Distribution and abundance of dinoflagellates from the coastal waters of Karachi, Pakistan, northern part of the Arabian Sea

The present study reports on seasonal and spatial variations in diversity, distribution and abundance of dinoflegellates and indicates the presence of HAB species in Pakistan waters. A total of 179 taxa, recorded in this study from offshore and near-shore waters, belong to 41 genera in 26 families and 10 orders. The high species count (149 species) was recorded from Manora Island offshore station (MI-1) and 105 spp, 109 spp and 115 spp were encountered from the Mubarak village offshore station (MV-1), Manora near shore station (MI-2) and Mubarak Village near-shore station (MV-2) respectively. Tripos furca was the dominant and frequently occurring species (> 1 x103 to > 25 x103 cells L-1 from coastal and >1x 105 cells L-l from near-shore stations) in addition to less abundant Alexandrium catenella, Alexandrium sp., Alexandrium minutum, and Prorocentrum micans (>103 to 25x 103cells/L). Another 44 species occurred in relatively low numbers (<103 cell L-l). Seventy species were found throughout the study period at all four stations. High number of species in three genera (Tripos (38), Protoperidinium (34) and Prorocentrum (20) was recorded. Potently toxic (16 genera 43 species) and HAB related (19 genera and 30 species) dinoflagellate taxa were also recorded. The percent contribution of dinoflagellates in total phytoplankton population generally remained below 20% except for a few instances. Manora Island stations had comparatively higher Shannon index and equitability and slightly lower dominance index. The PCA plot showed strong positive correlation among chlorophyll-a concentration, dissolved oxygen, total number of phytoplankton and dinoflagellates.

Finding Species-Specific Extracellular Surface-Facing Proteomes in Toxic Dinoflagellates

As a sequel to our previous report of the existence of species-specific protein/peptide expression profiles (PEPs) acquired by mass spectrometry in some dinoflagellates, we established, with the help of a plasma-membrane-impermeable labeling agent, a surface amphiesmal protein extraction method (SAPE) to label and capture species-specific surface proteins (SSSPs) as well as saxitoxins-producing-species-specific surface proteins (Stx-SSPs) that face the extracellular space (i.e., SSSPsEf and Stx-SSPsEf). Five selected toxic dinoflagellates, Alexandrium minutum, A. lusitanicum, A. tamarense, Gymnodinium catenatum, and Karenia mikimotoi, were used in this study. Transcriptomic databases of these five species were also constructed. With the aid of liquid chromatography linked-tandem mass spectrometry (LC-MS/MS) and the transcriptomic databases of these species, extracellularly facing membrane proteomes of the five different species were identified. Within these proteomes, 16 extracellular-facing and functionally significant transport proteins were found. Furthermore, 10 SSSPs and 6 Stx-SSPs were identified as amphiesmal proteins but not facing outward to the extracellular environment. We also found SSSPsEf and Stx-SSPsEf in the proteomes. The potential functional correlation of these proteins towards the production of saxitoxins in dinoflagellates and the degree of species specificity were discussed accordingly.

Insights into Alexandrium minutum Nutrient Acquisition, Metabolism and Saxitoxin Biosynthesis through Comprehensive Transcriptome Survey

The toxin-producing dinoflagellate Alexandrium minutum is responsible for the outbreaks of harmful algae bloom (HABs). It is a widely distributed species and is responsible for producing paralytic shellfish poisoning toxins. However, the information associated with the environmental adaptation pathway and toxin biosynthesis in this species is still lacking. Therefore, this study focuses on the functional characterization of A. minutum unigenes obtained from transcriptome sequencing using the Illumina Hiseq 4000 sequencing platform. A total of 58,802 (47.05%) unigenes were successfully annotated using public databases such as NCBI-Nr, UniprotKB, EggNOG, KEGG, InterPRO and Gene Ontology (GO). This study has successfully identified key features that enable A. minutum to adapt to the marine environment, including several carbon metabolic pathways, assimilation of various sources of nitrogen and phosphorus. A. minutum was found to encode homologues for several proteins involved in saxitoxin biosynthesis, including the first three proteins in the pathway of saxitoxin biosynthesis, namely sxtA, sxtG and sxtB. The comprehensive transcriptome analysis presented in this study represents a valuable resource for understanding the dinoflagellates molecular metabolic model regarding nutrient acquisition and biosynthesis of saxitoxin.

Electrophysiological Evaluation of Pacific Oyster (Crassostrea gigas) Sensitivity to Saxitoxin and Tetrodotoxin

Pacific oysters (Crassostrea gigas) may bio-accumulate high levels of paralytic shellfish toxins (PST) during harmful algal blooms of the genus Alexandrium. These blooms regularly occur in coastal waters, affecting oyster health and marketability. The aim of our study was to analyse the PST-sensitivity of nerves of Pacific oysters in relation with toxin bio-accumulation. The results show that C. gigas nerves have micromolar range of saxitoxin (STX) sensitivity, thus providing intermediate STX sensitivity compared to other bivalve species. However, theses nerves were much less sensitive to tetrodotoxin. The STX-sensitivity of compound nerve action potential (CNAP) recorded from oysters experimentally fed with Alexandrium minutum (toxic-alga-exposed oysters), or Tisochrysis lutea, a non-toxic microalga (control oysters), revealed that oysters could be separated into STX-resistant and STX-sensitive categories, regardless of the diet. Moreover, the percentage of toxin-sensitive nerves was lower, and the STX concentration necessary to inhibit 50% of CNAP higher, in recently toxic-alga-exposed oysters than in control bivalves. However, no obvious correlation was observed between nerve sensitivity to STX and the STX content in oyster digestive glands. None of the nerves isolated from wild and farmed oysters was detected to be sensitive to tetrodotoxin. In conclusion, this study highlights the good potential of cerebrovisceral nerves of Pacific oysters for electrophysiological and pharmacological studies. In addition, this study shows, for the first time, that C. gigas nerves have micromolar range of STX sensitivity. The STX sensitivity decreases, at least temporary, upon recent oyster exposure to dinoflagellates producing PST under natural, but not experimental environment.

Opposite Growth Responses of Alexandrium minutum and Alexandrium catenella to Photoperiods and Temperatures

Shift of phytoplankton niches from low to high latitudes has altered their experienced light exposure durations and temperatures. To explore this interactive effect, the growth, physiology, and cell compositions of smaller Alexandrium minutum and larger A. catenella, globally distributed toxic red tide dinoflagellates, were studied under a matrix of photoperiods (light:dark cycles of 8:16, 16:8, and 24:0) and temperatures (18 °C, 22 °C, 25 °C, and 28 °C). Under continuous growth light condition (L:D 24:0), the growth rate (µ) of small A. minutum increased from low to medium temperature, then decreased to high temperature, while the µ of large A. catenella continuously decreased with increasing temperatures. Shortened photoperiods reduced the µ of A. minutum, but enhanced that of A. catenella. As temperature increased, cellular Chl a content increased in both A. minutum and A. catenella, while the temperature-induced effect on RubisCO content was limited. Shortened photoperiods enhanced the Chl a but reduced RubisCO contents across temperatures. Moreover, shortened photoperiods enhanced photosynthetic capacities of both A. minutum and A. catenella, i.e., promoting the PSII photochemical quantum yield (FV/FM, ΦPSII), saturation irradiance (EK), and maximum relative electron transfer rate (rETRmax). Shortened photoperiods also enhanced dark respiration of A. minutum across temperatures, but reduced that of A. catenella, as well as the antioxidant activities of both species. Overall, A. minutum and A. catenella showed differential growth responses to photoperiods across temperatures, probably with cell size.

Longitudinal Study on Seasonal Variation of Marine Biotoxins and Related Harmful Algae in Bivalve Mollusks Bred in Sardinia (Italy, W Mediterranean Sea) from 2015 to 2020 and Assessment of Potential Public Health Risks

Annual and interannual dynamics of shellfish toxins and associated harmful algal species (HAS) were analyzed from 2015 to 2020 in Tortolì Lagoon (Sardinia, west Mediterranean Sea). Analysis of seasonal occurrence of different harmful algae, such as Dinophysis spp., Prorocentrum spp., Pseudo-nitzschia spp. and Alexandrium minutum, was performed. The species Dinophysis acuminata and Dinophysis sacculus were responsible for the accumulation of lipophilic toxins belonging to the okadaic acid group (OAs) and pectenotoxins2 (PTX2) in bivalve mollusks. The highest HAS detection was recorded in the winter months; in particular, Dinophysis spp. was mostly present in January–February. Out of 1090 analyzed mollusk samples, 39 were non-compliant, exceeding the legal limits (160 μg OA eq/kg e.p.) reported in Regulation 853/2004 of the European Commission. A statistical analysis related to the presence of OA and PTX2 in mollusks with various environmental parameters (pH, water temperature, dissolved oxygen, algal density) was implemented, proving a clear winter seasonality. The present study highlights the necessity to better understand the different factors able to influence the production and accumulation of toxins in bivalve mollusks bred in an important Sardinian production area. The contribution of this research is important not only from an environmental and productive point of view but also from the view of implementing management in order to mitigate any harm to human health.