Quantifying Karenia brevis bloom severity and respiratory irritation impact along the shoreline of Southwest Florida.

Nearly all annual blooms of the toxic dinoflagellate Karenia brevis (K. brevis) pose a serious threat to coastal Southwest Florida. These blooms discolor water, kill fish and marine mammals, contaminate shellfish, cause mild to severe respiratory irritation, and discourage tourism and recreational activities, leading to significant health and economic impacts in affected communities. Despite these issues, we still lack standard measures suitable for assessing bloom severity or for evaluating the efficacy of modeling efforts simulating bloom initiation and intensity. In this study, historical cell count observations along the southwest Florida shoreline from 1953 to 2019 were used to develop monthly and annual bloom severity indices (BSI). Similarly, respiratory irritation observations routinely reported in Sarasota and Manatee Counties from 2006 to 2019 were used to construct a respiratory irritation index (RI). Both BSI and RI consider spatial extent and temporal evolution of the bloom, and can be updated routinely and used as objective criteria to aid future socioeconomic and scientific studies of K. brevis. These indices can also be used to help managers and decision makers both evaluate the risks along the coast during events and design systems to better respond to and mitigate bloom impacts. Before 1995, sampling was done largely in response to reports of discolored water, fish kills, or respiratory irritation. During this timeframe, lack of sampling during the fall, when blooms typically occur, generally coincided with periods of more frequent-than-usual offshore winds. Consequently, some blooms may have been undetected or under-sampled. As a result, the BSIs before 1995 were likely underestimated and cannot be viewed as accurately as those after 1995. Anomalies in the frequency of onshore wind can also largely account for the discrepancies between BSI and RI during the period from 2006 to 2019. These findings highlighted the importance of onshore wind anomalies when predicting respiratory irritation impacts along beaches.

Proteome Analysis of Whole-Body Responses in Medaka Experimentally Exposed to Fish-Killing Dinoflagellate Karenia mikimotoi.

Karenia mikimotoi is a well-known harmful algal bloom species. Blooms of this dinoflagellate have become a serious threat to marine life, including fish, shellfish, and zooplanktons and are usually associated with massive fish death. Despite the discovery of several toxins such as gymnocins and gymnodimines in K. mikimotoi, the mechanisms underlying the ichthyotoxicity of this species remain unclear, and molecular studies on this topic have never been reported. The present study investigates the fish-killing mechanisms of K. mikimotoi through comparative proteomic analysis. Marine medaka, a model fish organism, was exposed to K. mikimotoi for a three-part time period (LT25, LT50 and LT90). Proteins extracted from the whole fish were separated by using two-dimensional gel electrophoresis, and differentially expressed proteins were identified with reference to an untreated control. The change in fish proteomes over the time-course of exposure were analyzed. A total of 35 differential protein spots covering 19 different proteins were identified, of which most began to show significant change in expression levels at the earliest stage of intoxication. Among the 19 identified proteins, some are closely related to the oxidative stress responses, energy metabolism, and muscle contraction. We propose that oxidative stress-mediated muscle damage might explain the symptoms developed during the ichthyotoxicity test, such as gasping for breath, loss of balance, and body twitching. Our findings lay the foundations for more in-depth studies of the mechanisms of K. mikimotoi's ichthyotoxicity.

Immune response of silver pomfret (Pampus argenteus) to Amyloodinium ocellatum infection.

Amyloodinium ocellatum (AO) infection in silver pomfret (Pampus argenteus) causes extensive mortality. Insufficient information exists on the molecular immune response of silver pomfret to AO infestation, so herein we simulated the process of silver pomfret being infected by AO. Translucent trophosomes were observed on the gills of AO-infected fish. Transcriptome profiling was performed to investigate the effects of AO infection on the gill, kidney complex and spleen. Overall, 404,412,298 clean reads were obtained, assembling into 96,341 unigenes, which were annotated against public databases. In total, 2730 differentially expressed genes were detected, and few energy- and immune-related genes were further assessed using RT-qPCR. Moreover, activities of three immune-related (SOD, AKP and ACP) and three energy-related (PKM, LDH and GCK) enzymes were determined. AO infection activated the immune system and increased interleukin-1 beta and immunoglobulin M heavy chain levels. Besides, the PPAR signalling pathway was highly enriched, which played a role in improving immunity and maintaining homeostasis. AO infection also caused dyspnoea, leading to extensive lactic acid accumulation, potentially contributing towards a strong immune response in the host. Our data improved our understanding regarding the immune response mechanisms through which fish coped with parasitic infections and may help prevent high fish mortality in aquaculture.

Transcriptome Analysis of Amyloodinium ocellatum Tomonts Revealed Basic Information on the Major Potential Virulence Factors.

The ectoparasite protozoan Amyloodinium ocellatum (AO) is the etiological agent of amyloodiniosis in European seabass (Dicentrarchus labrax) (ESB). There is a lack of information about basic molecular data on AO biology and its interaction with the host. Therefore, de novo transcriptome sequencing of AO tomonts was performed. AO trophonts were detached from infested ESB gills, and quickly becoming early tomonts were purified by Percoll® density gradient. Tomont total RNA was processed and quality was assessed immediately. cDNA libraries were constructed using TruSeq® Stranded mRNA kit and sequenced using Illumina sequencer. CLC assembly was used to generate the Transcriptome assembly of AO tomonts. Out of 48,188 contigs, 56.12% belong to dinophyceae wherein Symbiodinium microadriaticum had 94.61% similarity among dinophyceae. Functional annotations of contigs indicated that 12,677 had associated GO term, 9005 with KEGG term. The contigs belonging to dinophyceae resulted in the detection of several peptidases. A BLAST search for known virulent factors from the virulence database resulted in hits to Rab proteins, AP120, Ribosomal phosphoprotein, Heat-shock protein70, Casein kinases, Plasmepsin IV, and Brucipain. Hsp70 and casein kinase II alpha were characterized in-silico. Altogether, these results provide a reference database in understanding AO molecular biology, aiding to the development of novel diagnostics and future vaccines.

Passive acoustic listening stations (PALS) show rapid onset of ecological effects of harmful algal blooms in real time.

Monitoring ecological changes in marine ecosystems is expensive and time-consuming. Passive acoustic methods provide continuous monitoring of soniferous species, are relatively inexpensive, and can be integrated into a larger network to provide enhanced spatial and temporal coverage of ecological events. We demonstrate how these methods can be used to detect changes in fish populations in response to a Karenia brevis red tide harmful algal bloom by examining sound spectrum levels recorded by two land-based passive acoustic listening stations (PALS) deployed in Sarasota Bay, Florida, before and during a red tide event. Significant and temporally persistent decreases in sound spectrum levels were recorded in real time at both PALS in four frequency bands spanning 0.172-20 kHz after K. brevis cells were opportunistically sampled near the stations. The decrease in sound spectrum levels and increase in K. brevis cell concentrations also coincided with decreased catch per unit effort (CPUE) and species density per unit effort (SDPUE) data for non-clupeid fish and soniferous fish species, as well as increased reports of marine mammal mortalities in the region. These findings demonstrate how PALS can detect and report in real time ecological changes from episodic disturbances, such as harmful algal blooms.

Limnobacter alexandrii sp. nov., a thiosulfate-oxidizing, heterotrophic and EPS-bearing Burkholderiaceae isolated from cultivable phycosphere microbiota of toxic Alexandrium catenella LZT09.

A novel Gram-negative, aerobic, motile and short rod-shaped bacterium with exopolysaccharides production, designated as LZ-4T, was isolated from cultivable phycosphere microbiota of harmful algal blooms-causing marine dinoflagellate Alexandrium catenella LZT09 which produces paralytic shellfish poisoning toxins. Strain LZ-4T was able to use thiosulfate (optimum concentration 10 mM) as energy source for bacterial growth. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain LZ-4T belonged to the genus Limnobacter, showing high 16S rRNA gene sequences similarities with L. thiooxidans DSM 13612T (99.4%), L. humi NBRC 11650T (98.2%) and L. litoralis NBRC 105857T (97.2%), respectively. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between LZ-4T and L. thiooxidans DSM 13612T were 78.9 and 21.9%, respectively. Both values were far lower than the thresholds (95-96% for ANI and 70% for dDDH) generally accepted for new species delineation. The respiratory quinone of strain LZ-4T was Q-8. The dominant cellular fatty acids were determined as summed feature 3 (C16:1 ω6c/ω7c), summed feature 8 (C18:1 ω6c/ω7c) and C16:0. Polar lipids profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, two unidentified aminolipids and three unidentified polar lipids. The genomic DNA G+C content of strain LZ-4T was 52.5 mol%. Based on polyphasic characterization, strain LZ-4T represents a novel species of the genus Limnobacter, for which the name Limnobacter alexandrii sp. nov. is proposed. The type strain is LZ-4T (=CCTCC AB 2019004T =KCTC 72281T).

An assessment of trends in the frequency and duration of Karenia brevis red tide blooms on the South Texas coast (western Gulf of Mexico).

Limited data coverage on harmful algal blooms (HABs) in some regions makes assessment of long-term trends difficult, and also impedes understanding of bloom ecology. Here, observations reported in a local newspaper were combined with cell count and environmental data from resource management agencies to assess trends in Karenia brevis "red tide" frequency and duration in the Nueces Estuary (Texas) and adjacent coastal waters, and to determine relationships with environmental factors. Based on these analyses, the Coastal Bend region of the Texas coast has experienced a significant increase in the frequency of red tide blooms since the mid-1990s. Salinity was positively correlated with red tide occurrence in the Nueces Estuary, and a documented long-term increase in salinity of the Nueces Estuary may be a major factor in the long-term increase in bloom frequency. This suggests that freshwater inflow management efforts in Texas should consider impacts on red tide habitat suitability (i.e., salinity regime) in downstream estuaries. Natural climate variability such as the El Niño-Southern Oscillation, which is strongly related to rainfall and salinity in Central and South Texas, was also an influential predictor of red tide presence/absence. Though no significant change in the duration of blooms was detected, there was a negative correlation between duration and temperature. Specifically, summer-like temperatures were not favorable to K. brevis bloom development. The relationships found here between red tide frequency/duration and environmental drivers present a new avenue of research that will aid in refining monitoring and forecasting efforts for red tides on the Texas coast and elsewhere. Findings also highlight the importance of factors (i.e., salinity, temperature) that are likely to be altered in the future due to both population growth in coastal watersheds and anthropogenic climate change.

Inhibition to crucial enzymes in the lethal effects of the dinoflagellate Karenia mikimotoi on the rotifer Brachionus plicatilis.

Blooms of the dinoflagellate Karenia mikimotoi have cause great financial losses to the marine aquaculture industry. However, the toxicity mechanism of this species is still not fully known. In this study, we evaluated the short-term effects of K. mikimotoi on the rotifer Brachionus plicatilis by micro and sub micro observing and by measuring inhibition of crucial enzymes. Behaviour disorder, mucus production, corona and cilium damage, vesical production, and body shrinkage occurred within 1 h after rotifers were treated with K. mikimotoi at a density of 3 × 104 cells/mL. Enzyme activity assays showed that K. mikimotoi at low densities significantly inhibited multiple enzymes within 3 h, and obvious density-effect trends were also observed. For instance, activity of esterase and acetylcholinesterase of rotifers significantly decreased to 94.3/83.3% and 82.8/66.9% of control treatment values in 30 and 1000 cells/mL algal treatment, respectively. Total ATPase and Na+-K+-ATPase activities of rotifers also decreased to 82.3% and 68.6% of control values in 1000 cells/mL treatment. The LDH releasement test and MDA tests showed no significant difference between algae treatment and control. It suggested that K. mikimotoi might not cause significant cytolysis and oxidative damage to rotifers, but may cause mortality by inhibiting the activity of crucial enzymes, which may lead to cell permeability disorder and body shrinkage.

Effects of the DSP-toxic dinoflagellate Dinophysis acuta on clearance and respiration rate of the blue mussel, Mytilus edulis.

Diarrheic Shellfish Poisoning toxins (DST) are a severe health risk to shellfish consumers and can be a major problem for the shellfish industry. Bivalve molluscs can accumulate DST via ingestion of toxic dinoflagellates like Dinophysis spp., which are the most prominent producers of DST. The effects of DST-containing dinoflagellate Dinophysis acuta on bivalve clearance and respiration rate were investigated in the blue mussel (Mytilus edulis) exposed to different algal densities in a controlled laboratory study. Results showed that M. edulis exposed to D. acuta displayed a reduced clearance rate compared to M. edulis exposed to equivalent bio-volumes of the non-toxic cryptophyte Rhodomonas salina. Furthermore, M. edulis ceased to feed on D. acuta after 1 to 4 h, depending on D. acuta densities. The quickest response was observed at the highest densities of D. acuta. The estimated total amount of DST accumulated in the M. edulis exceeded the regulatory limit for human consumption and furthermore, intoxication of the M. edulis seemed to occur faster at high cell toxicity rather than at high cell density. However, respiration rates were, similar, irrespective of whether M. edulis were fed single diets of R. salina, D. acuta or a mixed diet of both algal species. In conclusion, the DST-containing D. acuta had a severe negative effect on the clearance of M. edulis, which can affect the conditions of the M. edulis negatively. Hence, DST may cause low quality M. edulis, due to reduced feeding when exposed to DST-containing D. acuta.

Exposure to the Florida red tide dinoflagellate, Karenia brevis, and its associated brevetoxins induces ecophysiological and proteomic alterations in Porites astreoides.

As reef-building corals are increasingly being exposed to persistent threats that operate on both regional and global scales, there is a pressing need to better understand the complex processes that diminish coral populations. This study investigated the impacts of the Florida red tide dinoflagellate Karenia brevis and associated brevetoxins on selected facets of coral biology using Porites astreoides as a model system. When provided with choice assays, P. astreoides larvae were shown to actively avoid seawater containing red tide (5×105 cells L-1-7.6×106 cells L-1) or purified brevetoxins (0.018 µg mL-1 brevetoxin-2 and 0.0018 µg mL-1 brevetoxin-3). However, forced exposure to similar treatments induced time-dependent physiological and behavioral changes that were captured by PAM fluorometry and settlement and survival assays, respectively. Adult fragments of P. astreoides exposed to red tide or associated brevetoxins displayed signs of proteomic alterations that were characterized by the use of an iTRAQ-based quantitative proteomic analysis. The novel use of this technique with P. astreoides demonstrated that protein regulation was highly contingent upon biological versus chemical treatment (i.e. live K. brevis vs. solely brevetoxin exposure) and that several broad pathways associated with cell stress were affected including redox homeostasis, protein folding, energy metabolism and reactive oxygen species production. The results herein provide new insight into the ecology, behavior and sublethal stress of reef-building corals in response to K. brevis exposure and underscore the importance of recognizing the potential of red tide to act as a regional stressor to these important foundation species.

Solute carriers in scallop genome: Gene expansion and expression regulation after exposure to toxic dinoflagellate.

The solute carriers (SLCs) are membrane proteins that transport many endogenous and exogenous substances such as xenobiotic toxins. Bivalve mollusks, mainly feeding on microalgae, show marked capacity to accumulate paralytic shellfish toxins (PSTs), the most common and hazardous marine biotoxins produced by dinoflagellates. Exploring the SLCs related to PST accumulation in bivalve could benefit our understanding about the mechanisms of PST bioavailability in bivalve and the adaptations of these species. Herein, we provided the first systematic analysis of SLC genes in mollusks, which identified 673 SLCs (PySLCs, 48 subfamilies) in Yesso scallop (Patinopecten yessoensis), 510 (48 subfamilies) in Pacific oyster (Crassostrea gigas), and 350 (47 subfamilies) in gastropod owl limpet (Lottia gigantea). Significant expansion of subfamilies SLC5, SLC6, SLC16, and SLC23 in scallop, and SLC46 subfamily in both scallop and oyster were revealed. Different PySLC members were highly expressed in the developmental stages and adult tissues, and hepatopancreas harboured more specifically expressed PySLCs than other tissues/organs. After feeding the scallops with PST-producing dinoflagellate, 131 PySLCs were regulated and more than half of them were from the expanded subfamilies. The trend of expression fold change in regulated PySLCs was consistent with that of PST changes in hepatopancreas, implying the possible involvement of these PySLCs in PST transport and homeostasis. In addition, the PySLCs from the expanded subfamily were revealed to be under positive selection, which might be related to lineage-specific adaptation to the marine environments with algae derived biotoxins.

Spatial and temporal disease dynamics of the parasite Hematodinium sp. in shore crabs, Carcinus maenas.

BACKGROUND: The parasitic dinoflagellates of the genus Hematodinium represent the causative agent of so-called bitter or pink crab disease in a broad range of shellfish taxa. Outbreaks of Hematodinium-associated disease can devastate local fishing and aquaculture efforts. The goal of our study was to examine the potential role of the common shore (green) crab Carcinus maenas as a reservoir for Hematodinium. Carcinus maenas is native to all shores of the UK and Ireland and the North East Atlantic but has been introduced to, and subsequently invaded waters of, the USA, South Africa and Australia. This species is notable for its capacity to harbour a range of micro- and macro-parasites, and therefore may act as a vector for disease transfer. METHODS: Over a 12-month period, we interrogated 1191 crabs across two distinct locations (intertidal pier, semi-closed dock) in Swansea Bay (Wales, UK) for the presence and severity of Hematodinium in the haemolymph, gills, hepatopancreas and surrounding waters (eDNA) using PCR-based methods, haemolymph preparations and histopathology. RESULTS: Overall, 13.6% were Hematodinium-positive via PCR and confirmed via tissue examination. Only a small difference was observed between locations with 14.4% and 12.8% infected crabs in the Dock and Pier, respectively. Binomial logistic regression models revealed seasonality (P < 0.002) and sex (P < 0.001) to be significant factors in Hematodinium detection with peak infection recorded in spring (March to May). Male crabs overall were more likely to be infected. Phylogenetic analyses of the partial ITS and 18S rRNA gene regions of Hematodinium amplified from crabs determined the causative agent to be the host generalist Hematodinium sp., which blights several valuable crustaceans in the UK alone, including edible crabs (Cancer pagurus) and langoustines (Nephrops norvegicus). CONCLUSIONS: Shore crabs were infected with the host generalist parasite Hematodinium sp. in each location tested, thereby enabling the parasite to persist in an environment shared with commercially important shellfish.

The blue mussel Mytilus edulis is vulnerable to the toxic dinoflagellate Karlodinium armiger-Adult filtration is inhibited and several life stages killed.

Blooms of the toxic dinoflagellates Karlodinium armiger and K. veneficum are frequently observed in Alfacs Bay, Spain, causing mass mortality to wild and farmed mussels. An isolate of K. armiger from Alfacs Bay was grown in the laboratory and exposed to adults, embryos and trochophore larvae of the blue mussel, Mytilus edulis. Adult mussels rejected to filter K. armiger at cell concentrations >1.5·103 cells ml-1. Exposure of adult mussels (23-33 mm shell length) to a range of K. armiger cell concentrations led to mussel mortality with LC50 values of 9.4·103 and 6.1·103 cells ml-1 after 24 and 48 h exposure to ~3.6·104 K. armiger cells ml-1, respectively. Karlodinium armiger also affected mussel embryos and trochophore larvae and feeding by K. armiger on both embryos and larvae was observed under the microscope. Embryos exposed to low K. armiger cell concentrations suffered no measurable mortality. However, at higher K. armiger cell concentrations the mortality of the embryos increased significantly with cell concentration and reached 97% at 1.8·103 K. armiger cells ml-1 after 29 h of exposure. Natural K. armiger blooms may not only have serious direct effects on benthic communities, but may also affect the recruitment of mussels in affected areas.

The relationship of blue crab (Callinectes sapidus) size class and molt stage to disease acquisition and intensity of Hematodinium perezi infections.

In the blue crab, Callinectes sapidus, early studies suggested a relationship between smaller crabs, which molt more frequently, and higher rates of infection by the dinoflagellate parasite, Hematodinium perezi. In order to better explore the influence of size and molting on infections, blue crabs were collected from the Maryland coastal bays and screened for the presence of H. perezi in hemolymph samples using a quantitative PCR assay. Molt stage was determined by a radioimmunoassay which measured ecdysteroid concentrations in blue crab hemolymph. Differences were seen in infection prevalence between size classes, with the medium size class (crabs 61 to 90 mm carapace width) and juvenile crabs (≤ 30 mm carapace width) having the highest infection prevalence at 47.2% and 46.7%, respectively. All size classes were susceptible to infection, although fall months favored disease acquisition by juveniles, whereas mid-sized animals (31-90 mm carapace width) acquired infection predominantly in summer. Disease intensity was also most pronounced in the summer, with blue crabs > 61 mm being primary sources of proliferation. Molt status appeared to be influenced by infection, with infected crabs having significantly lower concentrations of ecdysteroids than uninfected crabs in the spring and the fall. We hypothesize that infection by H. perezi may increase molt intervals, with a delay in the spring molt cycle as an evolutionary adaptation functioning to coincide with increased host metabolism, providing optimal conditions for H. perezi propagation. Regardless of season, postmolt crabs harbored significantly higher proportions of moderate and heavy infections, suggesting that the process of ecdysis, and the postmolt recovery period, has a positive effect on parasite proliferation.

The influence of temperature and salinity on mortality of recently recruited blue crabs, Callinectes sapidus, naturally infected with Hematodinium perezi (Dinoflagellata).

The parasitic dinoflagellate Hematodinium perezi is highly prevalent in juvenile blue crabs, Callinectes sapidus, along the eastern seaboard of the USA. Although the parasite is known to kill adult crabs, the mortality rate of naturally infected juvenile crabs remains unknown. We analyzed the influence of temperature and salinity on the mortality of recently recruited blue crabs that were naturally infected with H. perezi. Over 492 juvenile crabs (infected, n = 282; uninfected controls, n = 210) were held individually in six-well plates and held at six temperatures (4, 10, 15, 20, 25, and 30 °C) or three salinities (5, 15, and 30 psu) for a maximum of 90 days. Mortality of infected crabs was 10 times higher at elevated temperatures (25 and 30 °C) and salinity (30 psu) compared to uninfected control treatments. By contrast, infected crabs exposed to mild temperatures (10, 15, and 20 °C) showed a high survival (>80%), no different than uninfected control treatments. Infected crabs at the lowest temperature (4 °C) exhibited a high mortality, but the intensity of infection was lower than in the other temperature treatments. In addition, this study revealed the optimal temperature (25 °C) and salinity (30 psu) for H. perezi to progress in its life cycle leading to sporulation in juvenile crabs; 31.6% (19/60) of crabs held under these conditions released dinospores of H. perezi after 10 days. Crabs held at other temperatures did not release dinospores over the time course of the experiment. Infected crabs were capable of molting and in most cases molted at the same frequency as uninfected crabs serving as controls. The mortality observed in this study indicates that early benthic juveniles will experience significant mortality due to H. perezi with increasing ocean temperatures and that this mortality may be a significant factor in the recruitment of blue crabs to high salinity regions.

Carbonic anhydrase 2-like in the giant clam, Tridacna squamosa: characterization, localization, response to light, and possible role in the transport of inorganic carbon from the host to its symbionts.

The fluted giant clam, Tridacna squamosa, lives in symbiosis with zooxanthellae which reside extracellularly inside a tubular system. Zooxanthellae fix inorganic carbon (Ci) during insolation and donate photosynthate to the host. Carbonic anhydrases catalyze the interconversion of CO2 and HCO3-, of which carbonic anhydrase 2 (CA2) is the most ubiquitous and involved in many biological processes. This study aimed to clone a CA2 homolog (CA2-like) from the fleshy and colorful outer mantle as well as the thin and whitish inner mantle of T. squamosa, to determine its cellular and subcellular localization, and to examine the effects of light exposure on its gene and protein expression levels. The cDNA coding sequence of CA2-like from T. squamosa comprised 789 bp, encoding 263 amino acids with an estimated molecular mass of 29.6 kDa. A phenogramic analysis of the deduced CA2-like sequence denoted an animal origin. CA2-like was not detectable in the shell-facing epithelium of the inner mantle adjacent to the extrapallial fluid. Hence, CA2-like is unlikely to participate directly in light-enhanced calcification. By contrast, the outer mantle, which contains the highest density of tertiary tubules and zooxanthellae, displayed high level of CA2-like expression, and CA2-like was localized to the tubule epithelial cells. More importantly, exposure to light induced significant increases in the protein abundance of CA2-like in the outer mantle. Hence, CA2-like could probably take part in the increased supply of inorganic carbon (Ci) from the host clam to the symbiotic zooxanthellae when the latter conduct photosynthesis to fix Ci during light exposure.

X-Cells Are Globally Distributed, Genetically Divergent Fish Parasites Related to Perkinsids and Dinoflagellates.

"X-cells" have long been associated with tumor-like formations (xenomas) in marine fish, including many of commercial interest. The name was first used to refer to the large polygonal cells that were found in epidermal xenomas from flatfish from the Pacific Northwest [1]. Similar looking cells from pseudobranchial xenomas had previously been reported from cod in the Atlantic [2] and Pacific Oceans [3]. X-cell pathologies have been reported from five teleost orders: Pleuronectiformes (flatfish), Perciformes (perch-like fish), Gadiformes (cods), Siluriformes (catfish), and Salmoniformes (salmonids). Various explanations have been elicited for their etiology, including being adenomas or adenocarcinomas [4, 5], virally transformed fish cells [6-8], or products of coastal pollution [9, 10]. It was hypothesized that X-cells were protozoan parasites [1, 11-13], and although recent molecular analyses have confirmed this, they have failed to place them in any phylum [14-18], demonstrating weak phylogenetic associations with the haplosporidians [16] or the alveolates [15]. Here, we sequenced rRNA genes from European and Japanese fish that are known to develop X-cell xenomas. We also generated a metagenomic sequence library from X-cell xenomas of blue whiting and Atlantic cod and assembled 63 X-cell protein-coding genes for a eukaryote-wide phylogenomic analysis. We show that X-cells group in two highly divergent clades, robustly sister to the bivalve parasite Perkinsus. We formally describe these as Gadixcellia and Xcellia and provide a phylogenetic context to catalyze future research. We also screened Atlantic cod populations for xenomas and residual pathologies and show that X-cell infections are more prevalent and widespread than previously known.

Glutathione S-Transferase Regulation in Calanus finmarchicus Feeding on the Toxic Dinoflagellate Alexandrium fundyense.

The effect of the dinoflagellate, Alexandrium fundyense, on relative expression of glutathione S-transferase (GST) transcripts was examined in the copepod Calanus finmarchicus. Adult females were fed for 5-days on one of three experimental diets: control (100% Rhodomonas spp.), low dose of A. fundyense (25% by volume, 75% Rhodomonas spp.), and high dose (100% A. fundyense). Relative expression of three GST genes was measured using RT-qPCR on days 0.5, 1, 2 and 5 in two independent experiments. Differential regulation was found for the Delta and the Sigma GSTs between 0.5 to 2 days, but not on day 5 in both experiments. The third GST, a microsomal, was not differentially expressed in either treatment or day. RT-qPCR results from the two experiments were similar, even though experimental females were collected from the Gulf of Maine on different dates and their reproductive output differed. In the second experiment, expression of 39 GSTs was determined on days 2 and 5 using RNA-Seq. Global gene expression analyses agreed with the RT-qPCR results. Furthermore, the RNA-Seq measurements indicated that only four GSTs were differentially expressed under the experimental conditions, and the response was small in amplitude. In summary, the A. fundyense diet led to a rapid and transient response in C. finmarchicus in three cytosolic GSTs, while a fourth GST (Omega I) was significantly up-regulated on day 5. Although there was some regulation of GSTs in response the toxic dinoflagellate, the tolerance to A. fundyense by C. finmarchicus is not dependent on the long-term up-regulation of specific GSTs.

Intoxicated copepods: ingesting toxic phytoplankton leads to risky behaviour.

Understanding interactions between harmful algal bloom (HAB) species and their grazers is essential for determining mechanisms of bloom proliferation and termination. We exposed the common calanoid copepod, Temora longicornis to the HAB species Alexandrium fundyense and examined effects on copepod survival, ingestion, egg production and swimming behaviour. A. fundyense was readily ingested by T. longicornis and significantly altered copepod swimming behaviour without affecting copepod survival or fitness. A. fundyense caused T. longicornis to increase their swimming speed, and the straightness of their path long after the copepods had been removed from the A. fundyense treatment. Models suggest that these changes could lead to a 25-56% increase in encounter frequency between copepods and their predators. This work highlights the need to determine how ingesting HAB species alters grazer behaviour as this can have significant impacts on the fate of HAB toxins in marine systems.

The sxt Gene and Paralytic Shellfish Poisoning Toxins as Markers for the Monitoring of Toxic Alexandrium Species Blooms.

Paralytic shellfish poisoning (PSP) is a serious human illness caused by the ingestion of seafood contaminated with saxitoxin and its derivatives (STXs). These toxins are produced by some species of marine dinoflagellates within the genus Alexandrium. In the Mediterranean Sea, toxic Alexandrium spp. blooms, especially of A. minutum, are frequent and intense with negative impact to coastal ecosystem, aquaculture practices and other economic activities. We conducted a large scale study on the sxt gene and toxin distribution and content in toxic dinoflagellate A. minutum of the Mediterranean Sea using both quantitative PCR (qPCR) and HILIC-HRMS techniques. We developed a new qPCR assay for the estimation of the sxtA1 gene copy number in seawater samples during a bloom event in Syracuse Bay (Mediterranean Sea) with an analytical sensitivity of 2.0 × 10° sxtA1 gene copy number per reaction. The linear correlation between sxtA1 gene copy number and microalgal abundance and between the sxtA1 gene and STX content allowed us to rapidly determine the STX-producing cell concentrations of two Alexandrium species in environmental samples. In these samples, the amount of sxtA1 gene was in the range of 1.38 × 10(5) - 2.55 × 10(8) copies/L and the STX concentrations ranged from 41-201 nmol/L. This study described a potential PSP scenario in the Mediterranean Sea.