Environmental evaluation of the Reloncaví estuary in southern Chile based on lipophilic shellfish toxins as related to harmful algal blooms.

The Reloncaví estuary in southern Chile is famous for its aquaculture. However, recurring harmful algal blooms have adversely affected mussel production. Therefore, regular monitoring of algal toxins is urgently needed to better understand the contamination status of the estuary. In this study, we quantified 15 types of lipophilic shellfish toxins in Metri Bay in the Reloncaví estuary on a biweekly basis for 4 years. We identified algal species using microscopy and metabarcoding analysis. We also measured water temperature, salinity, chlorophyll-a, and dissolved oxygen to determine the potential relationships of these parameters with algal toxin production. Our results revealed the presence of a trace amount of pectenotoxin and the causal phytoplankton Dinophysis, as well as yessotoxin and the causal phytoplankton Protoceratium. Statistical analysis indicated that fluctuations in water temperature affected the detection of these toxins. Additionally, metabarcoding analysis detected the highly toxic phytoplankton Alexandrium spp. in some samples. Although our results suggest that the level of lipophilic shellfish toxins in Metri Bay during the study period was insignificantly low using our current LC-MS method, the confirmed presence of highly toxic algae in Metri Bay raises concerns, given that favorable environmental conditions could cause blooms.

Diversity and toxicity of the planktonic diatom genus Pseudo-nitzschia from coastal and offshore waters of the Southeast Pacific, including Pseudo-nitzschia dampieri sp. nov.

To expand knowledge of Pseudo-nitzschia species in the Southeast Pacific, we isolated specimens from coastal waters of central Chile (36°S-30°S), the Gulf of Corcovado, and the oceanic Robinson Crusoe Island (700 km offshore) and grew them into monoclonal strains. A total of 123 Pseudo-nitzschia strains were identified to 11 species based on sequencing of the ITS region of the nuclear rDNA and on ultrastructural and morphometric analyses of the frustule in selected representatives of each clade: P. australis, P. bucculenta, P. cf. chiniana, P. cf. decipiens, P. fraudulenta, P. hasleana, P. multistriata, P. plurisecta, P. cf. sabit, the new species P. dampieri sp. nov., and one undescribed species. Partial 18S and 28S rDNA sequences, including the hypervariable V4 and D1-D3 regions used for barcoding, were gathered from representative strains of each species to facilitate future metabarcoding studies. Results showed different levels of genetic, and at times ultrastructural, diversity among the above-mentioned entities, suggesting morphological variants (P. bucculenta), rapidly radiating complexes with ill-defined species boundaries (P. cf. decipiens and P. cf. sabit), and the presence of new species (P. dampieri sp. nov., Pseudo-nitzschia sp. 1, and probably P. cf. chiniana). Domoic acid (DA) was detected in 18 out of 82 strains tested, including those of P. australis, P. plurisecta, and P. multistriata. Toxicity varied among species mostly corresponding to expectations from previous reports, with the prominent exception of P. fraudulenta; DA was not detected in any of its 10 strains tested. In conclusion, a high diversity of Pseudo-nitzschia exists in Chilean waters, particularly offshore.

Emerging phycotoxins in the Chilean coast: First localized detection of the neurotoxic cyclic imine Pinnatoxin-G in shellfish banks.

Pinnatoxins (PnTXs) produced by the cosmopolitan dinoflagellate Vulcanodinium rugosum are highly potent cyclic imines that represent a risk for seafood consumers, artisanal fisheries, and the local aquaculture industry. Among the eight known PnTXs, pinnatoxin-G (PnTX-G) is the most frequent toxin analog detected in shellfish. Despite PnTX-G is still not internationally regulated, the French Agency for Food, Environmental and Occupational Health and Safety established that a risk for human consumers may exist when the accumulation of PnTX-G in shellfish exceeds 23 µg kg-1. This study reports the first detection of these fast-acting lipophilic toxins in localized shellfish banks (Mytilus chilensis) from the Chilean coast. Among 32 sentinel sampling stations monthly monitored for phytotoxins detection and quantification between 2021 and 2022 along the southern Chilean coast (from 36°25' S to 54°57'S), PnTx-G was only detected in shellfish from the southernmost region of Magallanes in concentrations that ranged between 15 and 100 µg kg-1, highlighting the binational (Chile/Argentina) Beagle Channel as a 'hotspot'. As Chile is one of the major mussel producers worldwide, this result raises concern about the potential adverse effect of PnTXs for human health and point to the need of governmental actions for an enhanced monitoring of these emerging toxins. To date, the production of PnTXs has not yet been associated with any microalgae species in Chilean waters.

Heterosigma akashiwo in Patagonian Fjords: Genetics, Growth, Pigment Signature and Role of PUFA and ROS in Ichthyotoxicity.

Heterosigma akashiwo is the only raphidophyte described for Chilean waters. A recent 2021 fish-killing bloom event of this raphidophyte ignited scientific research, but the ichthyotoxic mechanism and environmental conditions that promote its growth are still unclear. This is the first study confirming the occurrence of H. akashiwo in Chilean waters on the basis of the region D1/D2 of the 28S ribosomal gene. The pigment signature of the CREAN_HA03 strain revealed chlorophyll-a, fucoxanthin, and violaxanthin as the most abundant pigments, but profiles were variable depending on culture and field conditions. A factorial temperature−salinity growth experiment showed a maximal growth rate of 0.48 d−1 at 17 °C and 35 in salinity, but reached a maximal cell abundance of ~50,000 cells mL−1 at 12 °C and 25 in salinity. The fatty acid profile included high levels of saturated (16:0) and polyunsaturated (18:4 ω3; 20:5 ω3) fatty acids, but superoxide production in this strain was low (~0.3 pmol O2− cell−1 h−1). The RTgill-W1 bioassay showed that the H. akashiwo strain was cytotoxic only at high cell concentrations (>47,000 cells mL−1) and after cell rupture. In conclusion, salmon mortality during H. akashiwo bloom events in Patagonian fjords is likely explained by the high production of long-chain PUFAs at high cell densities, but only in the presence of high ROS production.

Toxic Alexandrium catenella expanding northward along the Chilean coast: New risk of paralytic shellfish poisoning off the Bío-Bío region (36° S).

In Chile, the toxic dinoflagellate A. catenella shows an apparent oceanic range expansion from south to the north since its first detection in 1972 in the Magallanes Region (56° S). Until 2017, A. catenella detections were restricted to a geographic area between Magallanes to Los Rios Regions (40° S). The establishment of a monitoring program in the offshore Pacific coast allowed the detection of A. catenella between 2018 and 2019 in northern areas off the Bío-Bío Region (36°S). Monoclonal cultures established from the Bío-Bío coast were genetically identified, and PSTs screened. Phylogenetic analyses determined that the Bío-Bío isolates aggregated in Group I ribotype (previously A. tamarense or A. fundyense) and the presence of PSTs analogs were confirmed. It is the northernmost detection of the toxic dinoflagellate A. catenella in the Pacific coast of Chile. These results have important implications for species monitoring and governmental management in the Bío-Bío Region.

Disentangling the environmental processes responsible for the world's largest farmed fish-killing harmful algal bloom: Chile, 2016.

The dictyochophyte microalga Pseudochattonella verruculosa was responsible for the largest farmed fish mortality ever recorded in the world, with losses for the Chilean salmon industry amounting to US$ 800 M in austral summer 2016. Super-scale climatic anomalies resulted in strong vertical water column stratification that stimulated development of a dynamic P. verruculosa thin layer (up to 38 µg chl a L-1) for several weeks in Reloncaví Sound. Hydrodynamic modeling (MIKE 3D) indicated that the Sound had extremely low flushing rates (between 121 and 200 days) in summer 2016. Reported algal cell densities of 7000-20,000 cells mL-1 generated respiratory distress in fish that was unlikely due to low dissolved oxygen (permanently >4 mg L-1). Histological examination of salmon showed that gills were the most affected organ with significant tissue damage and circulatory disorders. It is possible that some of this damage was due to a diatom bloom that preceded the Pseudochattonella event, thereby rendering the fish more susceptible to Pseudochattonella. No correlation between magnitude of fish mortality and algal cell abundance nor fish age was evident. Algal cultures revealed rapid growth rates and high cell densities (up to 600,000 cells mL-1), as well as highly complex life cycle stages that can be easily overlooked in monitoring programs. In cell-based bioassays, Chilean P. verruculosa was only toxic to the RTgill-W1 cell line following exposures to high cell densities of lysed cells (>100,000 cells mL-1). Fatty acid profiles of a cultured strain showed elevated concentrations of potentially ichthyotoxic, long-chain polyunsaturated fatty acids (PUFAs) (69.7% ± 1.8%)- stearidonic (SDA, 18:4ω3-28.9%), and docosahexaenoic acid (DHA, 22:6ω3-22.3%), suggesting that lipid peroxidation may help to explain the mortalities, though superoxide production by Pseudochattonella was low (< 0.21 ± 0.19 pmol O2- cell-1 h-1). It therefore remains unknown what the mechanisms of salmon mortality were during the Pseudochattonella bloom. Multiple mitigation strategies were used by salmon farmers during the event, with only delayed seeding of juvenile fish into the cages and towing of cages to sanctuary sites being effective. Airlift pumping, used effectively against other fish-killing HABs in the US and Canada was not effective, perhaps because it brought subsurface layers of Pseudochattonella to the surface, or and it also may have lysed the fragile cells, rendering them more lethal. The present study highlights knowledge gaps and inefficiency of contingency plans by the fish farming industry to overcome future fish-killing algal blooms under future climate change scenarios. The use of new technologies based on molecular methods for species detection, good farm practices by fish farms, and possible mitigation strategies are discussed.

Unraveling the Karenia selliformis complex with the description of a non-gymnodimine producing Patagonian phylotype.

Karenia selliformis is a bloom-forming toxic dinoflagellate known for production of gymnodimines (GYMs) and causing mass mortalities of marine fauna. Blooms have been reported from coastal waters of New Zealand, Mexico, Tunisia, Kuwait, Iran, China and Chile. Based on molecular phylogeny, morphology, toxin production, pigment composition and cell growth of Chilean K. selliformis isolated in 2018 (CREAN_KS01 and CREAN_KS02), this study revealed a more complex diversity within this species than previously thought. A phylogenetic reconstruction based on the large sub-unit ribosomal nucleotide (LSU rDNA) and Internal Transcriber Spacer (ITS) sequences of 12 worldwide isolates showed that within the K. selliformis clade there are at least two different phylotypes with clear phenotypic differences. Morphological differences related to the dorsal-ventral compression, shape of the hyposome and the presence of pores on the left lobe of the hyposome. A comparison of pigment signatures among worldwide isolates revealed the existence of both acyl-oxyfucoxanthin and fucoxanthin-rich strains within the phylotypes. A LC-MS/MS screening on both Chilean 2018 K. selliformis strains showed for first time no GYMs production among cultured clones of this species. However, both CREAN_KS01 and CREAN_KS02 contained two compounds with the same mass transition as brevenal, a brevetoxin related compound. A fish gill cell-based assay showed that the CREAN_KS02 strain was highly cytotoxic but pure GYM standard did not exhibit loss of cell viability, even at cell concentrations equivalent or exceeding those reported in nature. The fatty acid profile of CREAN_KS02 included high levels of saturated (14:0; 16:0) and polyunsaturated (18:3ω6+18:5ω3; 22:6ω3) fatty acids but superoxide production in this strain was low (0.86±0.53 pmol O2- cell-1 h-1). A factorial T-S growth experiment using the CREAN_KS02 strain showed a µmax of 0.41±0.03 d-1 at high salinity and temperature, which points to its optimal environmental niche in offshore waters during the summer season. In conclusion, the present study provides evidence for significant genetic and phenotypic variability among worldwide isolates, which points to the existence of a K. selliformis "species complex". The massive fauna mortality during K. selliformis bloom events in the Chilean coast cannot be explained by GYMs nor brevetoxins, but can to a large extent be accounted for by the high production of long-chain PUFAs and/or still uncharacterized highly toxic compounds.

Development and validation of a method for the simultaneous extraction and separate measurement of oxytetracycline, florfenicol, oxolinic acid and flumequine from marine sediments.

A simple and rapid method for the detection and extraction of oxolinic acid, flumequine, florfenicol and oxytetracycline from marine sediments was developed and validated. The analytes were extracted from the marine sediment using a solution of oxalic acid diluted in methanol with sonication before detection by HPLC using a diode-array detector (florfenicol and oxytetracycline) and fluorescence (oxolinic acid and flumequine). The quantification limits (QL) were 100 ng/g for oxytetracycline and florfenicol and 5 ng/g for oxolinic acid and flumequine. The coefficients of variation of the repeatability and intermediate precision were less than 10% in all of the analytes. The calibration curves were linear between 50 and 500 ng/ml for oxytetracycline and florfenicol and 1 and 20 ng/ml for oxolinic acid and flumequine. The recuperation rate for the analytes was above 86%.

Coexistencia de neoplasias intraepiteliales cérvico-uterinas y condiloma plano: análisis de la distorsión en diagnósticos provocada por condilomas / Association of cervical intrahepitelial neoplasia and condyloma latum: analysis of diagnostic mistake due to condyloma

Con el fin de establecer la importancia de la presencia de condilomas planos en biopsias diagnosticadas previamente a su conocimiento como entidad morfológica, se revisan 414 cuellos uterinos obtenidos por conización o histerectomía, hasta 1978. Se comprueba que la coexistencia de condiloma con diversos grados de neoplasias intraepiteliales (NIE) es alta; en NIE III hay condiloma concomitante en 43,4% de los casos; en NIE II es de 59,3% y en NIE I de 63,9%. Esta coexistencia provocó sobrevaloración diagnóstica en un número importante de casos y gran parte de ésta ocurrió a causa de haber interpretado lesiones condilomatosas como NIE I y II

Estudio morfológico en 152 mamas de autopsias médico-legales / Morphological study in 152 breasts from medico-legal autopsies

Con el objeto de estudiar la morfología del tejido mamario en nuestro medio, se examinaron 152 muestras obtenidas de autopsias médico-legales, separando el material por grupos de edad, con amplitud de 14 a 96 años. Se encontró fibrosis lobulillar en el 70% de los casos, con una presencia ligeramente superior al 50% en la tercera y cuartas décadas. Los microquistes estaban presentes en el 50%; los macroquistes en el 9%; la metaplasia apocrina en el 28%, y la hiperplasia epitelial en el 9%. Estos hallazgos concuerdan con la observación de otros autores, que han informado una alta presencia de rasgos morfológicos de la enfermedad fibroquística en tejido mamario aparentemente sano. El examen de nuestro material estaría indicando que la fibrosis, los microquistes y la metaplasia apocrina son muy frecuentes, y su presencia aislada no permite formular el diagnóstico de enfermedad fibroquística de la mama