Pseudo-nitzschia

Since 1983, San Pedro Bay in the Philippines had been reported to be the site of episodic Pyrodinium bahamense var. compressum blooms that caused paralytic shellfish poisoning in its nearby coastal communities. This bay is also subjected to numerous storms; the strongest was super typhoon Haiyan in November 8, 2013. For the first time, the seasonal dynamics of potentially toxic and harmful phytoplankton in this bay is elucidated. This is also the first record of a bloom of the cyanobacteria, Trichodesmium erythraeum that reached 70 000 colonies/L in April 2013 in this area. There were other 19 potentially toxic and harmful phytoplankton encountered during the sampling period. These consisted of a haptophyte, Phaeocystis globosa, the diatom Pseudo-nitzschia and 17 dinoflagellates. Seven of these harmful algae had densities high enough to be traced through time. Normally, diatoms abound during the dry season. But Pseudo-nitzschia increased in abundance during the wet season of 2012 and 2013. The dinoflagellates and Phaeocystis globosa behaved as expected and exhibited a relative increase in cell density during the rainy season of both years too. High nutrient availability during this season must have influenced the behavior of the phytoplankton despite differences in temperature and light intensity among seasons. Other notable but rare harmful species found only in plankton net tows during the study were Pyrodinium bahamense var. compressum, Alexandrium tamiyavanichii, Cochlodinium polykrikoides, and Noctiluca scintillans. Rev. Biol. Trop. 64 (2): 897-911. Epub 2016 June 01.

The genus Pseudo-nitzschia is one of the most well studied marine diatoms as fifteen of the species are known to produce the neurotoxin, domoic acid (DA) that is responsible for Amnesic Shellfish Poisoning (ASP). Thus far, little attention is given to the distribution of Pseudo-nitzschia spp. in Malaysia. In this study, three species of Pseudo-nitzschia were identified as P. pungens, P. brasiliana and P. dolorosa based on detailed ultrastructure under light and electron microscopy. Species identification was supported by LSU rDNA phylogenetic analyses. The application of sequence-structure alignment based on the second internal transcribed spacer (ITS2) RNA transcript was also inferred to improve the phylogeny which allows comparisons at deeper taxonomic levels. The highly conserved ITS2 secondary structures of Pseudo-nitzschia spp. were predicted through free energy minimization and homologous modeling. Results revealed two distinct types of ITS2 secondary structure models viz. Type A and Type B. This region of ITS2, together with the structural information obtained was further incorporated into our phylogenetic inference of Pseudo-nitzschia. The study revealed that the phylogenetic position of Pseudo-nitzschia spp. in the ITS2 sequence-structure tree is better well resolved compared to the LSU rDNA tree topology. On the other hand, mapping of 14 morphological characters on Pseudo-nitzschia tree showed that the character of central interspace (CIS) was of taxonomic diagnostic value. The other 13 characters and toxicity were somehow homoplastic. Intraspecific genetic variation of P. pungens and P. brasiliana from Borneo was also investigated and results showed that Bornean populations of P. pungens were distinctly isolated which could be due to the surface current pattern in the South China Sea. This study represents the first study of the Pseudo-nitzschia occurrence in the region with extensive morphological and genetic information.

Field sampling was undertaken to investigate the occurrence of Pseudo-nitzschia Peragallo species in eight locations along the coast of Malaysian Borneo. A total of 108 strains of Pseudo-nitzschia species were isolated, and their morphology examined with SEM and TEM. Additionally, molecular data from nuclear-encoded partial LSU rDNA, and ITS regions, were characterized. A total of five species were confidently identified based on a combination of distinct morphological characteristics and supporting molecular evidence: P. brasiliana Lundholm, Hasle & Fryxell, P. cuspidata (Hasle) Hasle, P. dolorosa Lundholm & Moestrup, P. micropora Priisholm, Moestrup & Lundholm, and P. pungens (Grunow) Hasle var. pungens. However, one morphotype from Sarawak, while somewhat similar to P. caciantha, showed significant morphological distinction from this and any other of the currently described species. Most notably this morphotype possessed a characteristic pore arrangement in the poroids, with the fine pores in each perforation sector arranged in circles. Pair-wise sequence comparison of the LSU rDNA between this unidentified morphotype and P. caciantha Lundholm, Moestrup & Hasle, revealed 2.7% genetic divergence. Phylogenetic analyses strongly supported the monophyly of the morphotype. Based upon these supporting data it is here described as a new species, Pseudo-nitzschia circumpora sp. nov. A key to the six species of Pseudo-nitzschia from Malaysian Borneo is presented. Molecular signatures for all species were established based on structural comparisons of ITS2 rRNA transcripts.

Three clades of Pseudo-nitzschia pungens,
determined by the internal transcribed space (ITS)
region, are distributed throughout the world. We
studied 15 P. pungens clones from various
geographical locations and confirmed the existence
of the three clades within P. pungens, based on ITS
sequencing and described the three subgroups
(IIIaa, IIIab, and IIIb) of clade III. Clade III (clade
IIIaa) populations were reported for the first time
in Korean coastal waters and the East China Sea. In
morphometric analysis, we found the ultrastructural
differences in the number of fibulae, striae, and
poroids that separate the three clades. We carried
out physiological tests on nine clones belonging to
the three clades growing under various culture
conditions. In temperature tests, only clade III
clones could not grow at lower temperatures (10°C
and 15°C), although clade I and II clones grew well.
The estimated optimal growth range of clade I
clones was wider than that of clades II and III.
Clade II clones were considered to be adapted to
lower temperatures and clade III to higher
temperatures. In salinity tests, clade II and III
clones did not grow well at a salinity of 40. Clade I
clones were regarded as euryhaline and clade II and
III clones were stenohaline. This supports the
hypothesis that P. pungens clades have different
ecophysiological characteristics based on their
habitats. Our data show that physiological and
morphological features are correlated with genetic
intraspecific differentiation in P. pungens.

The diatom Pseudo-nitzschia has been known for domoic acid poisoning, or better known as Amnesic Shellfish Poisoning (ASP). Accumulation of domoic acid (DA) in commercial bivalves during the bloom of this toxic diatom has caused economic losses to the industry. To clarify the status of ASP and the diversity of toxic or potentially toxic Pseudo-nitzschia species in the Malaysian waters, plankton samples were collected from 17 locations using 20 µm-mesh plankton net haul. Samples were subjected to acid wash treatment before detail observation under transmission electron microscope (TEM). Identification of Pseudo-nitzschia species was based on the frustules morphology with the morphometric measurements. In total, 22 species were identified, including 18 new records in Malaysia. and P. turgidula are known to be toxic and previously reported to associate with ASP events worldwide. This study provides useful information on the harmful algae species inventory of the country.

To clarify the genetic diversity of a potentially toxic pennate diatom, Pseudo-nitzschia brasiliana found in Malaysian waters, 30 strains of P. brasiliana were established into clonal culture since May 2008. The ultrastructure of these strains was examined for confirmation of species identification. The genetic marker, internal transcribed spacer (ITS) of nuclear-encoded ribosomal DNA was used to examine the genetic diversity of P. brasiliana isolated from different geographical localities. The ITS sequences of P. brasiliana were highly conserved in their secondary structures, with five helices in the first internal transcribe spacer (ITS1) and four universal helices in the second internal transcribe spacer (ITS2) with a pseudo-helix. No compensatory base change was observed among the strains examined. Genetic divergences among the Malaysian strains ranged from 0.07 to 0.54%. The present study revealed a high genetic homogeneity of Malaysian P. brasiliana strains.

Amnesic shellfish poisoning (ASP) is a type of shellfish poisoning due to the consumption of shellfish mollusks contaminated with domoic acid (DA). The toxin was first reported in the chain-forming pennate diatom, Pseudo-nitzschia and subsequently in other diatom species. In this study, clonal cultures of Pseudo-nitzschia were established from plankton samples collected from Sarawak and Sabah coastal waters. Clonal cultures were maintained in SWII medium with the addition of silicate at 25°C, 30 PSU and under 12:12 light-dark photoperiod. Fifteen milliliters of late exponential phase cultures were collected for toxin analysis and subsamples were taken for cell count. Cellular toxin was extracted by boiling in medium at 100°C for 5 minutes. The extracts were filtered to remove cell debris before being analyzed with HPLC using standard domoic acid, isodomoic A and B as reference toxins. All the 32 strains of Pseudo-nitzschia sp. analyzed in this study showed the absence of peaks corresponding to the three ASP toxins. This implies that non-toxic strains of Pseudo-nitzschia sp. are common in Malaysian waters. Further study will be carried out to include more strains along the coastal waters of Borneo as well as selected sites with shellfish farming activities in Peninsula Malaysia.

About one third of the species in the genus Pseudo-nitzschia has been known to produce domoic acid, which is responsible for amnesic shellfish poisoning, resulting in human intoxication and the death of marine fauna. Precise identification to species level was done based on ultrastructure of frustules marking, which includes fibulae, striae, poroids and central interspace, as well as the cell dimensions. In this study, detail morphological observation of field samples and clonal cultures of Pseudo-nitzschia from estuarine waters in Kuching, Samariang Batu, Santubong and Muara Tebas was carried out using electron microscopy (SEM and TEM). Diatom cells were treated with acid to remove organic cellular contents before observation under SEM and TEM. Two morphotypes were observed from these three locations. One morphotype possesses a rectangular valve shape, the apical and transapical axis of 31.9-36.8μm and 2.6-3.0μm, respectively, with 21-25 fibulae and 21-24 striae in 10μm. The other morphotype shows linear to lanceolate cell shape with the apical axis of 67.2-79.9μm and the transapical axis of 3.0-3.5μm. Numbers of fibulae and striae in 10μm are 12-13 and 11-14, respectively. Central interspace is absent in both morphotypes. Based on the ultrastructure, the two morphotypes of Pseudo-nitzschia is designated as P. brasiliana and P. pungens, respectively.