Raman Microspectroscopy Goes Viral: Infection Dynamics in the Cosmopolitan Microalga, Emiliania huxleyi

Abstract

Emiliania huxleyi is a cosmopolitan member of the marine phytoplankton. This species’ capacities for carbon sequestration and sulfur mobilization make it a key player in oceanic biogeochemical cycles that influence climate on a planetary scale. Seasonal E. huxleyi blooms are abruptly terminated by viral epidemics caused by a clade of large DNA viruses collectively known as coccolithoviruses (EhVs). EhVs thereby mediate a significant part of material and energy fluxes associated with E. huxleyi population dynamics. In this study, we use spontaneous Raman microspectroscopy to perform label-free and non-invasive measurements of the macromolecular composition of individual virions and E. huxleyi host cells. Our novel autofluorescence suppression protocol enabled spectroscopic visualization of evolving macromolecular redistributions in individual E. huxleyi cells at different stages of EhV infection. Material transfer from E. huxleyi hosts to single EhV-163 virions was confirmed by combining stable isotope probing (SIP) experiments with Raman microspectroscopy. Inheritance of the host cells’ 13C-enriched isotopic signature was quantified based on red shifts of Raman peaks characteristic of phenylalanine’s phenyl ring. Two-dimensional Raman mapping of EhV-infected E. huxleyi cells revealed that the compact region producing an intense Raman DNA signal (i.e., the nucleus) in healthy E. huxleyi cells becomes diffuse during the first hours of infection. Raman DNA emissions integrated throughout individual cells decreased during the infection cycle. Our observations are consistent with EhV-163 degrading the host’s nuclear DNA, scavenging released nucleotides for its own genome replication, and shedding newly-produced virions prior to host lysis via budding.

Structure and replication cycle of a virus infecting climate-modulating alga Emiliania huxleyi

“…4B ). Similar changes in the nuclear structure were reported previously for infections by NCVs that replicate in cell nuclei ( 55 , 56 ).…”

Viral infection arrests coccolithophore calcification and nutrient consumption, and triggers shifts in organic stoichiometry

“…However, a higher affinity for phosphate has been described during the night, likely due to higher requirements for cell division (Geider and La Roche 2002). Nutrients acquired up to this stage through the infection are naturally incorporated into EhV biosynthesis as has been recently demonstrated in isotopic labeling experiments with carbon and N (Pasulka et al 2018;Yakubovskaya et al 2021). The reliance of EhV on external phosphate has not been thus far assessed, but likely follows analogous trends.…”

“…. Nutrients acquired up to this stage through the infection are naturally incorporated into EhV biosynthesis as has been recently demonstrated in isotopic labeling experiments with carbon and N(53,54). The reliance of EhV on external phosphate has not been thus far assessed, but likely follows analogous trends facilitated by the activity of a viral phosphate permease gene encoded by some EhV strains(55,56).…”

One Cell at a Time: Advances in Single-Cell Methods and Instrumentation for Discovery in Aquatic Microbiology

“…SIP-Raman has also been applied to food web studies, tracing movement of carbon from prey to predator ( Li et al, 2013 ). Taking SIP-Raman a step further, Yakubovskaya et al (2021) demonstrated that individual viruses inherited the 13 C-isotopic signatures of their labeled host, Emiliania huxleyi . Similarly, a viral-BONCAT method was used in concert with nanoSIP to track N and C transfer from host to virus during lysis ( Pasulka et al, 2018 ).…”