Tissue Localization of Tetrodotoxin in the Flatworm Planocera multitentaculata (Platyhelminthes: Polycladida).

Tetrodotoxin (TTX), a pufferfish toxin, is a highly potent neurotoxin that has been found in a wide variety of animals. The TTX-bearing flatworm Planocera multitentaculata possesses a large amount of TTX and is considered responsible for the toxification of TTX-bearing animals such as pufferfish (Takifugu and Chelonodon) and the toxic goby Yongeichthys criniger. However, the mechanism underlying TTX accumulation in flatworms remains unclear. Previous studies have been limited to identifying the distribution of TTX in multiple organs, such as the digestive organs, genital parts, and the remaining tissues of flatworms. Here, we performed liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and immunohistochemical staining using a monoclonal anti-TTX antibody to elucidate the detailed localization of TTX in the tissues and organs of the flatworm P. multitentaculata. Immunohistochemical staining for P. multitentaculata showed that TTX-specific signals were detected not only in the ovaries and pharynx but also in many other tissues and organs, whereas no signal was detected in the brain, Lang's vesicle, and genitalia. In addition, combined with LC-MS/MS analysis, it was revealed for the first time that TTX accumulates in high concentrations in the basement membrane and epidermis. These findings robustly support the hypotheses of "TTX utilization protection from predators."

Transcriptional landscape of small non-coding RNAs reveals diversity of categories and functions in molluscs.

Small non-coding RNAs (sncRNAs) are non-coding RNA molecules that play various roles in metazoans. Among the sncRNAs, microRNAs (miRNAs) guide post-translational gene regulation during cellular development, proliferation, apoptosis, and differentiation, while PIWI-interacting RNAs (piRNAs) suppress transposon activity to safeguard the genome from detrimental insertion mutagenesis. While an increasing number of piRNAs are being identified in the soma and germlines of various organisms, they are scarcely reported in molluscs. To unravel the small RNA (sRNA) expression patterns and genomic function in molluscs, we generated a comprehensive sRNA dataset by sRNA sequencing (sRNA-seq) of eight mollusc species. Abundant miRNAs were identified and characterized in all investigated molluscs, and ubiquitous piRNAs were discovered in both somatic and gonadal tissues in six of the investigated molluscs, which are more closely associated with transposon silencing. Tens of piRNA clusters were also identified based on the genomic mapping results, which varied among different tissues and species. Our dataset serves as important reference data for future genomic and genetic studies on sRNAs in these molluscs and related species, especially in elucidating the ancestral state of piRNAs in bilaterians.

Japanese Planocerid Flatworms: Difference in Composition of Tetrodotoxin and Its Analogs and the Effects of Ingestion by Toxin-Bearing Fishes in the Ryukyu Islands, Japan.

Tetrodotoxin (TTX), known as pufferfish toxin, is a potent neurotoxin blocking sodium channels in muscle and nerve tissues. TTX has been detected in various taxa other than pufferfish, including marine polyclad flatworms, suggesting that pufferfish toxin accumulates in fish bodies via food webs. The composition of TTX and its analogs in the flatworm Planocera multitentaculata was identical to those in wild grass puffer Takifugu alboplumbeus. Previously, Planocera sp. from Okinawa Island, Japan, were reported to possess high level of TTX, but no information was available on TTX analogs in this species. Here we identified TTX and analogs in the planocerid flatworm using high-resolution liquid chromatography-mass spectrometry, and compared the composition of TTX and analogs with those of another toxic and non-toxic planocerid species. We show that the composition of TTX and several analogs, such as 5,6,11-trideoxyTTX, dideoxyTTXs, deoxyTTXs, and 11-norTTX-6(S)-ol, of Planocera sp. was identical to those of toxic species, but not to its non-toxic counterpart. The difference in the toxin composition was reflected in the phylogenetic relationship based on the mitochondrial genome sequence. A toxification experiment using predatory fish and egg plates of P. multitentaculata demonstrated that the composition of TTX and analogs in wild T. alboplumbeus juveniles was reproduced in artificially toxified pufferfish. Additionally, feeding on the flatworm egg plates enhanced the signal intensities of all TTX compounds in Chelonodon patoca and that of deoxyTTXs in Yongeichthys criniger.

Transcriptome analysis of Edwardsiella piscicida during intracellular infection reveals excludons are involved with the activation of a mitochondrion-like energy generation program.

Phylogenetic evidence suggests a shared ancestry between mitochondria and modern Proteobacteria, a phylum including several genera of intracellular pathogens. Studying these diverse pathogens, particularly during intracellular infection of their hosts, can reveal characteristics potentially representative of the mitochondrial-Proteobacterial ancestor by identifying traits shared with mitochondria. While transcriptomic approaches can provide global insights into intracellular acclimatization by pathogens, they are often limited by excess host RNAs in extracts. Here, we developed a method employing magnetic nanoparticles to enrich RNA from an intracellular Gammaproteobacterium, Edwardsiella piscicida, within zebrafish, Danio rerio, fin fibroblasts, enabling comprehensive exploration of the bacterial transcriptome. Our findings revealed that the intracellular E. piscicida transcriptome reflects a mitochondrion-like energy generation program characterized by the suppression of glycolysis and sugar transport, coupled with upregulation of the tricarboxylic acid (TCA) cycle and alternative import of simple organic acids that directly flux into TCA cycle intermediates or electron transport chain donors. Additionally, genes predicted to be members of excludons, loci of gene pairs antagonistically co-regulated by overlapping antisense transcription, are significantly enriched in the set of all genes with perturbed sense and antisense transcription, suggesting a general but important involvement of excludons with intracellular acclimatization. Notably, genes involved with the activation of the mitochondrion-like energy generation program, specifically with metabolite import and glycolysis, are also members of predicted excludons. Other intracellular Proteobacterial pathogens appear to employ a similar mitochondrion-like energy generation program, suggesting a potentially conserved mechanism for optimized energy acquisition from hosts centered around the TCA cycle.IMPORTANCEPhylogenetic evidence suggests that mitochondria and Proteobacteria, a phylum encompassing various intracellular pathogens, share a common ancestral lineage. In this study, we developed a novel method employing magnetic nanoparticles to explore the transcriptome of an aquatic Gammaproteobacterium, Edwardsiella piscicida, during intracellular infection of host cells. We show that the strategy E. piscicida uses to generate energy strikingly mirrors the function of mitochondria-energy generators devoid of glycolytic processes. Notably, several implicated genes are members of excludons-gene pairs antagonistically co-regulated by overlapping antisense transcription. Other intracellular Proteobacterial pathogens appear to adopt a similar mitochondrion-like energy generation program, indicating a possibly conserved strategy for optimized energy acquisition from hosts centered around the tricarboxylic acid cycle.

The highly developed symbiotic system between the solar-powered nudibranch Pteraeolidia semperi and Symbiodiniacean algae.

The intricate coexistence of Symbiodiniacean algae with a diverse range of marine invertebrates underpins the flourishing biodiversity observed within coral reef ecosystems. However, the breakdown of Symbiodiniaceae-host symbiosis endangers these ecosystems, necessitating urgent study of the symbiotic mechanisms. The symbiosis between nudibranchs and Symbiodiniaceae has been identified as an efficacious model for examining these mechanisms, yet a comprehensive understanding of their histological structures and cellular processes remains elusive. A meticulous histological exploration of the nudibranch Pteraeolidia semperi, employing optical, fluorescence, and electron microscopy, has revealed fine tubules extending to the body surface, with associated epithelial cells having been shown to adeptly encapsulate Symbiodiniaceae intracellularly. By tracing the stages of the "bleaching" in nudibranchs, it was inferred that algal cells, translocated via the digestive gland, are directly phagocytosed and expelled by these epithelial cells. Collectively, these insights contribute substantially to the scholarly discourse on critical marine symbiotic associations.

The complete mitochondrial genome of Spurilla braziliana MacFarland 1909 (Nudibranchia, Aeolidiidae).

Spurilla braziliana MacFarland 1909 is a morphologically diverse nudibranch found in the Pacific and Western Atlantic. The complete mitochondrial genome of S. braziliana has been constructed using next-generation sequencing technology. The mitochondrial genome is 14,291 bp and contains 13 protein-coding genes, 2 rRNA genes, and 23 tRNA genes. Molecular phylogenetic analysis using the maximum likelihood method revealed that S. braziliana is included in the superfamily Aeolidioidea and forms a monophyletic group with Berghia stephanieae, a nudibranch of the family Aeolidiidae. This study reinforces existing taxonomic insights and provides a basis for further molecular phylogenetic analysis.

Draft genome sequencing and assembly of Risso's dolphin, Grampus griseus.

The Risso's dolphin (Grampus griseus) is one of the migratory marine mammals and they have commonly dispersed in tropical and temperate seas. It is a least concerned species in the IUCN red list of threatened species. However, their population size and factors affecting their population structure are unknown. Due to the wide distribution of this species, their populations might be genetically stable and less structured. To support genetic studies on dolphins and other marine mammals, we assembled the draft genome of Risso's dolphin that was found in Japan. The tissue samples were used to extract high molecular DNA and subjected to sequencing by Illumina HiSeq X, Oxford Nanopore MinION, and Bionano Saphyr. The assembled hybrid genome was 75.9% of complete eukaryotic BUSCOs and the genome size was 2.256 Gb with 2.042 Mb of scaffold N50. De novo assembly of this genome by Bionano Saphyr recovered 2.036 Gb total genome map length and structural variations. The gene structures of this draft genome were identified by BRAKER2, and 9947 genes were recovered. The data will be useful for future studies of cetaceans.

Age-Associated Different Transcriptome Profiling in Zebrafish and Rats: an Insight into the Diversity of Vertebrate Aging.

Most mammals, including humans, show obvious aging phenotypes, for example, loss of tissue plasticity and sarcopenia. In this regard, fish can be attractive models to study senescence because of their unique aging characteristics. The lifespan of fish varies widely, and several species can live for over 200 years. Moreover, some fish show anti-aging features and indeterminate growth throughout their life. Therefore, exploring the aging mechanism in fish could provide new insights into vertebrate aging. To this end, we conducted RNA sequencing (RNA-seq) assays for various organs and growth stages of zebrafish and compared the data with previously published RNA-seq data of rats. Age-associated differentially expressed genes (DEGs) for all zebrafish tissue samples reveal the upregulation of circadian genes and downregulation of hmgb3a. On one hand, a comparative analysis of DEG profiles associated with aging between zebrafish and rats identifies upregulation of circadian genes and downregulation of collagen genes as conserved transcriptome changes. On the other hand, in zebrafish, upregulation of autophagy-related genes in muscles and AP-1 transcription factor genes in various tissues is observed, which may imply fish-specific anti-aging characteristics. Consistent with our knowledge of mammalian aging, DEG profiles related to tissue senescence are observed in rats. We also detect age-associated downregulation of muscle homeostasis and differentiation-related genes in zebrafish gills, indicating a fish-specific senescence phenotype. Our results indicate both common and different aging profiles between fish and mammals, which could be used for future translational research.

Zebrafish Danio rerio myotomal muscle structure and growth from a spatial transcriptomics perspective.

Fish exhibit different muscle structures and growth characteristics compared with mammals. We used a spatial transcriptomics approach and examined myotomal muscle sections from zebrafish. Adult muscles were divided into eight regions according to spatial gene expression characteristics. Slow muscle was located in the wedge-shaped region near the lateral line and at the base of the dorsal fin, intermediate muscle was located in a ribbon-shaped region adjacent to slow muscle, and fast muscle was located in the deep region of the trunk, surrounded by intermediate muscle; the interior of fast muscle was further divided into 6 parts by their transcriptomic features. Combined analysis of adult and larval data revealed that adult muscles contain specific regions similar to larval muscles. These regions showed active myogenesis and a high expression of genes associated with muscle hyperplasia. This is the first study to apply spatial transcriptomics to fish myotomal muscle structure and growth.

Evolution of nacre- and prisms-related shell matrix proteins in the pen shell, Atrina pectinata.

The molluscan shell is a good model for understanding the mechanisms underlying biomineralization. It is composed of calcium carbonate crystals and many types of organic molecules, such as the matrix proteins, polysaccharides, and lipids. The pen shell Atrina pectinata (Pterioida, Pinnidae) has two shell microstructures: an outer prismatic layer and an inner nacreous layer. Similar microstructures are well known in pearl oysters (Pteriidae), such as Pinctada fucata, and many kinds of shell matrix proteins (SMPs) have been identified from their shells. However, the members of SMPs that consist of the nacreous and prismatic layers of Pinnidae bivalves remain unclear. In this study, we identified 114 SMPs in the nacreous and prismatic layers of A. pectinata, of which only seven were found in both microstructures. 54 of them were found to bind calcium carbonate. Comparative analysis of nine molluscan shell proteomes showed that 69 of 114 SMPs of A. pectinata were found to have sequential similarity with at least one or more SMPs of other molluscan species. For instance, nacrein, tyrosinase, Pif/BMSP-like, chitinase (CN), chitin-binding proteins, CD109, and Kunitz-type serine proteinase inhibitors are widely shared among bivalves and gastropods. Our results provide new insights for understanding the complex evolution of SMPs related to nacreous and prismatic layer formation in the pteriomorph bivalves.

Induction of endoplasmic reticulum stress markers in an acromegaly model.

Acromegaly is a growth hormone (GH) excess pathological condition in humans. Acromegaly is associated with somatic disfigurement and a wide range of systemic manifestations such as arthritis, neuropathy, carpal tunnel syndrome, reproductive disorders, metabolic disorders, and gastrointestinal complications. The influence of excess GH on the cellular level could aid in understanding the root causes of acromegaly-related health complications. Previously, we found that GH excess induces DNA damage to somatic cells and reduces the stem cells number and causes premature aging. In this study, an in-depth analysis of the acromegaly RNAseq data revealed the disruption of important biological cellular processes. Gene set enrichment analysis, heatmap, and enrichment analysis of acromegaly RNAseq data revealed induction of endoplasmic reticulum (ER) stress markers in various organs. Interestingly, the induction of ER stress was even more apparent than in aged zebrafish. Splicing of box-binding protein-1 (XBP1) mRNA is a hallmark of ER stress. Therefore, we quantified spliced XBP1 mRNA in different organs of our acromegaly model. Thus, our study emphasizes the importance of ER stress in GH oversecretion, which is important for understanding the health complications of acromegaly.

Exosome-derived small non-coding RNAs reveal immune response upon grafting transplantation in Pinctada fucata (Mollusca).

Exosomes, a subset of small extracellular vesicles, carry various nucleic acids, proteins, lipids, amino acids and metabolites. They function as a mode of intercellular communication and molecular transfer. Exosome cargo molecules, including small non-coding RNAs (sncRNAs), are involved in the immune response in various organisms. However, the role of exosome-derived sncRNAs in immune responses in molluscs remains unclear. Here, we aimed to reveal the sncRNAs involved in the immune response during grafting transplantation by the pearl oyster Pinctada fucata. Exosomes were successfully extracted from the P. fucata haemolymph during graft transplantation. Abundant microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs) were simultaneously discovered in P. fucata exosomes by small RNA sequencing. The expression patterns of the miRNAs and piRNAs at the grafting and initial stages were not substantially different, but varied significantly between the initial and later stages. Target prediction and functional analysis indicate that these miRNAs and piRNAs are related to immune response upon grafting transplantation, whereas piRNAs may also be associated with transposon silencing by targeting with genome transposon elements. This work provides the basis for a functional understanding of exosome-derived sncRNAs and helps to gain further insight into the PIWI/piRNA pathway function outside of germline cells in molluscs.

Construction of a chromosome-level Japanese stickleback species genome using ultra-dense linkage analysis with single-cell sperm sequencing.

It is still difficult to construct the genomes of higher organisms as their genome sequences must be extended to the length of the chromosome by linkage analysis. In this study, we attempted to provide an innovative alternative to conventional linkage analysis by devising a method to genotype sperm using 10× Genomics single-cell genome sequencing libraries to generate a linkage map without interbreeding individuals. A genome was assembled using sperm from the Japanese stickleback Gasterosteus nipponicus, with single-cell genotyping yielding 1 864 430 very dense hetero-SNPs and an average coverage per sperm cell of 0.13×. In total, 1665 sperm were used, which is an order of magnitude higher than the number of recombinations used for conventional linkage analysis. We then improved the linkage analysis tool scaffold extender with low depth linkage analysis (SELDLA) to analyze the data according to the characteristics of the single-cell genotyping data. Finally, we were able to determine the chromosomal location (97.1%) and orientation (64.4%) of the contigs in the 456 Mb genome of G. nipponicus, sequenced using nanopores. This method promises to be a useful tool for determining the genomes of non-model organisms for which breeding systems have not yet been established by linkage analysis.

Selection of a reference gene for studies on lipid-related aquatic adaptations of toothed whales (Grampus griseus).

Toothed whales are one group of marine mammals that has developed special adaptations, such as echolocation for predation, to successfully live in a dynamic aquatic environment. Their fat metabolism may differ from that of other mammals because toothed whales have acoustic fats. Gene expression in the metabolic pathways of animals can change with respect to their evolution and environment. A real-time quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for studying the relative expressions of genes. However, since the accuracy of RT-qPCR data is totally dependent on the reference gene, the selection of the reference gene is an essential step. In this study, 10 candidate reference genes (ZC3H10, FTL, LGALS1, RPL27, GAPDH, FTH1, DCN, TCTP, NDUS5, and UBIM) were initially tested for amplification efficiency using RT-qPCR. After excluding DCN, the remaining nine genes, which are nearly 100% efficient, were selected for the gene stability analysis. Stable reference genes across eight different fat tissue, liver, and muscle samples from Grampus griseus were identified by four algorithms, which were provided in Genorm, NormFinder, BestKeeper, and Delta CT. Finally, a RefFinder comprehensive ranking was performed based on the stability values, and the nine genes were ranked as follows: LGALS1 > FTL > GAPDH > ZC3H10 > FTH1 > NDUS5 > TCTP > RPL27 > UBIM. The LGALS1 and FTL genes were identified as the most stable novel reference genes. The third-ranked gene, GAPDH, is a well-known housekeeping gene for mammals. Ultimately, we suggest the use of LGALS1 as a reliable novel reference gene for genomics studies on the lipid-related aquatic adaptations of toothed whales.

Metagenomic analysis provides functional insights into seasonal change of a non-cyanobacterial prokaryotic community in temperate coastal waters.

The taxonomic compositions of marine prokaryotic communities are known to follow seasonal cycles, but functional metagenomic insights into this seasonality is still limited. We analyzed a total of 22 metagenomes collected at 11 time points over a 14-month period from two sites in Sendai Bay, Japan to obtain seasonal snapshots of predicted functional profiles of the non-cyanobacterial prokaryotic community. Along with taxonomic composition, functional gene composition varied seasonally and was related to chlorophyll a concentration, water temperature, and salinity. Spring phytoplankton bloom stimulated increased abundances of putative genes that encode enzymes in amino acid metabolism pathways. Several groups of functional genes, including those related to signal transduction and cellular communication, increased in abundance during the mid- to post-bloom period, which seemed to be associated with a particle-attached lifestyle. Alternatively, genes in carbon metabolism pathways were generally more abundant in the low chlorophyll a period than the bloom period. These results indicate that changes in trophic condition associated with seasonal phytoplankton succession altered the community function of prokaryotes. Our findings on seasonal changes of predicted function provide fundamental information for future research on the mechanisms that shape marine microbial communities.

Regulation of the Expression of the Myosin Heavy Chain (MYH) Gene myh14 in Zebrafish Development.

The human sarcomeric myosin heavy chain gene MYH14 contains an intronic microRNA, miR-499. Our previous studies demonstrated divergent genomic organization and expression patterns of myh14/miR-499 among teleosts; however, the regulatory mechanism is partly known. In this study, we report the regulation of myh14 expression in zebrafish, Danio rerio. Zebrafish myh14 has three paralogs, myh14-1, myh14-2, and myh14-3. Detailed promoter analysis suggested that a 5710-bp 5'-flanking region of myh14-1 and a 5641-bp region of myh14-3 contain a necessary regulatory region to recapitulate specific expression during embryonic development. The 5'-flanking region of zebrafish myh14-1 and its torafugu ortholog shared two distal and a single proximal conserved region. The two distal conserved regions had no effect on zebrafish myh14-1 expression, in contrast to torafugu expression, suggesting an alternative regulatory mechanism among the myh14 orthologs. Comparison among the 5'-flanking regions of the myh14 paralogs revealed two conserved regions. Deletion of these conserved regions significantly reduced the promoter activity of myh14-3 but had no effect on myh14-1, indicating different cis-regulatory mechanisms of myh14 paralogs. Loss of function of miR-499 resulted in a marked reduction in slow muscle fibers in embryonic development. Our study identified different cis-regulatory mechanisms controlling the expression of myh14/miR-499 and an indispensable role of miR-499 in muscle fiber-type specification in zebrafish.

Another polymorphic mitochondrial genome of Grampus griseus and phylogeny of family Delphinidae.

Risso's dolphin (Grampus griseus Cuvier, 1812) is the only species of genus Grampus and a cosmopolitan marine inhabitant. Here, we report a polymorphic complete mitochondrial genome of G. griseus. The size of the total mitochondrial genome was 16,386 bp in length and contains 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and a control region. 37 single nucleotide polymorphic sites (SNPs) were identified compared to the references. Based on the available total mitochondrial dolphin genomes' phylogenetics, G. griseus has formed a clade with 0.1415 distance, sister to the following species of the subfamily Globicephalinae and the taxonomy of Orcinus orca still needs further investigations.

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

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

An Acromegaly Disease Zebrafish Model Reveals Decline in Body Stem Cell Number along with Signs of Premature Aging.

In our previous publication, it was shown that growth hormone (GH) excess in acromegaly affects the cell integrity of somatic cells through increased DNA damage throughout the body and impaired DNA repair pathways. Acromegaly is a hormone disorder pathological condition that develops as a result of growth hormone over-secretion from the pituitary gland. We produced a zebrafish acromegaly model to gain a better understanding of the excess GH effects at the cellular level. Here we show that the acromegaly zebrafish model progressively reduced the number of stem cells in different organs and increased oxidative stress in stem cells. Importantly, the decline in the stem cells was even more apparent than in aged fish. The controversy and debate over the use of GH as an anti-aging therapy have been going on for several years. In this study, excess GH induced aging signs such as increased senescence-associated (SA)-ß-galactosidase staining of abdominal skin and similarity of the pattern of gene expression between aged and acromegaly zebrafish. Thus, this study highlights the role of excess GH in acromegaly stem cells.

Trivalent Iron Is Responsible for the Yellow Color Development in the Nacre of Akoya Pearl Oyster Shells.

The gold and cream colors of cultured Akoya pearls, as well as natural yellow nacre of pearl oyster shells, are thought to arise from intrinsic yellow pigments. While the isolation of the yellow pigments has been attempted using a large amount of gold pearls, the substance concerned is still unknown. We report here on the purification and characterization of yellow pigments from the nacre of Akoya pearl oyster shells. Two yellow components, YC1 and YC2, were isolated from the HCl-methanol (HCl-MeOH) extract from nacreous organic matrices obtained by decalcification of the shells with ethylenediaminetetraacetic acid (EDTA). Energy-dispersive X-ray and infrared spectroscopy analyses suggested that YC1 and YC2 precipitated under basic conditions are composed of Fe-containing inorganic and polyamide-containing organic compounds, respectively. YC1 solubilized under acidic conditions exhibited positive reactions to KSCN and K4[Fe(CN)6] reagents, showing the same ultraviolet-visible absorption spectrum as those of Fe(III)-containing compounds. In addition, X-ray absorption fine structure analysis supported the compound in the form of Fe(III). The total amount of Fe was approximately 2.6 times higher in the yellow than white nacre, and most Fe was fractionated into the EDTA-decalcifying and HCl-MeOH extracts. These results suggest that Fe(III) coordinated to EDTA-soluble and insoluble matrix compounds are mainly associated with yellow color development not only in the Akoya pearl oyster shells but also in the cultured Akoya pearls.