Rapid quantification of viable cells of the planktonic diatom <i>Chaetoceros tenuissimus</i> and associated RNA viruses in culture

Abstract

Abstract:To understand the ecological relationships between diatoms and viruses in marine environments, extensive culture experiments under diverse environmental conditions are essential. Previous methods developed for enumeration of diatom cells and viruses in culture experiments, i.e., manual counting using a microscope and the most probable number (MPN) method, are time consuming and labour intensive. In this study, we developed a rapid enumeration method for cells of the planktonic diatom Chaetoceros tenuissimus and the genome copy numbers of the RNA virus CtenRNAV using image-based cytometry (IBC) and real-time quantitative polymerase chain reaction (qPCR), respectively. The live cell numbers counted using IBC were similar to those obtained by direct microscopic counts within the range from 10 4 to 10 6 mL −1. The genome copy numbers of the viruses, as determined by qPCR, were almost 2-20 times higher than those determined by the MPN method owing to the presence of defective particles and virion aggregations in the samples. The dynamic patterns of virus abundances were similar for both methods; therefore, qPCR could be applied as an alternative method for enumeration of infectious virus units. The IBC and qPCR methods developed for diatom cells and viruses in this study were rapid and accurate and should therefore have applications as powerful tools for improving our understanding of diatom-virus ecological relationships.

Growth Rate-dependent Cell Death of Diatoms due to Viral Infection and Their Subsequent Coexistence in a Semi-continuous Culture System

“…They inhibit the production of normal wild-type viruses and lytic infections (Thyrhaug et al, 2003;Brown and Bidle, 2014). CtenRNAV-II abundances in titer only explain 8-42% of qPCR copy numbers (Tomaru and Kimura, 2016). In the present study, RNA virus copy numbers assessed with the qPCR assay were 2-175-fold higher than the number of infectious viruses estimated using the extinction dilution method (Fig.…”

“…Live cell numbers were counted using an image-based cytometer (Tali ® Image Cytometer; Thermo Fisher Scientific) (Tomaru and Kimura, 2016). The SYTOX ® Green nucleic acid stain (excitation, 504 nm; emission, 523 nm; Thermo Fisher Scientific) was added to the cell suspension (1% [v/v]) at a final concentration of 0.5 μM.…”

Virus‐induced spore formation as a defense mechanism in marine diatoms

“…A serial dilution of a viral reverse transcript sample was employed to construct a standard curve to quantify products. The viral copy number was calculated with empirical formulas which have been described previously (Tomaru & Kimura, 2016). Virus burst size was calculated by dividing the total number of viruses produced by the number of host vegetative cells lost (i.e.…”

Chronological age-related metabolome responses in the dinoflagellate Karenia mikimotoi, can predict future bloom demise

“…For cell counting, a 25 μL aliquot of culture was placed on a disposable counting slide (Thermo Fisher Scientific). After incubation for 10 min in the dark at 20 °C, the cells were counted in the same manner as that for K. mikimotoi except that size range was set at 3-20 μm and the red fluorescent threshold at 1200 39 . Cell numbers were counted for up to 28 days at intervals of 5−8 days (day 0, 2, 7, 13, 21, and 28).…”