RESUMEN

Dinoflagellates within the genus Karenia are well known for their potential to cause harmful algal blooms and induce detrimental ecological consequences. In this study, five Karenia species, Karenia longicanalis, Karenia papilionacea, Karenia mikimotoi, Karenia selliformis, and a new species, Karenia hui sp. nov., were isolated from Chinese coastal waters. The new species exhibits the typical characteristics of the genus Karenia, including a linear apical groove and butanoyl-oxyfucoxanthin as the major accessory pigment. It is distinguished from the other Karenia species by a wide-open sulcal intrusion onto the epicone, a conical epicone with an apical crest formed by the rim of the apical groove, and a hunchbacked hypocone. It is most closely related to Karenia cristata, with a genetic divergence of 3.16 % (22 bp out of 883 bp of LSU rDNA). Acute toxicity tests indicated that the five Karenia species from China are all toxic to marine medaka Oryzias melastigma. Karenia selliformis and K. hui were very toxic to O. melastigma, resulting in 100 % mortality within 4 h and 24 h, respectively. Further analysis by high-performance liquid chromatography revealed that four species, K. selliformis, K. longicanalis, K. papilionacea and K. mikimotoi were capable of producing Gymnodimine-A (GYM-A). The highest GYM-A content was in K. selliformis (strain HK-43), in which the value was 889 fg/cell. No GYM-A was detected in the new species K. hui, however and its toxin remains unknown. Below we provide a comprehensive report of the morphology, phylogeny, pigment composition, and toxicity profiles of Karenia species along the Chinese coast. These findings contribute new insights for monitoring of Karenia species, with important toxicological and ecological implications.

Asunto(s)

RESUMEN

Harmful algal blooms kill fish populations worldwide, as exemplified by the haptophyte microalga Prymnesium parvum. The suspected causative agents are prymnesins, categorized as A-, B-, and C-types based on backbone carbon atoms. Impacts of P. parvum extracts and purified prymnesins were tested on the epithelial rainbow trout fish gill cell line RTgill-W1 and on the human colon epithelial cells HCEC-1CT. Cytotoxic potencies ranked A > C > B-type with concentrations spanning from low (A- and C-type) to middle (B-type) nM ranges. Although RTgill-W1 cells were about twofold more sensitive than HCEC-1CT, the cytotoxicity of prymnesins is not limited to fish gills. Both cell lines responded rapidly to prymnesins; with EC50 values for B-types in RTgill-W1 cells of 110 ± 11 nM and 41.5 ± 0.6 nM after incubations times of 3 and 24 h. Results of fluorescence imaging and measured lytic effects suggest plasma membrane interactions. Postulating an osmotic imbalance as mechanisms of toxicity, incubations with prymnesins in media lacking either Cl-, Na+, or Ca2+ were performed. Cl- removal reduced morphometric rearrangements observed in RTgill-W1 and cytotoxicity in HCEC-1CT cells. Ca2+-free medium in RTgill-W1 cells exacerbated effects on the cell nuclei. Prymnesin composition of different P. parvum strains showed that analog composition within one type scarcely influenced the cytotoxic potential, while analog type potentially dictate potency. Overall, A-type prymnesins were the most potent ones in both cell lines followed by the C-types, and lastly B-types. Disturbance of Ca2+ and Cl- ionoregulation may be integral to prymnesin toxicity.

Asunto(s)

RESUMEN

Dinoflagellates of the genus Karlodinium are ichthyotoxic species that produce toxins including karlotoxins and karmitoxins. Karlotoxins show hemolytic and cytotoxic activities and have been associated with fish mortality. This study evaluated the effect of toxins released into the environment of Karlodinium veneficum strain K10 (Ebro Delta, NW Mediterranean) on the early stages of Danio rerio (zebrafish). Extracts of the supernatant of K10 contained the mono-sulfated KmTx-10, KmTx-11, KmTx-12, KmTx-13, and a di-sulfated form of KmTx-10. Total egg mortality was observed for karlotoxin concentration higher than 2.69 µg L-1. For 1.35 µg L-1, 87% of development anomalies were evidenced (all concentrations were expressed as KmTx-2 equivalent). Larvae of 8 days postfertilization exposed to 1.35 µg L-1 presented epithelial damage with 80% of cells in the early apoptotic stage. Our results indicate that supernatants with low concentration of KmTxs produce both lethal and sublethal effects in early fish stages. Moreover, apoptosis was induced at concentrations as low as 0.01 µg L-1. This is of great relevance since detrimental long-term effects due to exposure to low concentrations of these substances could affect wild and cultured fish.

Asunto(s)

RESUMEN

Mitigation of fish-killing algal toxins by clay minerals offers great promise as an emergency strategy for fish farms threatened by harmful algal blooms, but its efficiency is highly clay and algal species (i.e. ichthyotoxin) specific. We here screened several different clay types (kaolin, zeolite, Korean loess and six bentonites) for their adsorptive capacity of extracellular Karlodinium veneficum and Karenia mikimotoi ichthyotoxins as quantified with the rainbow trout RTgill-W1 cell line assay. Treatment with Korean loess, zeolite (0-0.5 g L - 1), polyaluminium chloride (0-0.1 g L - 1) and clays modified with this flocculant (0-0.25 g L - 1) could not significantly improve gill cell viability compared to toxic controls. Kaolin only demonstrated effective removal in case of K. mikimotoi, but concentrations required for complete removal of cytotoxicity were at least 2 x those required for bentonite. Bentonites of high swelling capacity and ideally small particle size (<2 µm) proved best suited for ichthyotoxin removal against both algal species (100% removal at concentrations as low as 0.1 g L - 1). Complete elimination of K. veneficum and K. mikimotoi toxicity towards the rainbow trout gill cell line was achieved by bentonite clay, demonstrating the potential to control ichthyotoxicity in an aquaculture setting through targeted clay application.

Asunto(s)

RESUMEN

Amphidinols are polyketides produced by dinoflagellates suspected of causing fish kills. Here, we demonstrate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the identification and quantification of amphidinols (AM). Novel AM were detected by neutral loss (NL) scan and then quantified together with known AM by selection reaction monitoring (SRM). With the new method, AM were detected in four of eight analyzed strains with a maximum of 3680 fg toxin content per cell. In total, sixteen novel AM were detected by NL scan and characterized via their fragmentation patterns. Of these, two substances are glycosylated forms. This is the first detection of glycosylated AM.

Asunto(s)

RESUMEN

Harmful algal blooms of Prymnesium parvum have recurrently been associated with the killing of fish. The causative ichthyotoxic agents of this haptophyte are believed to be prymnesins, a group of supersized ladder-frame polyether compounds currently divided into three types. Here, the development of a quantitative method to assess the molar sum of prymnesins in water samples and in algal biomass is reported. The method is based on the derivatization of the primary amine group and subsequent fluorescence detection using external calibrants. The presence of prymnesins in the underivatized sample should be confirmed by liquid chromatography mass spectrometry. The method is currently only partly applicable to water samples due to the low amounts that are present. The growth and cellular toxin content of two B-type producing strains were monitored in batch cultures eventually limited by an elevated pH. The cellular toxin contents varied by a factor of ~2.5 throughout the growth cycle, with the highest amounts found in the exponential growth phase and the lowest in the stationary growth/death phases. The strain K-0081 contained ~5 times more toxin than K-0374. Further investigations showed that the majority of prymnesins were associated with the biomass (89% ± 7%). This study provides the basis for further investigations into the toxicity and production of prymnesins.

Asunto(s)

RESUMEN

Colorectal cancer (CRC) remains one of the most commonly diagnosed cancers and the 3rd leading cause of cancer-related mortality. The emergence of drug resistance poses a major challenge in CRC care or treatment. This can be addressed by determining cancer mechanisms, discovery of druggable targets, and development of new drugs. In search for novel agents, aquatic microorganisms offer a vastly untapped pharmacological source that can be developed for cancer therapeutics. In this study, we characterized the anti-colorectal cancer potential of euglenophycin, a microalgal toxin from Euglena sanguinea. The toxin (49.1-114.6 µM) demonstrated cytotoxic, anti-proliferative, anti-clonogenic, and anti-migration effects against HCT116, HT29, and SW620 CRC cells. We identified G1 cell cycle arrest and cell type - dependent modulation of autophagy as mechanisms of growth inhibition. We validated euglenophycin's anti-tumorigenic activity in vivo using CRL:Nu(NCr)Foxn1nu athymic nude mouse CRC xenograft models. Intraperitoneal toxin administration (100 mg/kg; 5 days) decreased HCT116 and HT29 xenograft tumor volumes (n=10 each). Tumor inhibition was associated with reduced expression of autophagy negative regulator mechanistic target of rapamycin (mTOR) and decreased trend of serum pro-inflammatory cytokines. Together, these results provide compelling evidence that euglenophycin can be a promising anti-colorectal cancer agent targeting multiple cancer-promoting processes. Furthermore, this study supports expanding natural products drug discovery to freshwater niches as prospective sources of anti-cancer compounds.

RESUMEN

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the detection and quantitation of karlotoxins in the selected reaction monitoring (SRM) mode. This novel method was based upon the analysis of purified karlotoxins (KcTx-1, KmTx-2, 44-oxo-KmTx-2, KmTx-5), one amphidinol (AM-18), and unpurified extracts of bulk cultures of the marine dinoflagellate Karlodinium veneficum strain CCMP2936 from Delaware (Eastern USA), which produces KmTx-1 and KmTx-3. The limit of detection of the SRM method for KmTx-2 was determined as 2.5 ng on-column. Collision induced dissociation (CID) spectra of all putative karlotoxins were recorded to present fragmentation patterns of each compound for their unambiguous identification. Bulk cultures of K. veneficum strain K10 isolated from an embayment of the Ebro Delta, NW Mediterranean, yielded five previously unreported putative karlotoxins with molecular masses 1280, 1298, 1332, 1356, and 1400 Da, and similar fragments to KmTx-5. Analysis of several isolates of K. veneficum from the Ebro Delta revealed small-scale diversity in the karlotoxin spectrum in that one isolate from Fangar Bay produced KmTx-5, whereas the five putative novel karlotoxins were found among several isolates from nearby, but hydrographically distinct Alfacs Bay. Application of this LC-MS/MS method represents an incremental advance in the determination of putative karlotoxins, particularly in the absence of a complete spectrum of purified analytical standards of known specific potency.

Asunto(s)