Molecular probing on Pfiesteria piscicida to assess variability.

Pfiesteria piscida is a marine dinoflagellate related to toxic algal blooms. They pose threats to areas such as public health and natural resources. They’re also related to large-scale fish kills, which are well documented, being recorded since 1991. They’re particularly well documented in the North Carolina Albermale-Pam-Lico estuaries. In some cases they have caused deaths of one billion fish in a single event. These will have clear ecological and economic impacts, as well as possible threats to humans (Conye et al., 2001)

There are high levels of P.piscicida found along the Atlantic coast of the US. These estuaries in the mid-Atlantic are at a particularly high risk of toxic blooms. Therefore, the levels of P.piscicida need to be correctly assessed in order to develop monitoring strategies of the dinoflagellates. Monitoring strategies are in place, using such techniques as light microscopy. However, these methods aren’t sensitive or accurate enough aren’t species-specific and are unable to detect numbers. With monitoring of such importance, more sensitive techniques are needed.

A study done by Conye et al., 2007, used 2 different techniques for detection and enumeration of P.piscicida in the Delaware inland bays and Pokomoke River. Samples were collected from shallow water estuaries in this area. DNA was then extracted from each water sample. The samples were cultured in f/2 medium. The first technique used on the samples was denaturing gradient gel electrophoresis (DGGE). This technique was performed as in Muyzer et al., 1993. The second technique used to quantitatively enumerate samples was PCR fluorescent fragment detection (PCR-FFD). This was done by firstly doing PCR amplification, followed by detection of HEX-labeled PCR products on an ABI prism 310 genetic analyzer using genescan software (Conye et al., 2009). Positive PCR products were shown by retention time during electropheresis, and quantified by peak area (Conye et al., 2009).

The DGCE confirmed that the spread of P.piscicida in the mid-Atlantic estuaries. On top of the already known toxic strain, 5 other strains were also identified, which wasn’t an unexpected result. They also found that the use of PCR-FFD had a huge increase in sensitivity in comparison to microscope techniques, up to 1000 times more so.

The study by Conye et al., 2007 demonstrated the utility of PCR-FFD by doing a diel study in relation to other physical, chemical and biological factors. It indicates that P.piscicida is present in low levels in channels in highly turbid conditions and in areas of high water exchange, not just in surface waters. Overall they state P.piscicida as a minor but important part of the phytoplankton.

As a whole, I see this study as being a huge help to the identification of the dinoflagellates responsible for harmful algal blooms. This is also of huge importance due to the impacts that these algal blooms have, especially in such areas as human health and fish kills. It can then be linked to other aspects of algal blooms, in areas such as global warming and its effects on bloom severity and frequency. So the techniques used in this study have definite potential when looking at bloom forming dinoflagellates, and further research may be done that looks at particular areas within harmful algal blooms.

Coyne, K., Hutchins, D., Hare, C., Cary, S. (2001). Assessing temporal and spatial variability in Pfiesteria piscicida distributions using molecular probing techniques. Aquatic Microbial Ecology. 24, 275-285

Muyzer ,G., De Waal, E., Uitterlinden, A.. (1993). Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA.. Applied and Environmental Microbiology. 59, 695-700