An assessment of RNA content inPrymnesium parvum,Prymnesium polylepis,cf.Chattonellasp. andKarlodinium veneficumunder varying environmental conditions for calibrating an RNA microarray for species detection

Abstract

Traditional methods of identification and enumeration can be somewhat ambiguous when identifying phytoplankton that requires electron microscopic examination to verify specific morphological features. Members of the genus Prymnesium (division Haptophyta), members of the Raphidophyceae and naked dinoflagellates are examples of such phytoplankton whose identification can be difficult. One alternative to traditional microscopy-based methods of identification is to use molecular protocols to detect target species. Methods that measure cellular DNA and RNA content can be used to estimate the number of cells present in a sample. This study investigated the variation of RNA yields in Prymnesium parvum, P. polylepis, cf. Chattonella sp. and Karlodinium veneficum cells grown under different light, temperature, salinity and inorganic nutrient conditions. This information was used to calibrate the signal intensity of a variety of oligonucleotide probes spotted onto the microarrays for the detection of toxic algae (MIDTAL), which is being developed to aid national monitoring agencies and to provide a faster means of identifying and quantifying harmful phytoplankton in water column samples.

Stress Response in Toxic Diatoms: The Effects of Abiotic Factors on Growth and RNA Content in Pseudo-nitzschia calliantha and in Pseudo-nitzschia multistriata

“…RNA increase was particularly evident in cultures exposed to low salinities, in Pm clones in hypersaline media, and under low-temperature stress and silicon depletion. This could be associated with the stenohaline nature of the test species [16]. The same low-temperature effect was observed in the model freshwater species Chlamydomonas reinhardtii by Hessen and co-workers, who hypothesized that RNA increase was due to a compensatory mechanism aimed at avoiding a reduction in protein synthesis [33].…”

“…Moreover, identification with an LM is not accurate, since it does not allow us to distinguish between toxic and similar non-toxic strains [14]. For these reasons, the use of RNA probes was approved in the EU Seventh Framework Programme (FP7), and regulated in the "microarrays for the detection of toxic algae" (MIDTAL) project [13,15,16], which employs microarrays designed with SSU and LSU rRNA specific probes for species molecular identification [17]. RNA-based microarrays had been successfully applied for the identification of Pseudo-nitzschia species in the natural environment, as well as for the detection of cryptic species.…”

Quantitative real‐time PCR detection of a harmful unarmoured dinoflagellate, Karlodinium australe (Dinophyceae)

“…Improper fixation for unarmored dinoflagellates that lack the protective theca may cause cell shape distortion and shrinkage (Sherr & Sherr ; McCoy et al . ). The morphological characteristics such as cell size, position of nucleus, and the arrangement of chloroplasts that used to identify species of Karlodinium are also subtle and often misleading.…”

Molecular Techniques for the Detection of Organisms in Aquatic Environments, with Emphasis on Harmful Algal Bloom Species

“…The laser in a microarray scanner scans the slides and the hybridization pattern captured via fluorescent excitation indicates which species are present [ 60 ]. DNA microarrays, or phylochips as they have been termed, have been used to identify phytoplankton [ 63 ], toxic algae [ 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 ], bacteria [ 78 , 79 , 80 , 81 , 82 , 83 , 84 ], and eggs and larvae from fish species [ 85 ]. Phylochip ® , a universal microarray for all prokaryotic organisms is commercially available and circumvents the long analysis time to perform community analysis for the prokaryotes using other molecular tools.…”