3.2.5. Species Unfound by Cell Counts but Identified with Microarray and Hierarchy File

Fish Killing Species
Lugol’s-fixed cells of Pseudochattonella are difficult to identify by light microscopy because the cell shape changes and the discharge of mucocysts gives them a warty appearance [26]. It is possible to distinguish the two sister species P. farcimen and P. verruculosa molecularly, because the two differ in several bases in the large ribosomal subunit [26]. In sample 6A, all genus-level probes of Pseudochattonella (PschGS01_25_dT, PschGS04_25_dT, PschGS05_25_dT) and the two species-level probes PfarD01_25_dT (P. farcimen) and PverD01_25_dT (P. verruculosa) were detected with a signal-to-noise above 2 (Figure 5(a)). The integrated calculation of cells L−1 in the GPR-Analyzer [20] revealed for Pseudochattonella farcimen 19,463 cells·L−1 and for Pseudochattonella verruculosa 48,428 cells·L−1, which is likely to be overestimated. Indeed, this species has only been identified in fjords and open waters of the North Sea, Skagerrak, and Kattegat, whose temperatures are below 10 °C [27]. During the summer–fall season, waters in Arcachon Bay are typically >25 °C [28]. Our results suggest perhaps another very closely related species as yet undetected could be in Arcachon Bay if the distribution of this species is exclusively in cold temperate waters. If this probe continues to show positive results, the probe could be used as a FISH probe to retrieve the cells giving the signal on the microarray for further investigations. Cells hybridized by the probe could be sorted by flow cytometry and investigated morphologically or molecularly. Once identified, the cells could later be brought into culture and their toxicity tested with bioassays.
Figure 5  (a) Normalized signal intensity of the genus-level probes (PschGS01_25_dT, PschGS04_25_dT, PschGS05_25_dT) of Pseudochattonella and the two species-level probes PfarD01_25_dT (Pseudochattonella farcimen) and PverD01_25_dT (Pseudochattonella verruculosa) for sample 6A (20.10.2011) only. (b) Normalized signal intensity of the class-level probes (PrymS03_25_dT, PrymS01_25_dT) and the clade-level probe (Clade01old_25_dT) of Prymnesium spp. (c) Normalized signal intensity of the genus-level probe of Karlodinium spp. (KargeD01_25_Dt).   No Prymnesiophyta were identified by cell counts, but the higher group probe for Prymnesiophyta (PrymS01_25_dT), the class level for Prymnesiophyceae (PrymS03_25_dT) and the clade-level probe for Prymnesium clade B1 (Clade01old_25_dT) were detected throughout the sampling period (Figure 5(b)). The second clade-level probe for Prymnesium clade B1 (Clade01new25_dT) was detected in samples 3A, 4A and 6A. Furthermore, the species-level probe for P. polylepis (CpolyS01_25_dT) was detected in sample 6A and in sample 4A the species-level probe for P. parvum (PparvD01_25_dT). This indicates the potential for a fish-killing event in Arcachon Bay under the appropriate conditions for growth. Although the Prymnesiophyta group is taken into account within the harmful phytoplankton monitoring program, the small size of this genus (<10 µm) as well as the smaller volume of water used for Utermöhl sedimentation and observation (100 mL maximum) than the volume of filtered seawater for RNA extraction, avoid any faithful microscopy identification and counting. The microarray can detect Prymnesium above 5 ng, which is equivalent to 3,800 cells for P. polylepis and 8,800 cells for P. parvum [29]. In our case (3 L filtered) it means 1,100 and 2,500 cells·L−1, respectively, which are high enough to be counted in a 10- or 100-mL sedimented subsample.
The genus Karlodinium (KargeD01_25_dT) was first detected in sample 3A and then with decreasing signals onwards (Figure 5(c)). Karlodinium veneficum is a high-biomass producer and the collapse of a bloom leads to the production of a surface scum that is visible as an oily, brownish discoloration of the water and kills fish and other gill-breathing animals [30]. However, no signals were detected for the six species-specific probes of K. veneficum present on the microarray. Based on their calibration curves (data not shown), the detection limit for four of the six probes is around 247 cells. The species-specific level probes are more sensitive than the genus-level probe. Therefore, we can exclude this species as a potential candidate being present in the bay. This is another example of how the microarray can detect potentially toxic species that are not counted or identified as being potentially toxic.

Azaspiracid Shellfish Poisoning (AZP) Toxins Producer
Azadinium spp. (AzaGS01_25_dT) was detected in sample 4A but only in two out of five spots on two different microarray slides. This may not be a genuine signal, but this species has only recently described [31] and it is also a relatively arduous species to identify based on light microscopy. Not all monitoring agencies are able to adjust their cell counts routinely to account for this toxic species. At least three more toxic species have been recently isolated and described [32,33,34].

Other PSP Toxins
One species-level probe out of four for Gymnodinium catenatum (LSGcat0270A24_dT) was detected in samples 1A, 4A and 6A. In samples 4A and 6A, the microarray also detected another species-level probe for G. catenatum (SSGcat0826A27_dT). The signals were not very high (S/N ratio between 2.2 and 4.9). G. catenatum is known to cause PSP and could therefore contribute, besides Alexandrium, to its detection via the ELISA and Multi SPR.