Salinity-Growth Response and Ichthyotoxic Potency of the Chilean Pseudochattonella verruculosa

Abstract

Despite salmon farmers suffering the worst damage from a harmful algal bloom in Chile's history (US$800M) due to a massive outbreak of the dictyochophyte Pseudochattonella verruculosa in 2016 (∼7000-20,000 cells ml −1), the effect of environmental drivers and the potency of lytic toxins produced by the local clones of this species remain still largely unexplored. Based on the drastic oceanographic anomalies observed in the Chilean fjords during the 2016-El Niño "Godzilla" event, the role of salinity (15 to 35 psu) on Pseudochattonella cell growth and cytotoxicity was studied by culturing, scanning electron microscopy (SEM) and using a rainbow trout cell line RTgill-W1 assay to define: (1) vegetative growth rates, (2) cell taxonomy, (3) ichthyotoxicity of monoclonal cultures at 25 and 35 psu in salinity, (4) differences in toxicity of lysed cells and supernatant at different cell concentrations (from 10 to 100,000 cells ml −1), and (5) temporal stability of lytic compounds. This study formally confirms the presence of P. verruculosa in Chilean waters using the large subunit (LSU) of the nuclear ribosomal RNA gene. The Chilean P. verruculosa ARC498 strain showed maximum cell densities at 30 psu (max. 84,333 ± 4,833 cells ml −1) and maximum growth rates (µ max) at 20 psu (1.44 cells d −1). Cultures at 15 psu showed suppressed maximum cell density (max. 269 ± 71 cell ml −1) but high µ max were recorded at the beginning of the exponential growth (1.31 cells d −1). No significant differences were observed between lysed cells and supernatant treatments in the two salinity levels, suggesting that the most lytic portion is released into the cell-free media instead of remaining cell bound. Cytotoxicity was correlated to cell abundance, reducing gill cell viability down to 80 and 65% compared to controls at 10,000 and 100,000 cells ml −1 , respectively. Unexpectedly, lytic compounds from P. verruculosa ARC498 at 35 psu showed to be less toxic than cultures at 25, where a noticeable presence of peripheral mucocysts were observed by SEM. Lytic compounds from in vitro experiments are weakly toxic even at high cell concentrations, highly unstable and rapidly degraded in the light after 5 days of storage at 15 • C. Our results point to the important effect of salinity on growth and ichthyotoxic potency of Pseudochattonella species and highlight the need for a deeper insight into the role of mucocysts in fish gill damage, which would provide a greater understanding as to the harmful modes of action of this species.

The State of Knowledge of Harmful Algal Blooms of Margalefidinium polykrikoides (a.k.a. Cochlodinium polykrikoides) in Latin America

“…The higher amount of reports from Mexico might be because this country possesses a longer coastal area, as well as having a high number of experts on harmful algae, or simply because M. polykrikoides has formed more blooms in the Mexican coastline than in other Latin America countries. The presence of Margalefidinium as well as other species of marine fish-killing microalgae in Latin America will require constant monitoring mainly in fish-growing areas to avoid severe economic impacts, such as the recent cases in Chile and Mexico (Clement et al, 2016;García-Mendoza et al, 2018;León-Muñoz et al, 2018;Mardones et al, 2019).…”

Blooms of Alexandrium catenella in Coastal Waters of Chilean Patagonia: Is Subantarctic Surface Water Involved?

“…High cell densities of P. verruculosa were noted at relatively high temperatures (>15 • C) during previous bloom episodes (Trainer et al, 2020), but in culture, growth rates and maximum cell densities do not vary strongly between temperatures of 12-18 • C (Mardones et al, 2021). Cell growth of Chilean P. verruculosa is enhanced under moderate-high salinity conditions (Mardones et al, 2019), and cells aggregate into thin layers within the halocline in Reloncaví Sound (Mardones et al, 2021) with a vertical migration behavior that might allow access to DIN and DIP in MSAW (León-Muñoz et al, 2018) and promote retention in a fjord with low flushing rates (Mardones et al, 2021). The apparent convergence of blooms of P. verruculosa and A. catenella in 2016 from opposite latitudes suggests a possible overlap of differential preferences along the MSAW-EW spectrum.…”

Screening of Chilean fish-killing microalgae using a gill cell-based assay

“…pacted coasts (Tanneberger et al, 2013;Dorantes-Aranda et al, 2015;Natsch et al, 2018). Through this cell line assay, ichthyotoxic compounds produced by Chilean strains of A. catenella and P. verruculosa have been initially explored (Mardones et al, 2015(Mardones et al, , 2018(Mardones et al, , 2019. Synergism between the long-chain (C20) polyunsaturated fatty acids (PUFA) docosahexaenoic acid (DHA, 22:63; 16-20% of total fatty acids) and reactive oxygen species (ROS -as superoxide anion) has been suggested as a primary ichthyotoxic mechanism in A. catenella, but cannot be explained by paralytic shellfish toxins (Mardones et al, 2015(Mardones et al, , 2018.…”

“…Synergism between the long-chain (C20) polyunsaturated fatty acids (PUFA) docosahexaenoic acid (DHA, 22:63; 16-20% of total fatty acids) and reactive oxygen species (ROS -as superoxide anion) has been suggested as a primary ichthyotoxic mechanism in A. catenella, but cannot be explained by paralytic shellfish toxins (Mardones et al, 2015(Mardones et al, , 2018. Furthermore, the role of mucocysts has been pointed for a more in-depth research insight, which could provide a greater understanding as to the harmful mode of action of P. verruculosa (Mardones et al, 2019).…”

“…First, Karenia selliformis has been observed to bloom in offshore areas (2-10 NM), barely affecting inshore salmon farming activities (<2 cells mL -1 ). Exceptional outbreaks of this toxic dinoflagellate, in inner channels, might, however, be even more damaging than the Pseudochattonella bloom recorded in 2016 (Mardones et al, 2019); thus, early warning systems should be activated when the presence of this dinoflagellate species. Second, according to the present historical analysis, there is an apparent increment in the occurrence of phytoflagellates and their ichthyotoxic potency during the last decade in southern Chile (see the ranking in Table 1).…”