothesized as: 1) formation of initial extracellular matrix between epithelial cells and substrate, employing pre-existing transmembrane proteins; 2) additional extracellular matrix formation using novel proteins that have emerged by domain shuffling and rapid molecular evolution and; 3) calcification controlled by coral-specific SOMPs.

Two HAP2-GCS1 homologs responsible for gamete interactions in the cellular slime mold with multiple mating types: Implication for common mechanisms of sexual reproduction shared by plants and protozoa and for male-female differentiation.

Fertilization is a central event in sexual reproduction, and understanding its molecular mechanisms has both basic and applicative biological importance. Recent studies have uncovered the molecules that mediate this process in a variety of organisms, making it intriguing to consider conservation and evolution of the mechanisms of sexual reproduction across phyla. The social amoeba Dictyostelium discoideum undergoes sexual maturation and forms gametes under dark and humid conditions. It exhibits three mating types, type-I, -II, and -III, for the heterothallic mating system. Based on proteome analyses of the gamete membranes, we detected expression of two homologs of the plant fertilization protein HAP2-GCS1. When their coding genes were disrupted in type-I and type-II strains, sexual potency was completely lost, whereas disruption in the type-III strain did not affect mating behavior, suggesting that the latter acts as female in complex organisms. Our results demonstrate the highly conserved function of HAP2-GCS1 in gamete interactions and suggest the presence of additional allo-recognition mechanisms in D. discoideum gametes.

Transcriptional Framework of Male Gametogenesis in the Liverwort Marchantia polymorpha L.

In land plants, there are two types of male gametes: one is a non-motile sperm cell which is delivered to the egg cell by a pollen tube, and the other is a motile sperm cell with flagella. The molecular mechanism underlying the sexual reproduction with the egg and pollen-delivered sperm cell is well understood from studies using model plants such as Arabidopsis and rice. On the other hand, the sexual reproduction with motile sperm has remained poorly characterized, due to the lack of suitable models. Marchantia polymorpha L. is a model basal land plant with sexual reproduction involving an egg cell and bi-flagellated motile sperm. To understand the differentiation process of plant motile sperm, we analyzed the gene expression profile of developing antheridia of M. polymorpha. We performed RNA-sequencing experiments and compared transcript profiles of the male sexual organ (antheridiophore and antheridium contained therein), female sexual organ (archegoniophore) and a vegetative organ (thallus). Transcriptome analysis showed that the antheridium expresses nearly half of the protein-coding genes predicted in the genome, but it also has unique features. The antheridium transcriptome shares some common features with male gamete transcriptomes of angiosperms and animals, and homologs of genes involved in male gamete formation and function in angiosperms and animals were identified. In addition, we showed that some of them had distinct expression patterns in the spermatogenous tissue of developing antheridia. This study provides a transcriptional framework on which to study the molecular mechanism of plant motile sperm development in M. polymorpha as a model.

The Lingula genome provides insights into brachiopod evolution and the origin of phosphate biomineralization.

The evolutionary origins of lingulid brachiopods and their calcium phosphate shells have been obscure. Here we decode the 425-Mb genome of Lingula anatina to gain insights into brachiopod evolution. Comprehensive phylogenomic analyses place Lingula close to molluscs, but distant from annelids. The Lingula gene number has increased to ∼34,000 by extensive expansion of gene families. Although Lingula and vertebrates have superficially similar hard tissue components, our genomic, transcriptomic and proteomic analyses show that Lingula lacks genes involved in bone formation, indicating an independent origin of their phosphate biominerals. Several genes involved in Lingula shell formation are shared by molluscs. However, Lingula has independently undergone domain combinations to produce shell matrix collagens with EGF domains and carries lineage-specific shell matrix proteins. Gene family expansion, domain shuffling and co-option of genes appear to be the genomic background of Lingula's unique biomineralization. This Lingula genome provides resources for further studies of lophotrochozoan evolution.

Evidence for participation of GCS1 in fertilization of the starlet sea anemone Nematostella vectensis: implication of a common mechanism of sperm-egg fusion in plants and animals.

It has been reported that GCS1 (Generative Cell Specific 1) is a transmembrane protein that is exclusively expressed in sperm cells and is essential for gamete fusion in flowering plants. The GCS1 gene is present not only in angiosperms but also in unicellular organisms and animals, implying the occurrence of a common or ancestral mechanism of GCS1-mediated gamete fusion. In order to elucidate the common mechanism, we investigated the role of GCS1 in animal fertilization using a sea anemone (Cnidaria), Nematostella vectensis. Although the existence of the GCS1 gene in N. vectensis has been reported, the expression of GCS1 in sperm and the role of GCS1 in fertilization are not known. In this study, we showed that the GCS1 gene is expressed in the testis and that GCS1 protein exists in sperm by in situ hybridization and proteomic analysis, respectively. Then we made four peptide antibodies against the N-terminal extracellular region of NvGCS1. These antibodies specifically reacted to NvGCS1 among sperm proteins on the basis of Western analysis and potently inhibited fertilization in a concentration-dependent manner. These results indicate that sperm GCS1 plays a pivotal role in fertilization, most probably in sperm-egg fusion, in a starlet sea anemone, suggesting a common gamete-fusion mechanism shared by eukaryotic organisms.

Identification of the augmin complex in the filamentous fungus Aspergillus nidulans.

Augmin is a protein complex that binds to spindle microtubules (MTs), recruits the potent MT nucleator, γ-tubulin, and thereby promotes the centrosome-independent MT generation within mitotic and meiotic spindles. Augmin is essential for acentrosomal spindle assembly, which is commonly observed during mitosis in plants and meiosis in female animals. In many animal somatic cells that possess centrosomes, the centrosome- and augmin-dependent mechanisms work cooperatively for efficient spindle assembly and cytokinesis. Yeasts have lost the augmin genes during evolution. It is hypothesized that their robust MT nucleation from the spindle pole body (SPB), the centrosome-equivalent structure in fungi, compensates for the lack of augmin. Intriguingly, however, a gene homologous to an augmin subunit (Aug6/AUGF) has been found in the genome of filamentous fungi, which has the SPB as a robust MT nucleation centre. Here, we aimed to clarify if the augmin complex is present in filamentous fungi and to identify its role in mitosis. By analysing the Aug6-like gene in the filamentous fungus Aspergillus nidulans, we found that it forms a large complex with several other proteins that share weak but significant homology to known augmin subunits. In A. nidulans, augmin was enriched at the SPB and also associated with spindle MTs during mitosis. However, the augmin gene disruptants did not exhibit growth defects under normal, checkpoint-deficient, or MT-destabilised conditions. Moreover, we obtained no evidence that A. nidulans augmin plays a role in γ-tubulin recruitment or in mitotic cell division. Our study uncovered the conservation of the augmin complex in the fungal species, and further suggests that augmin has several functions, besides mitotic spindle MT nucleation, that are yet to be identified.

Integrative omics analysis reveals differentially distributed proteins in dimorphic euspermatozoa of the squid, Loligo bleekeri.

In the coastal squid Loligo bleekeri, each male produces one of two types of fertilization-competent spermatozoa (eusperm) that exhibit morphological and behavioral differences. Large "consort" males produce short-tailed spermatozoa that display free-swimming behavior when ejaculated into seawater. Small "sneaker" males, on the other hand, produce long-tailed spermatozoa that exhibit a self-swarming trait after ejaculation. To understand the molecular basis for adaptive traits employed by alternative male mating tactics, we performed the transcriptome deep sequencing (RNA-seq) and proteome analyses to search for differences in testicular mRNAs and sperm proteins, respectively. From mature male testes we identified a total of 236,455 contigs (FPKM ≧1) where 3789 and 2789 were preferentially (≧10-fold) expressed in consort and sneaker testes, respectively. A proteomic analysis detected 4302 proteins in the mature sperm as post-translational products. A strongly biased (≧10-fold) distribution occurred in 55 consort proteins and 61 sneaker proteins. There was no clear mRNA-protein correlation, making a ballpark estimate impossible for not only overall protein abundance but also the degree of biased sperm type expressed in the spermatozoa. A family encoding dynein heavy chain gene, however, was found to be biased towards sneakers, whereas many enzymes involving energy metabolism were heavily biased towards consort spermatozoa. The difference in flagellar length matched exactly the different amount of tubulins. From these results we hypothesize that discrete differential traits in dimorphic eusperm arose from a series of innovative alterations in the intracellular components of spermatozoa.

Glutathione transferase theta in apical ciliary tuft regulates mechanical reception and swimming behavior of Sea Urchin Embryos.

An apical tuft, which is observed in a wide range of embryos/larvae of marine invertebrates, is composed of a group of cilia that are longer and less motile than the abundant lateral cilia covering the rest of the embryonic surface. Although the apical tuft has been thought to function as a sensory organ, its molecular composition and roles are poorly understood. Here, we identified a glutathione transferase theta (GSTT) as an abundant and specific component of the apical tuft in sea urchin embryos. The expression of GSTT mRNA increases and becomes limited to the animal plate of the mesenchyme blastula, gastrula, and prism larva. Electron microscopy and tandem mass spectrometry demonstrated that the apical tuft contains almost every axonemal component for ciliary motility. Low concentrations of an inhibitor of glutathione transferase bromosulphophthalein (BSP) induce bending of apical tuft, suggesting that GSTT regulates motility of apical tuft cilia. Embryos treated with BSP swim with normal velocity and trajectories but show less efficiency of changing direction when they collide with an object. These results suggest that GSTT in the apical tuft plays an important role in the mechanical reception for the motility regulation of lateral motile cilia in sea urchin embryos.

Differential gene expression in notochord and nerve cord fate segregation in the Ciona intestinalis embryo.

During early embryogenesis, embryonic cells gradually restrict their developmental potential and are eventually destined to give rise to one type of cells. Molecular mechanisms underlying developmental fate restriction are one of the major research subjects within developmental biology. In this article, this subject was addressed by combining blastomere isolation with microarray analysis. During the 6th cleavage of the Ciona intestinalis embryo, from the 32-cell to the 64-cell stage, four mother cells divide into daughter cells with two distinct fates, one giving rise to notochord precursor cells and the other to nerve cord precursors. Approximately 2,200 each of notochord and nerve cord precursor cells were isolated, and their mRNA expression profiles were compared by microarray. This analysis identified 106 and 68 genes, respectively, that are differentially expressed in notochord and nerve cord precursor cells. These included not only genes for transcription factors and signaling molecules but also those with generalized functions observed in many types of cells. In addition, whole-mount in situ hybridization showed dynamic spatial expression profiles of these genes during segregation of the two fates: partitioning of transcripts present in the mother cells into either type of daughter cells, and initiation of preferential gene expression in either type of cells.

cDNA cloning, localization, and candidate binding partners of acid-extractable vitelline-coat protein Ci-v-Themis-like in the ascidian Ciona intestinalis.

Ascidians are hermaphrodites, although several ascidian species show self-sterility because of the occurrence of a self/nonself-recognition system called the self-incompatibility system. We previously reported that two pairs of sperm polycystin 1-like receptors, s-Themis-A and s-Themis-B, and egg fibrinogen-like ligands, v-Themis-A and v-Themis-B, are responsible for self-incompatibility in the ascidian Ciona intestinalis. Our previous results showed that v-Themis-A and v-Themis-B were hardly extracted from the vitelline coat (VC) by acid treatment, which is not in accordance with a report that an acid-extractable VC factor has the ability to distinguish self- from nonself-sperm. These results led us to explore a novel factor from acid-extractable VC proteins that could be involved in self-incompatibility. Here, we report cDNA cloning, expression, and localization of Ci-v-Themis-like, a major acid-extractable VC protein. This protein has a fibrinogen-like domain, as do v-Themis-A and v-Themis-B, but it showed no polymorphisms. Phylogenic analysis suggested that Ci-v-Themis-like is an ancestral protein of v-Themis-A and v-Themis-B. Whole mount in situ hybridization revealed that Ci-v-Themis-like mRNA is expressed in the ovary and testis. Western blotting and immunocytochemistry showed the occurrence of Ci-v-Themis-like in developing oocytes and on the VC of mature eggs. Yeast two-hybrid screenings using testis and ovary libraries revealed candidate interacting proteins; among these candidates, we succeeded in identifying several testis-specific proteins, including sperm proteases and coiled-coil-domain-containing proteins. The results suggest that Ci-v-Themis-like and its binding partners are involved in sperm binding to the VC prior to the allorecognition process during C. intestinalis fertilization.

Efficient targeted mutagenesis of the chordate Ciona intestinalis genome with zinc-finger nucleases.

Zinc-finger nucleases (ZFNs) are engineered nucleases that induce DNA double-strand breaks (DSBs) at target sequences. They have been used as tools for generating targeted mutations in the genomes of multiple organisms in both animals and plants. The DSB induced by ZFNs is repaired by non-homologous end joining (NHEJ) or by homologous recombination (HR) mechanisms. Non-homologous end joining induces some errors because it is independent of a reference DNA sequence. Through the NHEJ mechanism, ZFNs generate insertional or deletional mutations at the target sequence. We examined the usability, specificity and toxicity of ZFNs in the basal chordate Ciona intestinalis. As the target of ZFNs, we chose an enhanced green fluorescent protein (EGFP) gene artificially inserted in the C. intestinalis genome because this locus is neutral for the development and growth of C. intestinalis, and the efficiency of mutagenesis with ZFNs can thus be determined without any bias. We introduced EGFP -ZFN mRNAs into the embryos of an EGFP -transgenic line and observed the mutation frequency in the target site of EGFP . We also examined the effects of the EGFP -ZFNs at off-target sites resembling the EGFP target sequence in the C. intestinalis genome in order to examine the specificity of ZFNs. We further investigated the influence of ZFNs on embryogenesis, and showed that adequate amounts of ZFNs, which do not disrupt embryogenesis, can efficiently induce mutations on the on-target site with less effect on the off-target sites. This suggests that target mutagenesis with ZFNs will be a powerful technique in C. intestinalis.

A surface glycoprotein indispensable for gamete fusion in the social amoeba Dictyostelium discoideum.

Sexual reproduction is essential for the maintenance of species in a wide variety of multicellular organisms, and even unicellular organisms that normally proliferate asexually possess a sexual cycle because of its contribution to increased genetic diversity. Information concerning the molecules involved in fertilization is accumulating for many species of the metazoan, plant, and fungal lineages, and the evolutionary consideration of sexual reproduction systems is now an interesting issue. Macrocyst formation in the social amoeba Dictyostelium discoideum is a sexual process in which cells become sexually mature under dark and submerged conditions and fuse with complementary mating-type cells. In the present study, we isolated D. discoideum insertional mutants defective in sexual cell fusion and identified the relevant gene, macA, which encodes a highly glycosylated, 2,041-amino-acid membrane protein (MacA). Although its overall similarity is restricted to proteins of unknown function within dictyostelids, it contains LamGL and discoidin domains, which are implicated in cell adhesion. The growth and development of macA-null mutants were indistinguishable from those of the parental strain. The overexpression of macA using the V18 promoter in a macA-null mutant completely restored its sexual defects. Although the macA gene encoded exactly the same protein in a complementary mating-type strain, it was expressed at a much lower level. These results suggest that MacA is indispensable for gamete interactions in D. discoideum, probably via cell adhesion. There is a possibility that it is controlled in a mating-type-dependent manner.

Self-incompatibility response induced by calcium increase in sperm of the ascidian Ciona intestinalis.

Many hermaphroditic organisms possess a self-incompatibility system to avoid self-fertilization. Recently, we identified the genes responsible for self-sterility in a hermaphroditic primitive chordate (ascidian), Ciona intestinalis: sperm-side polycystin 1-like receptors s-Themis-A/B and egg-side fibrinogen-like ligands on the vitelline coat (VC) v-Themis-A/B. Here, we investigated the sperm behavior and intracellular Ca(2+) concentration ([Ca(2+)](i)) in response to self/nonself-recognition. We found that sperm motility markedly decreased within 5 min after attachment to the VC of self-eggs but not after attachment to the VC of nonself-eggs and that the apparent decrease in sperm motility was suppressed in low Ca(2+) seawater. High-speed video analysis revealed that sperm detached from the self-VC or stopped motility within 5 min after binding to the self-VC. Because s-Themis-B contains a cation channel domain in its C terminus, we monitored sperm [Ca(2+)](i) by real-time [Ca(2+)](i) imaging using Fluo-8H-AM (AAT Bioquest, Inc.). Interestingly, we found that sperm [Ca(2+)](i) rapidly and dramatically increased and was maintained at a high level in the head and flagellar regions when sperm interacted with the self-VC but not when the sperm interacted with the nonself-VC. The increase in [Ca(2+)](i) was also suppressed by low-Ca(2+) seawater. These results indicate that the sperm self-recognition signal triggers [Ca(2+)](i) increase and/or Ca(2+) influx, which elicits a self-incompatibility response to reject self-fertilization in C. intestinalis.