Pseudo-nitzschia Challenged with Co-occurring Viral Communities Display Diverse Infection Phenotypes

Abstract

Viruses are catalysts of biogeochemical cycling, architects of microbial community structure, and terminators of phytoplankton blooms. Viral lysis of diatoms, a key group of eukaryotic phytoplankton, has the potential to impact carbon export and marine food webs. However, the impact of viruses on diatom abundance and community composition is unknown. Diatom-virus dynamics were explored by sampling every month at two coastal and estuarine locations in Washington state, USA resulting in 41 new isolates of the pennate diatom Pseudo-nitzschia and 20 environmental virus samples. We conducted a total of 820 pair-wise crosses of the Pseudo-nitzschia isolates and viral communities. Viral communities infected Pseudo-nitzschia isolates in 8% of the crosses overall and 16% of crosses when the host and viral communities were isolated from the same sample. Isolates ranged in their permissivity to infection with some isolates not infected by any viral samples and others infected by up to 10 viral communities. Isolates that were infected by the most viral communities also had the highest maximum observed viral titers (as high as 16000 infectious units ml(-1)). Titers of the viral communities were host dependent, as titers for one viral sample on eight different hosts spanned four orders of magnitude. Sequencing of the Pseudo-nitzschia Internal Transcribed Spacer 1 (ITS1) of the revealed multiple subgroups of hosts with 100% ITS1 identities that were infected by different viral communities. Indeed, we repeatedly isolated groups of isolates with identical ITS1 sequences from the same water sample that displayed different viral infection phenotypes. The interactions between Pseudo-nitzschia and the viral communities highlight the diversity of diatoms and emphasize the complexity and variability of diatom-virus dynamics in the ocean.

Keywords: Pseudo-nitzschia; diatom; harmful algal bloom; microdiversity; phytoplankton; titer; virus.

FIGURE 1

Locations of sampling. Penn Cove, located in the Puget Sound estuary, and Grays Harbor located on the coast of Washington state, USA. Inset map of North America shows the region of sampling.

FIGURE 2

Pseudo-nitzschia isolates obtained and community composition at (A,C) Penn Cove and (B,D) Grays Harbor from April 2013 to April 2014. Time of sampling is shown in Julian day and monthly increments on the x-axis. Solid lines are water temperature and dashed lines are nitrate concentration. Pseudo-nitzschia species are colored by phylogenetic clade (Lundholm et al., 2002, Guannel, unpublished data) with members of clade 1 represented by warm colors and clade 2 represented by cool colors. Unidentifiable ARISA fragments are represented in grayscale. Black bars represent ARISA fragments for isolated Pseudo-nitzschia with no species identification. ^∗ indicates months with no detectable Pseudo-nitzschia by ARISA or in net tows.

FIGURE 3

Percent of crosses between Pseudo-nitzschia isolates and environmental viral communities that were infectious based on the time and location of host isolation and virus community sample collection. ^∗ Denotes a significant of p-value = 0.009 as determined by a Chi-square test.

FIGURE 4

Total number of viral community samples that resulted in an infection for each host strain. Colors correspond to the number of replicates that were lysed and the corresponding range of infectious units based on most probable number tables for each infectious cross. Infectious units ml^-1 of seawater were calculated assuming 100% retention of infectivity and accounting for the effect of concentrating virus from 20 L of seawater and volume of viral concentrates added to host cultures in crosses.

FIGURE 5

Titers of infectious units over time in Julian days with monthly increments at (A) Penn Cove and (B) Grays Harbor. Each of the nine strains was crossed with each of the Penn Cove or Grays Harbor viral communities. Cool colors are hosts isolated from Penn Cove, warm colors are hosts isolated from Grays Harbor. Error bars are 95% confidence intervals from 5 well MPN tables. Values below the limit of detection of 1.8 infectious units ml^-1 are not shown.

FIGURE 6

Pseudo-nitzschia – virus infection network. Filled boxes represent infectious crosses. Black outlines delineate groups of hosts that share identical ITS1 sequences, which are labeled underneath.