Wheat germ agglutinin affinity chromatography enrichment and glyco-proteomic characterization of tetrodotoxin-binding proteins from the plasma of cultured tiger pufferfish (Takifugu rubripes).

Efficient enrichment of tetrodotoxin (TTX)-binding proteins from the plasma of cultured tiger pufferfish (Takifugu rubripes) was achieved by ammonium sulfate fractionation and wheat germ agglutinin (WGA) affinity chromatography. The enrichment efficiency was validated by ultrafiltration-LC/MS-based TTX-binding assay and proteomics. Major proteins in the WGA-bound fraction were identified as isoform X1 (125 kDa) and X2 variants (88 and 79 kDa) derived from pufferfish saxitoxin and tetrodotoxin-binding protein (PSTBP) 1-like gene (LOC101075943). The 125-kDa X1 protein was found to be a novel member of the lipocalin family, having three tandemly repeated domains. X2 variants, X2α and X2ß, were estimated to have two domains, and X2ß is structurally related to Takifugu pardalis PSTBP2 in their domain type and arrangement. Among 11 potential N-glycosylation sites in the X2 precursor, 5 N-glycosylated Asn residues (N55, N89, N244, N308, and N449) were empirically determined. Structural relationships among PSTBP homologs and complexity of their proteoforms are discussed.

Homophymamide A, Heterodetic Cyclic Tetrapeptide from a Homophymia sp. Marine Sponge: A Cautionary Note on Configurational Assignment of Peptides That Contain a Ureido Linkage.

A previously unreported heterodetic cyclic peptide, homophymamide A (1), was isolated from a Homophymia sp. marine sponge. The structure of homophymamide A was determined to be a lower homologue of anabaenopeptins by spectroscopic analysis, chemical degradation, and chemical synthesis. Analysis of the acidic hydrolysate showed that the racemization of Lys took place, leading us to pose a cautionary note on the configurational assignment of peptides that contain a ureido bond.

Geographic Variations in the Toxin Profile of the Xanthid Crab Zosimus aeneus in a Single Reef on Ishigaki Island, Okinawa, Japan.

Toxic crabs of the family Xanthidae contain saxitoxins (STXs) and/or tetrodotoxin (TTX), but the toxin ratio differs depending on their habitat. In the present study, to clarify within reef variations in the toxin profile of xanthid crabs, we collected specimens of the toxic xanthid crab Zosimus aeneus and their sampling location within a single reef (Yoshihara reef) on Ishigaki Island, Okinawa Prefecture, Japan, in 2018 and 2019. The STXs/TTX content within the appendages and viscera or stomach contents of each specimen was determined by instrumental analyses. Our findings revealed the existence of three zones in Yoshihara reef; one in which many individuals accumulate extremely high concentrations of STXs (northwestern part of the reef; NW zone), another in which individuals generally have small amounts of TTX but little STXs (central part of the reef; CTR zone), and a third in which individuals generally exhibit intermediate characteristics (southeastern part of the reef; SE zone). Furthermore, light microscopic observations of the stomach contents of crab specimens collected from the NW and CTR zones revealed that ascidian spicules of the genus Lissoclinum were dominant in the NW zone, whereas those of the genus Trididemnum were dominant in the CTR zone. Although the toxicity of these ascidians is unknown, Lissoclinum ascidians are considered good candidate source organisms of STXs harbored by toxic xanthid crabs.

Cytotoxic Glycosylated Fatty Acid Amides from a Stelletta sp. Marine Sponge.

We have discovered new glycosylated fatty acid amides, stellettosides, from a Stelletta sp. marine sponge. They were detected through LC-MS analysis of the extract combined with the cytotoxicity assay of the prefractionated sample. Their planar structures were determined by analyses of the NMR and tandem FABMS data. Stellettosides A1 and A2 (1 and 2) as well as stellettosides B1-B4 (3-6) were obtained as inseparable mixtures. Careful analysis of the NMR and tandem FABMS data of each mixture, along with comparison of the tandem FABMS data with that of a synthetic model compound, permitted us to assign the structure of the constituents in the mixture. The absolute configuration of the monosaccharide unit was determined by LC-MS after chiral derivatization. The relative configurations of the vicinal oxygenated methines in the fatty acid chains were assigned by the (1)H NMR data of the isopropylidene derivative. The mixture of stellettosides B1-B4 (3-6) exhibit moderate cytotoxic activity against HeLa cells with an IC50 value of 9 µM, whereas the mixture of stellettosides A1 and A2 (1 and 2) was not active at a concentration of 10 µM.

Peace, a MYB-like transcription factor, regulates petal pigmentation in flowering peach 'Genpei' bearing variegated and fully pigmented flowers.

Flowering peach Prunus persica cv. Genpei bears pink and variegated flowers on a single tree. The structural genes involved in anthocyanin biosynthesis were expressed strongly in pink petals but only very weakly or not at all in variegated petals. A cDNA clone encoding a MYB-like gene, isolated from pink petals was strongly expressed only in pink petals. Introduction of this gene, via biolistics gave magenta spots in the white areas of variegated petals, therefore this gene was named as Peace (peach anthocyanin colour enhancement). Differences in Peace expression determine the pattern of flower colouration in flowering peach. The R2R3 DNA-binding domain of Peace is similar to those of other plant MYBs regulating anthocyanin biosynthesis. Key amino acids for tertiary structure and the motif for interaction with bHLH proteins were conserved in Peace. Phylogenetic analysis indicates that Peace is closely related to AtMYB123 (TT2), which regulates proanthocyanidin biosynthesis in Arabidopsis, and to anthocyanin regulators in monocots rather than to regulators in dicots. This is the first report that a TT2-like R2R3 MYB has been shown to regulate anthocyanin biosynthesis.

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."

Spatial and temporal instability of local biotic community mediate a form of aposematic defense in newts, consisting of carotenoid-based coloration and tetrodotoxin.

Most animals advertise their unprofitability to potential predators via conspicuous signals. Whether the strength of this aposematic signal indicates the quality and quantity of chemical defenses in animals is controversial. Here, we investigated the relationship between the conspicuousness of an aposematic signal and toxicity, which likely depends, at least in part, on dietary sources, in the newt Cynops pyrrhogaster. Our results indicate that the magnitude of the aposematic signal was not correlated with the amount of tetrodotoxin (TTX) and 6-epi TTX of wild individuals among populations. Using atoxic newts, reared from eggs, we compared the ability to accumulate TTX from diets between mainland and island populations. Newts of a mainland population that exhibited a less conspicuous signal accumulated more TTX than did equivalent newts of an insular population that displayed a more conspicuous signal; this was unrelated to variation in the toxicity of wild individuals of these two populations. We also found toxicity of wild newts changed over approximately one generation (10 years) in both populations. These results indirectly suggest that environmental variance, such as fluctuations in TTX resources in nature, may obscure differences in the ability of wild newts to accumulate TTX, and that this variation may be responsible for a lack of correlation between the strength of a newt's signal and its toxicity in the wild. These results imply that toxicity of wild individuals likely is a phenotypic trait largely dependent on environmental conditions.

RT-PCR- and MALDI-TOF mass spectrometry-based identification and discrimination of isoforms homologous to pufferfish saxitoxin- and tetrodotoxin-binding protein in the plasma of non-toxic cultured pufferfish (Takifugu rubripes).

Four genes of Takifugu rubripes, tentatively designated Tr1-Tr4, encoding homologs of pufferfish saxitoxin- and tetrodotoxin-binding protein, were identified by BLAST search and 3'-RACE. RT-PCR and MALDI-TOF mass spectrometry allowed the identification and discrimination of Tr isoforms from the non-toxically cultured specimens. The expression of Tr1 and Tr3 mRNAs exclusively in the liver and the presence of their products as 120-kDa plasma proteins were confirmed.

Tetrodotoxin Profiles in Xanthid Crab Atergatis floridus and Blue-Lined Octopus Hapalochlaena cf. fasciata from the Same Site in Nagasaki, Japan.

The xanhid crab Atergatis floridus and the blue-lined octopus Hapalochlaena cf. fasciata have long been known as TTX-bearing organisms. It has been speculated that the TTX possessed by both organisms is exogenously toxic through the food chain, since they are reported to have geographic and individual differences. The source and supply chain of TTX for both of these organisms, however, remain unclear. On the other hand, since crabs are one of the preferred prey of octopuses, we focused our attention on the relationship between the two species living in the same site. The aim of this study was to determine TTX concentrations and TTX profiles of A. floridus and H. cf. fasciata, collected simultaneously in the same site, and examine the relationship between them. Although there were individual differences in the TTX concentration in both A. floridus and H. cf. fasciata, the toxin components commonly contained 11-norTTX-6(S)-ol in addition to TTX as the major components, with 4-epiTTX, 11-deoxyTTX, and 4,9-anhydroTTX as the minor components. The results suggest that octopuses and crabs in this site acquire TTX from common prey, including TTX-producing bacteria and/or may have a predator-prey relationship.

Tetrodotoxin/Saxitoxin Accumulation Profile in the Euryhaline Marine Pufferfish Chelonodontops patoca.

Marine Takifugu pufferfish, which naturally possess tetrodotoxins (TTXs), selectively take up and accumulate TTXs, whereas freshwater Pao pufferfish, which naturally possess saxitoxins (STXs), selectively take up and accumulate STXs. To further clarify the TTXs/STXs selectivity in pufferfish, we conducted a TTX/STX administration experiment using Chelonodontops patoca, a euryhaline marine pufferfish possessing both TTXs and STXs. Forty nontoxic cultured individuals of C. patoca were divided into a seawater group (SW, acclimated/reared at 33‱ salinity; n = 20) and a brackish water group (BW, acclimated/reared at 8‱ salinity; n = 20). An aqueous TTX/STX mixture was intrarectally administered (both at 7.5 nmol/fish), and five individuals/group were analyzed after 1-48 h. Instrumental toxin analyses revealed that both TTX and STX were taken up, transferred, and retained, but more STX than TTX was retained in both groups. TTX gradually decreased and eventually became almost undetectable in the intestinal tissue, while STX was retained at ~5-10% of the dose level, and only STX showed transient transfer in the liver. The BW group showed a faster decrease/disappearance of TTX, greater STX retention in the intestine, and greater STX transient transfer to the liver. Thus, C. patoca appears to more easily accumulate STXs than TTXs, especially under hypoosmotic conditions.

The quite low cross-reactivity of Kawatsu's anti-tetrodotoxin monoclonal antibody to 5,6,11-trideoxytetrodotoxin, 11-nortetrodotoxin-6(S)-ol, and 11-oxotetrodotoxin, the major tetrodotoxin analogues in pufferfish.

The monoclonal antibody against tetrodotoxin (TTX), prepared by Kawatsu et al. (1997), has been used in several TTX-related studies. Herein, we confirmed the quite low cross-reactivity of this antibody to three major TTX analogues in pufferfish using competitive ELISA: 5,6,11-trideoxyTTX (<2.2%), 11-norTTX-6(S)-ol (<0.3%), and 11-oxoTTX (<1.5%), with reactivity against TTX being 100%. We further confirmed that the presence of these analogues did not cause a marked overestimation of TTX in pufferfish extracts using competitive ELISA.

Local Differences in the Toxin Amount and Composition of Tetrodotoxin and Related Compounds in Pufferfish (Chelonodon patoca) and Toxic Goby (Yongeichthys criniger) Juveniles.

Tetrodotoxin (TTX)-bearing fish ingest TTX from their preys through the food chain and accumulate TTX in their bodies. Although a wide variety of TTX-bearing organisms have been reported, the missing link in the TTX supply chain has not been elucidated completely. Here, we investigated the composition of TTX and 5,6,11-trideoxyTTX in juveniles of the pufferfish, Chelonodon patoca, and toxic goby, Yongeichthys criniger, using LC-MS/MS, to resolve the missing link in the TTX supply chain. The TTX concentration varied among samples from different localities, sampling periods and fish species. In the samples from the same locality, the TTX concentration was significantly higher in the toxic goby juveniles than in the pufferfish juveniles. The concentration of TTX in all the pufferfish juveniles was significantly higher than that of 5,6,11-trideoxyTTX, whereas the compositional ratio of TTX and 5,6,11-trideoxyTTX in the goby was different among sampling localities. However, the TTX/5,6,11-trideoxyTTX ratio in the goby was not different among samples collected from the same locality at different periods. Based on a species-specific PCR, the detection rate of the toxic flatworm (Planocera multitentaculata)-specific sequence (cytochrome c oxidase subunit I) also varied between the intestinal contents of the pufferfish and toxic goby collected at different localities and periods. These results suggest that although the larvae of the toxic flatworm are likely to be responsible for the toxification of the pufferfish and toxic goby juveniles by TTX, these fish juveniles are also likely to feed on other TTX-bearing organisms depending on their habitat, and they also possess different accumulation mechanisms of TTX and 5,6,11-trideoxyTTX.

A novel spatially resolved interactance spectroscopy system to estimate degree of red coloration in red-fleshed apple.

Reliable information about degree of red coloration in fruit flesh is essential for grading and sorting of red-fleshed apples. We propose a spatially resolved interactance spectroscopy approach as a new rapid and non-destructive technique to estimate degree of red coloration in the flesh of a red-fleshed apple cultivar 'Kurenainoyume'. A novel measurement system was developed to obtain spatially resolved interactance spectra (190-1070 nm) for apple fruits at eight different light source-detector separation (SDS) distances on fruit surface. Anthocyanins in apple were extracted using a solvent extraction technique, and their contents were quantified with a spectrophotometer. Partial least squares (PLS) regression analyses were performed to develop estimation models for anthocyanin content from spatially resolved interactance spectra. Results showed that the PLS models based on interactance spectra obtained at different SDS distances achieved different predictive accuracy. Further, the system demonstrated the possibility to detect the degree of red coloration in the flesh at specific depths by identifying an optimal SDS distance. This might contribute to provide a detailed profile of the red coloration (anthocyanins) that is unevenly distributed among different depths of the flesh. This new approach may be potentially applied to grading and sorting systems for red-fleshed apples in fruit industry.

Tetrodotoxin/Saxitoxins Selectivity of the Euryhaline Freshwater Pufferfish Dichotomyctere fluviatilis.

The present study evaluated differences in the tetrodotoxin (TTX)/saxitoxins (STXs) selectivity between marine and freshwater pufferfish by performing in vivo and in vitro experiments. In the in vivo experiment, artificially reared nontoxic euryhaline freshwater pufferfish Dichotomyctere fluviatilis were intrarectally administered a mixture of TTX (24 nmol/fish) and STX (20 nmol/fish). The amount of toxin in the intestine, liver, muscle, gonads, and skin was quantified at 24, 48, and 72 h. STX was detected in the intestine over a long period of time, with some (2.7-6.1% of the given dose) being absorbed into the body and temporarily located in the liver. Very little TTX was retained in the body. In the in vitro experiments, slices of intestine, liver, and skin tissue prepared from artificially reared nontoxic D. fluviatilis and the marine pufferfish Takifugu rubripes were incubated in buffer containing TTX and STXs (20 nmol/mL each) for up to 24 or 72 h, and the amount of toxin taken up in the tissue was quantified over time. In contrast to T. rubripes, the intestine, liver, and skin tissues of D. fluviatilis selectively took up only STXs. These findings indicate that the TTX/STXs selectivity differs between freshwater and marine pufferfish.

Evidence of accumulation of tetrodotoxin (TTX) in tissues and body parts of ectoparasitic copepods via their feeding on mucus of TTX-bearing pufferfish.

Adults of the ectoparasitic copepod Caligus fugu found on tetrodotoxin (TTX)-bearing pufferfish such as Takifugu alboplumbeus and Takifugu flavipterus are known to accumulate TTX in body tissues and parts other than the ovaries, oviducts, eggs, and cuticles. This study aimed to demonstrate, using immunoenzymatic staining techniques, that the TTX-free planktonic/infective copepodid stage of C. fugu could accumulate TTX in the tissues after molting into the parasitic stage (chalimus I) and then fed on mucus of host puffers. All the tissues of the planktonic copepodids were completely TTX-free, whereas chalimus I copepods accumulated TTX in parts other than the cuticles, guts, and some muscles. Chalimus IV and adult copepods retained TTX in these body parts but not in the reproductive organs, which were TTX-resistant, indicating that TTX was not vertically transmitted via eggs. Non-cellular TTX-positive contents found in the guts of some chalimi and adults indicated that the copepods potentially accumulated TTX by feeding on host mucus rather than skin tissues and blood. This study revealed that the presence or absence of TTX in some body parts differed among individuals of the parasite.

Complete mitochondrial genomes of the Southeast Asian freshwater pufferfishes, Pao abei (Roberts, 1998) and Pao suvattii (Sontirat and Soonthornsatit, 1985) (Tetraodontiformes: Tetraodontidae) and an insight into the taxonomic status of Pao species.

The complete mitochondrial genomes of the Southeast Asian freshwater pufferfishes, Pao abei and Pao suvattii, were reconstructed using the MGISEQ platform. The genomes were 16,448 bp and 16,449 bp in length, each made up of 37 mitochondrial genes (13 CDSs, 22 tRNAs, and two rRNAs) and putative control region. It is suggested that an accumulation of complete mitochondrial genome sequences can contribute to resolve the taxonomic status of Pao species.

Ultrasonic-Assisted Extraction and Structural Characterization of Chondroitin Sulfate Derived from Jumbo Squid Cartilage.

Chondroitin sulfate (ChS) is usually used as an oral nutraceutical supplement, and has been popular in Asia, Europe, and United States for many years. In this study, a potential and sustainable source of ChS from jumbo squid (Dosidicus gigas) cartilage was explored; ultrasound-assisted extraction (UAE) was used to extract ChS from jumbo squid cartilage. The result of mass transfer coefficients based on Fick's law showed that UAE had higher mass transfer efficacy. The response surface methodology (RSM) combined with Box-Behnken design (BBD) was employed to evaluate the effects of the extraction parameters. The optimal conditions were extraction temperature of 52 °C, extraction time of 46 min, and NaOH concentration of 4.15%. The crude extract was precipitated by 50% ethanol, which obtained a purified ChS with 23.7% yield and 82.3% purity. The purified ChS measured by energy-dispersive X-ray spectroscopy (EDX) had a carbon to sulfur molar ratio of approximately 14:1. The FTIR, 1H, and 13C NMR confirmed jumbo squid ChS were present in the form of chondroitin-4-sulfate and chondroitin-6-sulfate, with a 4S/6S ratio of 1.62. The results of this study provide an efficient process for production and purification of ChS, and are significant for the development and utilization of ChS from jumbo squid cartilage in the nutrient food or pharmaceutical industries.

Myrindole A, an Antimicrobial Bis-indole from a Marine Sponge Myrmekioderma sp.

Myrindole A, a bis-indole alkaloid, was isolated from the deep-sea sponge Myrmekioderma sp. The high degree of unsaturation of the molecule complicated the assignment of its structure by standard 2D-NMR experiments but was ultimately achieved by a combination of 1H-15N-HMBC and 1,n-ADEQUATE experiments as well as the comparison of measured and calculated CD spectra. Myrindole A showed antimicrobial activity against Gram-positive and Gram-negative bacteria.

The role of toxic planocerid flatworm larvae on tetrodotoxin accumulation in marine bivalves.

Tetrodotoxin (TTX), also known as pufferfish toxin, has been detected in marine edible bivalves worldwide. In this study, several bivalve species, Azumapecten farreri subsp. akazara, Patinopecten yessoensis and Mytilus galloprovincialis, collected from the Pacific side of the northern Japanese Islands, were studied for the accumulation of TTX in the presence of toxic planocerid larvae. LC-MS/MS analysis demonstrated that TTX was detected only in the midgut gland of A. farreri subsp. akazara. Toxic flatworm-specific PCR and direct sequencing of the amplicons showed that the DNA fragments of the Planocera multitentaculata COI gene were detected in the gut contents of the toxified bivalves. The planocerid larvae were also detected in the environmental seawaters. Toxification experiments in the aquarium demonstrated that the mussel M. galloprovincialis was also toxified by feeding on the toxic flatworm larvae. These results suggest that the source of TTX accumulation in edible bivalves is toxic flatworm larvae.

Co-Occurrence of Tetrodotoxin and Saxitoxins and Their Intra-Body Distribution in the Pufferfish Canthigaster valentini.

Pufferfish of the family Tetraodontidae possess tetrodotoxin (TTX) and/or saxitoxins (STXs), but the toxin ratio differs, depending on the genus or species. In the present study, to clarify the distribution profile of TTX and STXs in Tetraodontidae, we investigated the composition and intra-body distribution of the toxins in Canthigaster valentini. C. valentini specimens (four male and six female) were collected from Amami-Oshima Island, Kagoshima Prefecture, Japan, and the toxins were extracted from the muscle, liver, intestine, gallbladder, gonads, and skin. Analysis of the extracts for TTX by liquid chromatography tandem mass spectrometry and of STXs by high-performance liquid chromatography with post-column fluorescence derivatization revealed TTX, as well as a large amount of STXs, with neoSTX as the main component and dicarbamoylSTX and STX itself as minor components, in the skin and ovary. The toxins were also detected in the other tissues, but in much lower amounts than in the skin and ovary. The TTX/STX ratio varied greatly, depending on the tissue, but TTX was the major toxin component in the whole body, and STXs accounted for 25% and 13% of the total toxin amount in males and females, respectively. Like the marine pufferfish of the genus Arothron, C. valentini should be considered a pufferfish with considerable amounts of both TTX and STXs present simultaneously.