Gambierdiscus (Gonyaulacales, Dinophyceae) diversity in Vietnamese waters with description of G. vietnamensis sp. nov.

Viet Nam has a coastline of 3200 km with thousands of islands providing diverse habitats for benthic harmful algal species including species of Gambierdiscus. Some of these species produce ciguatera toxins, which may accumulate in large carnivore fish potentially posing major threats to public health. This study reports five species of Gambierdiscus from Vietnamese waters, notably G. australes, G. caribaeus, G. carpenteri, G. pacificus, and G. vietnamensis sp. nov. All species are identified morphologically by LM and SEM, and identifications are supported by molecular analyses of nuclear rDNA (D1-D3 and D8-D10 domains of LSU, SSU, and ITS1-5.8S-ITS2 region) based on cultured material collected during 2010-2021. Statistical analyses of morphometric measurements may be used to differentiate some species if a sufficiently large number of cells are examined. Gambierdiscus vietnamensis sp. nov. is morphologically similar to other strongly reticulated species, such as G. belizeanus and possibly G. pacificus; the latter species is morphologically indistinguishable from G. vietnamensis sp. nov., but they are genetically distinct, and molecular analysis is deemed necessary for proper identification of the new species. This study also revealed that strains denoted G. pacificus from Hainan Island (China) should be included in G. vietnamensis sp. nov.

Costs and benefits of predator-induced defence in a toxic diatom.

Phytoplankton employ a variety of defence mechanisms against predation, including production of toxins. Domoic acid (DA) production by the diatom Pseudo-nitzschia spp. is induced by the presence of predators and is considered to provide defence benefits, but the evidence is circumstantial. We exposed eight different strains of P. seriata to chemical cues from copepods and examined the costs and the benefits of toxin production. The magnitude of the induced toxin response was highly variable among strains, while the costs in terms of growth reduction per DA cell quota were similar and the trade-off thus consistent. We found two components of the defence in induced cells: (i) a 'private good' in terms of elevated rejection of captured cells and (ii) a 'public good' facilitated by a reduction in copepod feeding activity. Induced cells were more frequently rejected by copepods and rejections were directly correlated with DA cell quota and independent of access to other food items. By contrast, the public-good effect was diminished by the presence of alternative prey suggesting that it does not play a major role in bloom formation and that its evolution is closely associated with the grazing-deterrent private good.

Diversity and emendation of the genus Planktoniella (Bacillariophyceae), with descriptions of P. tubulata sp. nov. and P. trifurcata sp. nov.

For the first time, LSU and SSU sequences were obtained from four species of the marine planktonic diatom Planktoniella. Samples were collected and cultured from Chinese coastal waters, and morphological observations were made using light and scanning electron microscopy. Combined morphological and DNA sequence data revealed two new species herein described as Planktoniella tubulata and Planktoniella trifurcata. Planktoniella tubulata is characterized by the prominent external tubes of two rimoportulae and the long external tubes of the marginal fultoportulae. The latter are organized regularly in a ring, externally on the mantle, and protrude parallel to the valvar plane. Planktoniella trifurcata is unique in having mucilaginous extensions that usually expand into three lobes, and external extensions of the marginal fultoportulae being either tubes or spines. In the phylogenetic analyses inferred from SSU and LSU sequences, the four analyzed Planktoniella taxa formed a well-supported monophyletic group. A suite of three characters, mucilage membranes or wings, two marginal rimoportulae, and mantle ribs are diagnostic for Planktoniella. Based on these detailed ultrastructural observations, the description of Planktoniella is emended.

Two New and Nontoxigenic Pseudo-nitzschia species (Bacillariophyceae) from Chinese Southeast Coastal Waters.

To explore the species diversity and toxin profile of Pseudo-nitzschia, monoclonal strains were established from Chinese southeast coastal waters. The morphology was examined under light and transmission electron microscopy. The internal transcribed spacer region of ribosomal DNA was sequenced for phylogenetic analyses, and the secondary structure of ITS2 was predicted and compared among allied taxa. A combination of morphological and molecular data showed the presence of two new species, Pseudo-nitzschia hainanensis sp. nov. and Pseudo-nitzschia taiwanensis sp. nov. Pseudo-nitzschia hainanensis was characterized by a dumpy-lanceolate valve with slightly blunt apices and a central nodule, as well as striae comprising two rows of poroids. Pseudo-nitzschia taiwanensis was characterized by a slender-lanceolate valve, and striae comprising one row of split poroids. The poroid structure mainly comprised two sectors. Both taxa constituted their own monophyletic lineage in the phylogenetic analyses inferred from ITS2 rDNA and were well differentiated from other Pseudo-nitzschia species. Morphologically, P. hainanensis and P. taiwanensis could be assigned to the Pseudo-nitzschia delicatissima and the Pseudo-nitzschia pseudodelicatissima complex, respectively. Particulate domoic acid was measured using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), but no detectable pDA was found. With the description of the two new species, the species diversity of genus Pseudo-nitzschia reaches 58 worldwide, among which 31 have been recorded from Chinese coastal waters.

Morphological and genetic analyses of Ostreopsis (Dinophyceae, Gonyaulacales, Ostreopsidaceae) species from Vietnamese waters with a re-description of the type species, O. siamensis.

Identification of species of the dinoflagellate genus Ostreopsis is difficult because several species have been poorly described, others misidentified in the literature, and the type species, O. siamensis, has not been described by contemporary taxonomic methods. In the present study, it is argued that Ostreopsis sp. 6 as described by previous authors is similar to the type species, and we offer an emended description of O. siamensis by LM, SEM, and molecular analyses of nuclear LSU and ITS rDNA based on material collected a few hundred kilometers from the type locality in the Gulf of Thailand and along the Vietnamese east coast. Ostreopsis siamensis is genetically different from the species reported as O. cf. siamensis in the literature and the latter should be described as a separate species. It is also concluded that with the poor knowledge of the morphological variability of many species of Ostreopsis, O. siamensis may not be distinguished from other similar-sized species by its morphological features, and hence molecular data are needed for reliable identification. The species Ostreopsis lenticularis and Ostreopsis cf. ovata were also found and described.

Molecular Phylogeny and Taxonomy of the Genus Minidiscus (Bacillariophyceae), with Description of Mediolabrus gen. nov.

The genus Minidiscus comprises a group of ecologically-important and globally-distributed planktonic diatoms that are characterized by their small cell size, high mantle and processes more or less concentrated in the valve center. Monoclonal strains were established from collections along the Chinese coast. In the phylogenetic analyses inferred from a LSU and SSU dataset, six Minidiscus species clustered into two well-supported clades. The first clade was located within a larger clade formed mainly by Thalassiosira taxa, and the second clade appeared as sister to a clade comprising the genus Skeletonema. Hence, presently known Minidiscus do not form a monophyletic clade, but rather make up a phenotypic grouping. Based on the morphology of the type species, M. trioculatus, as well as morphological characters of all taxa in the clade with M. trioculatus, Minidiscus is characterized by having fultoportula(e) in the valve center or sub-centered close to a single rimoportula, and the cells are usually cylindrical. Mediolabrus gen. nov. is proposed to accommodate species in the second clade. The main difference between Minidiscus and Mediolabrus is the type of process found in the valve (sub-)center, with fultoportula(e) close to a rimoportula in the former, and only a single rimoportula in the latter. According to the above criteria, previously described Minidiscus taxa were re-examined, and either retained in Minidiscus or transferred to Mediolabrus.

A Study of Chaetoceros debilis Sensu Lato Species (Bacillariophyceae), with Ememdation of C. debilis and Description of C. galeatus Sp. Nov.1.

Chaetoceros debilis is considered one of the most abundant and widespread diatoms in the coastal marine phytoplankton, and is often used in research studies ranging from ecophysiology and molecular biology to oceanography and aquaculture. To clarify the species delineation of C. debilis and explore the diversity among C. debilis sensu lato taxa, monoclonal strains were established from different geographical regions, including the Danish coast, close to the type locality of C. debilis, the Denmark Strait (between Iceland and Greenland) as well as the Taiwan Strait, East China Sea and Daya Bay, South China Sea. Vegetative cells and resting spores were examined using light and electron microscopy. The hypervariable D1-D3 region of the nuclear large subunit ribosomal gene and the small subunit ribosomal gene were sequenced to address phylogenetic relationships. In both SSU and LSU trees, the C. debilis sensu lato strains clustered in four distinct clades. Culture material from the type locality was, along with molecular data, used to delineate and emend the description of C. debilis. Based on molecular data and detailed morphological features, one of the clades originating from Chinese waters was described as C. galeatus sp. nov., characterized by curved and helical chains, elliptical valves, and smooth and helmet-shaped primary valves of the resting spores. The remaining two clades, which also represent two novel taxa, C. cf. debilis-1 and C. cf. debilis-2, were not formally described as new species due to the insufficient information on their morphology.

Acclimation of the Marine Diatom Pseudo-nitzschia australis to Different Salinity Conditions: Effects on Growth, Photosynthetic Activity, and Domoic Acid Content1.

Toxic Pseudo-nitzschia australis strains isolated from French coastal waters were studied to investigate their capacity to adapt to different salinities. Their acclimation to different salinity conditions (10, 20, 30, 35, and 40) was studied on growth, photosynthetic capacity, cell biovolume, and domoic acid (DA) content. The strains showed an ability to acclimate to a salinity range from 20 to 40, with optimal growth rates between salinities 30 and 40. The highest cell biovolume was observed at the lowest salinity 20 and was associated with the lowest growth rate. Salinity did not affect the photosynthetic activity; Fv /Fm values and the pigment contents remained high with no significant difference among salinities. An enhanced production of zeaxanthin was, however, observed in the late stationary and decline phases in all cultures except for those acclimated to salinity 20. In terms of cellular toxin content, DA concentrations were 2 to 3-fold higher at the lowest salinity (20) than at the other salinities and were combined with a low amount of dissolved DA. The fact that P. australis accumulate more DA per cell in less saline waters, illustrates that climate-related changes in salinity may affect Pseudo-nitzschia physiology through direct effects on growth, physiology, and toxin content.

Transcriptomic responses to grazing reveal the metabolic pathway leading to the biosynthesis of domoic acid and highlight different defense strategies in diatoms.

BACKGROUND: A major cause of phytoplankton mortality is predation by zooplankton. Strategies to avoid grazers have probably played a major role in the evolution of phytoplankton and impacted bloom dynamics and trophic energy transport. Certain species of the genus Pseudo-nitzschia produce the neurotoxin, domoic acid (DA), as a response to the presence of copepod grazers, suggesting that DA is a defense compound. The biosynthesis of DA comprises fusion of two precursors, a C10 isoprenoid geranyl pyrophosphate and L-glutamate. Geranyl pyrophosphate (GPP) may derive from the mevalonate isoprenoid (MEV) pathway in the cytosol or from the methyl-erythritol phosphate (MEP) pathway in the plastid. L-glutamate is suggested to derive from the citric acid cycle. Fragilariopsis, a phylogenetically related but nontoxic genus of diatoms, does not appear to possess a similar defense mechanism. We acquired information on genes involved in biosynthesis, precursor pathways and regulatory functions for DA production in the toxigenic Pseudo-nitzschia seriata, as well as genes involved in responses to grazers to resolve common responses for defense strategies in diatoms. RESULTS: Several genes are expressed in cells of Pseudo-nitzschia when these are exposed to predator cues. No genes are expressed in Fragilariopsis when treated similarly, indicating that the two taxa have evolved different strategies to avoid predation. Genes involved in signal transduction indicate that Pseudo-nitzschia cells receive signals from copepods that transduce cascading molecular precursors leading to the formation of DA. Five out of seven genes in the MEP pathway for synthesis of GPP are upregulated, but none in the conventional MEV pathway. Five genes with known or suggested functions in later steps of DA formation are upregulated. We conclude that no gene regulation supports that L-glutamate derives from the citric acid cycle, and we suggest the proline metabolism to be a downstream precursor. CONCLUSIONS: Pseudo-nitzschia cells, but not Fragilariopsis, receive and respond to copepod cues. The cellular route for the C10 isoprenoid product for biosynthesis of DA arises from the MEP metabolic pathway and we suggest proline metabolism to be a downstream precursor for L-glutamate. We suggest 13 genes with unknown function to be involved in diatom responses to grazers.

Diversity in the section Compressa of the genus Chaetoceros (Bacillariophyceae), with description of two new species from Chinese warm waters.

Monoclonal strains of Chaetoceros species were collected from Chinese warm waters. Vegetative cells and resting spores were examined using light microscopy as well as scanning and transmission electron microscopy. Combined morphological and DNA sequence data from the hypervariable D1-D3 region of the nuclear ribosomal large subunit showed the presence of two new species within the section Compressa, herein described as Chaetoceros bifurcatus sp. nov. and C. millipedarius sp. nov. Both species possessed features typical of the section Compressa, but C. bifurcatus was characterized by the heavy intercalary setae fusing and extending together for a distance before diverging. The heavy setae were not visually contorted, lacking poroids and spines, in contrast with how the section Compressa has been defined. Chaetoceros millipedarius was characterized by a horizontal stagger of ordinary intercalary setae when the chain was seen in broad girdle view, making the chain look like a millipede. The two new species were supported by molecular phylogenetic analyses with C. bifurcatus sister to C. contortus var. ornatus, while C. millipedarius was sister to C. contortus var. contortus. Based on the morphological features exhibited in C. bifurcatus, the diagnosis of the section Compressa was emended to exclude "contorted" heavy setae and compressed valve faces.

Phylogeny and species delineation in the marine diatom Pseudo-nitzschia (Bacillariophyta) using cox1, LSU, and ITS2 rRNA genes: A perspective in character evolution.

Analyses of the mitochondrial cox1, the nuclear-encoded large subunit (LSU), and the internal transcribed spacer 2 (ITS2) RNA coding region of Pseudo-nitzschia revealed that the P. pseudodelicatissima complex can be phylogenetically grouped into three distinct clades (Groups I-III), while the P. delicatissima complex forms another distinct clade (Group IV) in both the LSU and ITS2 phylogenetic trees. It was elucidated that comprehensive taxon sampling (sampling of sequences), selection of appropriate target genes and outgroup, and alignment strategies influenced the phylogenetic accuracy. Based on the genetic divergence, ITS2 resulted in the most resolved trees, followed by cox1 and LSU. The morphological characters available for Pseudo-nitzschia, although limited in number, were overall in agreement with the phylogenies when mapped onto the ITS2 tree. Information on the presence/absence of a central nodule, number of rows of poroids in each stria, and of sectors dividing the poroids mapped onto the ITS2 tree revealed the evolution of the recently diverged species. The morphologically based species complexes showed evolutionary relevance in agreement with molecular phylogeny inferred from ITS2 sequence-structure data. The data set of the hypervariable region of ITS2 improved the phylogenetic inference compared to the cox1 and LSU data sets. The taxonomic status of P. cuspidata and P. pseudodelicatissima requires further elucidation.

Effects of abiotic factors on the nanostructure of diatom frustules-ranges and variability.

The intricate patterning of diatom silica frustules at nanometer-to-micrometer scales makes them of interest for a wide range of industrial applications. For some of these applications, a specific size range in nanostructure is required and may be achieved by selecting species with the desired properties. However, as all biological materials, diatom frustules exhibit variability in their morphological parameters and this variability can to some extent be affected and controlled by environmental conditions. In this review, we explore the effects of different environmental factors including salinity, heavy metals, temperature, pH, extracellular Si(OH)4 or Ge(OH)4 concentration, light regime, UV irradiance, long-term cultivation, and biotic factors on the nanostructure of diatom frustules. This compilation of studies illustrates that it is possible to affect the nanostructure of diatom frustules in vivo by controlling different environmental factors as well as by direct chemical modification of frustules. We compare these methods and present examples of how these changes affect the range of variability as well as comparing the magnitude of size changes of the most promising methods.

Isolation by Time During an Arctic Phytoplankton Spring Bloom.

The arctic phytoplankton spring bloom, which is often diatom-dominated, is a key event that provides the high latitude communities with a fundamental flux of organic carbon. During a bloom, phytoplankton may increase its biomass by orders of magnitude within days. Yet, very little is known about phytoplankton bloom dynamics, including for example how blooming affects genetic composition and diversity of a population. Here, we quantified the genetic composition and temporal changes of the diatom Fragilariopsis cylindrus, which is one of the most important primary producers in the Arctic, during the spring bloom in western Greenland, using 13 novel microsatellite markers developed for this study. We found that genetic differentiation (quantified using sample-specific FST ) decreased between time points as the bloom progressed, with the most drastic changes in FST occurring at the start of the bloom; thus the genetic structure of the bloom is characterized by isolation by time. There was little temporal variation in genetic diversity throughout the bloom (mean HE  = 0.57), despite marked fluctuations in F. cylindrus cell concentrations and the temporal change in sample-specific FST . On the basis of this novel pattern of genetic differentiation, we suggest that blooming behavior may promote genetic diversity of a phytoplankton population.

Pseudo-nitzschia arctica sp. nov., a new cold-water cryptic Pseudo-nitzschia species within the P. pseudodelicatissima complex.

A new nontoxic Pseudo-nitzschia species belonging to the P. pseudodelicatissima complex, P. arctica, was isolated from different areas of the Arctic. The erection of P. arctica is mainly supported by molecular data, since the species shares identical ultrastructure with another species in the complex, P. fryxelliana, and represents a new case of crypticity within the genus. Despite their morphological similarity, the two species are not closely related in phylogenies based on LSU, ITS and rbcL. Interestingly, P. arctica is phylogenetically most closely related to P. granii and P. subcurvata, from which the species is, however, morphologically different. P. granii and P. subcurvata lack the central larger interspace which is one of the defining features of the P. pseudodelicatissima complex. The close genetic relationship between P. arctica and the two species P. granii and P. subcurvata is demonstrated by analysis of the secondary structure of ITS2 which revealed no compensatory base changes, two hemi-compensatory base changes, and two deletions in P. arctica with respect to the other two species. These findings emphasize that rates of morphological differentiation, molecular evolution and speciation are often incongruent for Pseudo-nitzschia species, resulting in a restricted phylogenetic value for taxonomic characters used to discriminate species. The description of a new cryptic species, widely distributed in the Arctic and potentially representing an endemic component of the Arctic diatom flora, reinforces the idea of the existence of noncosmopolitan Pseudo-nitzschia species and highlights the need for combined morphological and molecular analyses to assess the distributional patterns of phytoplankton species.

Morphology and molecular phylogeny of Chaetoceros dayaensis sp. nov. (Bacillariophyceae), characterized by two 90° rotations of the resting spore during maturation.

Recent studies suggest that a high species diversity of Chaetoceros exists in tropical waters. Based on plankton samples from Chinese tropical waters, Daya Bay of the South China Sea, a new species, Chaetoceros dayaensis sp. nov., is described. Vegetative cells and resting spores were examined by light microscopy as well as transmission and scanning electron microscopy. The nuclear rDNA molecular markers SSU, ITS and D1-D3 of LSU, and the chloroplast rbcL gene were sequenced for information on phylogenetic relationships. The species is characterized by the presence of two 90° rotations of the resting spore during maturation. First a rotation in the apical plane of the mother cell resulting in the valvar plane of the resting spore turning from parallel to perpendicular to that of the mother cell, and a second 90° rotation in the valvar plane of the mother cell resulting in the valve faces of the resting spore turning from facing narrow girdle view to broad girdle view of the mother cell. It is the first report of two 90° rotations of resting spores during maturation in Chaetoceros. Based on this, the maturation rotation of the resting spore of C. rotosporus was reevaluated. C. dayaensis belongs to the subgenus Hyalochaete Gran and we suggest placing it in the section Laciniosa, although a new subdivision of Chaetoceros is needed. The description of C. dayaensis was based on analyses of the phylogenetic relationships combined with morphological comparisons with other similar species.

Dangerous Relations in the Arctic Marine Food Web: Interactions between Toxin Producing Pseudo-nitzschia Diatoms and Calanus Copepodites.

Diatoms of the genus Pseudo-nitzschia produce domoic acid (DA), a toxin that is vectored in the marine food web, thus causing serious problems for marine organisms and humans. In spite of this, knowledge of interactions between grazing zooplankton and diatoms is restricted. In this study, we examined the interactions between Calanus copepodites and toxin producing Pseudo-nitzschia. The copepodites were fed with different concentrations of toxic P. seriata and a strain of P. obtusa that previously was tested to be non-toxic. The ingestion rates did not differ among the diets (P. seriata, P. obtusa, a mixture of both species), and they accumulated 6%-16% of ingested DA (up to 420 µg per dry weight copepodite). When P. seriata was exposed to the copepodites, either through physical contact with the grazers or separated by a membrane, the toxicity of P. seriata increased (up to 3300%) suggesting the response to be chemically mediated. The induced response was also triggered when copepodites grazed on another diatom, supporting the hypothesis that the cues originate from the copepodite. Neither pH nor nutrient concentrations explained the induced DA production. Unexpectedly, P. obtusa also produced DA when exposed to grazing copepodites, thus representing the second reported toxic polar diatom.

Transition from a mixotrophic/heterotrophic protist community during the dark winter to a photoautotrophic spring community in surface waters of Disko Bay, Greenland.

Unicellular eukaryotic plankton communities (protists) are the major basis of the marine food web. The spring bloom is especially important, because of its high biomass. However, it is poorly described how the protist community composition in Arctic surface waters develops from winter to spring. We show that mixotrophic and parasitic organisms are prominent in the dark winter period. The transition period toward the spring bloom event was characterized by a high relative abundance of mixotrophic dinoflagellates, while centric diatoms and the haptophyte Phaeocystis pouchetii dominated the successive phototrophic spring bloom event during the study. The data shows a continuous community shift from winter to spring, and not just a dormant spring community waiting for the right environmental conditions. The spring bloom initiation commenced while sea ice was still scattering and absorbing the sunlight, inhibiting its penetration into the water column. The initial increase in fluorescence was detected relatively deep in the water column at ~55 m depth at the halocline, at which the photosynthetic cells accumulated, while a thick layer of snow and sea ice was still obstructing sunlight penetration of the surface water. This suggests that water column stratification and a complex interplay of abiotic factors eventually promote the spring bloom initiation.

A Pseudo-nitzschia metabarcoding approach with a calibrated ITS1 reference sequence database applied in the Taiwan Strait.

Pseudo-nitzschia is a cosmopolitan phytoplankton genus of which some species can form blooms and produce the neurotoxin domoic acid (DA). Identification of Pseudo-nitzschia is generally based on field material or strains followed by morphological and/or molecular characterization. However, this process is time-consuming and laborious, and can not obtain a relatively complete and reliable profile of the Pseudo-nitzschia community, because species with low abundance in the field or potentially unavailable for culturing may easily be overlooked. In the present study, specific ITS primer sets were designed and evaluated using in silico matching. The primer set ITS-84F/456R involving the complete ITS1 region was found optimal. Based on matching with a Pseudo-nitzschia ITS1 reference sequence database carefully-calibrated in this study, a metabarcoding approach using annotated amplicon sequence variants (ASV) was applied in the Taiwan Strait of the East China Sea during two cruises in the spring and summer of 2019. In total, 48 Pseudo-nitzschia species/phylotypes including 36 known and 12 novel were uncovered, and verified by haplotype networks, ITS2 secondary structure comparisons and divergence analyses. Correlation analyses revealed that temperature was a key factor affecting the seasonal variation of the Pseudo-nitzschia community. This study provides an overview of the Pseudo-nitzschia community in the Taiwan Strait, with new insights into the diversity. The developed metabarcoding approach may be used elsewhere as a standard reference for accurate annotation of Pseudo-nitzschia.

Did algal toxin and Klebsiella infections cause the unexplained 2007 mass mortality event in Danish and Swedish marine mammals?

An unusual mass mortality event (MME) of harbour seals (Phoca vitulina) and harbour porpoises (Phocoena phocoena) occurred in Denmark and Sweden in June 2007. Prior to this incident, the region had experienced two MMEs in harbour seals caused by Phocine Distemper Virus (PDV) in 1988 and 2002. Although epidemiology and symptoms of the 2007 MME resembled PDV, none of the animals examined for PDV tested positive. Thus, it has been speculated that another - yet unknown - pathogen caused the June 2007 MME. To shed new light on the likely cause of death, we combine previously unpublished veterinary examinations of harbour seals with novel analyses of algal toxins and algal monitoring data. All harbour seals subject to pathological examination showed pneumonia, but were negative for PDV, influenza and coronavirus. Histological analyses revealed septicaemia in multiple animals, and six animals tested positive for Klebsiella pneumonia. Furthermore, we detected the algal Dinophysis toxin DTX-1b (1-115 ng g-1) in five seals subject to toxicology, representing the first time DTX-1b has been detected in marine vertebrates. However, no animals tested positive for both Klebsiella and toxins. Thus, while our relatively small sample size prevent firm conclusions on causative agents, we speculate that the unexplained MME may have been caused by a chance incidence of multiple pathogens acting in parallel in June 2007, including Dinophysis toxin and Klebsiella. Our study illustrates the complexity of wildlife MMEs and highlights the need for thorough sampling during and after MMEs, as well as additional research on and monitoring of DTX-1b and other algal toxins in the region.