Cell concentrations associated with genomics sampling to characterize bacterial responses to parasite-host metabolites from bottle incubation experiments in June of 2024

Website: https://www.bco-dmo.org/dataset/962727
Data Type: experimental
Version: 1
Version Date: 2025-05-29

Project
» Characterizing plankton parasite-host metabolites and the response of heterotrophic bacteria (Plankton parasite-host metabolites)

Program
» Center for Chemical Currencies of a Microbial Planet (C-CoMP)
ContributorsAffiliationRole
Anderson, SeanUniversity of New Hampshire (UNH)Principal Investigator
Harvey, ElizabethUniversity of New Hampshire (UNH)Co-Principal Investigator
Poulson-Ellestad, KelseyRoosevelt University (RU)Co-Principal Investigator
Place, PhilipUniversity of New Hampshire (UNH)Scientist
Gray, LauraWoods Hole Oceanographic Institution (WHOI)Data Manager
York, Amber D.Woods Hole Oceanographic Institution (WHOI BCO-DMO)BCO-DMO Data Manager

Abstract
Bottle-based incubation experiments were conducted with coastal seawater that was filtered to include only the bacterial fraction. Fresh filtrate from Scrippsiella acuminata cultures infected by Amoebophrya sp. were collected and added into coastal seawater to expose heterotrophic bacterial communities to parasite-host metabolites and explore changes in bacterial community composition and gene expression over time. Data reported here are cell concentrations of bulk heterotrophic bacteria measured via flow cytometry that accompanied samples collected for DNA metabarcoding and metatranscriptomics from the incubation bottles over time.


Coverage

Location: Laboratory incubation experiments with coastal New Hampshire seawater
Spatial Extent: Lat:43.072 Lon:-70.712
Temporal Extent: 2024-06-04 - 2024-10-04

Methods & Sampling

Location description:
Collected water and conducted bottle incubations off the UNH Coastal Marine Laboratory pier in June 2024.

Cultures and incubation set-up:

Amoebophrya sp. (RCC 4401) parasite spores were maintained by adding fresh host (Scrippsiella acuminata, strain RCC 1627)

every 2-3 d at a ratio of 1:1 spore to host by volume. Parasite and host cultures were kept at 18 °C on a 12:12 hour light: dark cycle at 80-100 µmol photon m-2 s-1. Prior to the incubation experiment, exudates from freshly lysed S. acuminata were collected by filtering parasite cultures through 0.2 µm filters (Long et al. 2021). As controls, exudates were also collected from healthy host cultures and f/2 media used for culturing. All exudates were stored at 4 °C for <1 h before being combined with natural seawater for incubation experiments.

Surface seawater (20 L) was collected at high tide from the UNH Coastal Marine Laboratory (CML) pier that is located at the mouth of Portsmouth Harbor in New Castle, NH. Seawater was immediately brought back to the lab and filtered through a 1.2-µm mesh filter to isolate natural bacterial communities. Filtered seawater (500 mL) was combined with 1.5 L of either filtrate from infected, healthy host, or the f/2 culture media in triplicate 2-L polycarbonate bottles (Maas et al. 2020) to explore the bacterial response to sources of dissolved organic carbon (DOC). These treatments are represented as "Infected", "Host", or "Media" within the data file in the Treatment column. The baseline community is represented by "Natural" and reflects the bacterial community at the initial time of sampling (Time zero = T0). A total of 9 bottles were allowed to incubate at ambient sweater conditions for a total of 48 h in a crab trap fixed to the dock. Bottles were sampled at 6, 24, and 48 h for DNA/RNA filtration (~500 ml per bottle) and flow cytometry. Triplicate flow cytometry samples were also taken from the natural bacterial community at T0.

Flow cytometry:

Samples (1.8 mL) from each bottle and time point were fixed with 1% glutaraldehyde, stored at 4 °C, and run on a Guava easyCyte HT (Millipore) flow cytometer within 4 months of collection to estimate bulk bacterial abundances. To prepare for the run, 198 µl per sample was loaded onto a 96-well plate, stained with 100x SYBR Green (Thermo Fisher), and set in the dark at room temperature for 30 min (Nunn et al. 2024). Samples were run on the flow cytometer at low flow rate (0.24 µl s-1) for 3 min per well. Bulk heterotrophic bacteria populations were distinguished from pre-defined gates based on plots of forward scatter, which provides an indication of cell size, and green fluorescence (512 nm). In this case, green fluorescence at an emission wavelength of 512 nm reflects the binding of double-stranded DNA  to the SYBR Green dye that emits fluorescence upon excitation. 

Organism information:

TaxonomicName, Life Science Identifier (LSID), strain_identifier, organism_type
Scrippsiella acuminata, urn:lsid:marinespecies.org:taxname:1321853, RCC 1627, host
Amoebophrya sp., urn:lsid:marinespecies.org:taxname:109448, RCC 4401, parasite


Data Processing Description

Cell concentrations (cells mL-1) were estimated within the Guava InCyte software based on abundances measured per gate and the volume of sample analyzed.


BCO-DMO Processing Description

* Submitted data file "bacteria_counts.csv" renamed to "bacteria_counts.xlsx" so it could be imported into the BCO-DMO data system for this dataset. File was in xlsx format not plain text.
* Sheet 1 within file "bacteria_counts.xlsx" was imported into the BCO-DMO data system for this dataset. Values "nd" imported as missing data values.   Table will appear as Data File: 962727_v1_bacterial_fcm_data.csv (along with other download format options).

Missing Data Identifiers:
* In the BCO-DMO data system missing data identifiers are displayed according to the format of data you access. For example, in csv files it will be blank (null) values. In Matlab .mat files it will be NaN values. When viewing data online at BCO-DMO, the missing value will be shown as blank (null) values.

* Taxonomic identifiers added from name matches at the World Register of Marine Species (WoRMS), they exactly matched known names there as of 2025-05-29.
* References added for the RCC strains cited in metadata.


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Data Files

File
962727_v1_bacterial_fcm_data.csv
(Comma Separated Values (.csv), 1.28 KB)
MD5:bce45c05496aa72eaee98ac296478c62
Primary data file for dataset ID 962727, version 1

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Supplemental Files

File
bacteria_stained.xml
(Extensible Markup Language (.xml), 6.98 KB)
MD5:de4e5979e4c5442e749ada7457517adc
Raw .xml file for flow cytometry samples that were measured as part of the bacterial response experiment to quantify cell concentrations of heterotrophic bacteria. Samples were stained prior to running with 100x SYBR Green (Thermo Fisher). A single run was performed on a 96-well plate on a Guava easyCyte HT (Millipore) flow cytometer.
Flow Cytometry Standard (FCS) File
filename: bacteria_stained.fcs
(Flow Cytometry Standard (FCS), 6.68 MB)
MD5:7b8fe512792e1aed337becd52909be36
Flow Cytometry Standard (FCS) file for flow cytometry samples that were measured as part of the bacterial response experiment to quantify cell concentrations of heterotrophic bacteria. Samples were stained prior to running with 100x SYBR Green (Thermo Fisher). A single run was performed on a 96-well plate on a Guava easyCyte HT (Millipore) flow cytometer.

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Related Publications

Carlson, C. A., Hansell, D. A., Nelson, N. B., Siegel, D. A., Smethie, W. M., Khatiwala, S., Meyers, M. M., Halewood, E. (2010). Dissolved organic carbon export and subsequent remineralization in the mesopelagic and bathypelagic realms of the North Atlantic basin. Deep Sea Research Part II: Topical Studies in Oceanography, 57(16), 1433–1445. doi:10.1016/j.dsr2.2010.02.013
Methods
Chen, T., Liu, Y., Hu, Z., Song, S., & Li, C. (2021). Chloroplast Ultrastructure and Photosynthetic Response of the Dinoflagellate Akashiwo sanguinea Throughout Infection by Amoebophrya sp. Frontiers in Marine Science, 8. https://doi.org/10.3389/fmars.2021.742498
Methods
Guillard, R. R. L. (1975). Culture of Phytoplankton for Feeding Marine Invertebrates. Culture of Marine Invertebrate Animals, 29–60. doi:10.1007/978-1-4615-8714-9_3
Methods
Kayal, E., Alves-de-Souza, C., Farhat, S., Velo-Suarez, L., Monjol, J., Szymczak, J., Bigeard, E., Marie, D., Noel, B., Porcel, B. M., Corre, E., Six, C., & Guillou, L. (2020). Dinoflagellate Host Chloroplasts and Mitochondria Remain Functional During Amoebophrya Infection. Frontiers in Microbiology, 11. https://doi.org/10.3389/fmicb.2020.600823
Methods
Long, M., Marie, D., Szymczak, J., Toullec, J., Bigeard, E., Sourisseau, M., Le Gac, M., Guillou, L., & Jauzein, C. (2021). Dinophyceae can use exudates as weapons against the parasite Amoebophrya sp. (Syndiniales). ISME Communications, 1(1). https://doi.org/10.1038/s43705-021-00035-x
Methods
Station Biologique de Roscoff (n.d.). RCC1627: Scrippsiella_acuminata. Roscoff Culture Collection. https://www.roscoff-culture-collection.org/rcc-strain-details/1627
Methods
Station Biologique de Roscoff (n.d.). RCC4401: Amoebophrya_sp. Roscoff Culture Collection. https://www.roscoff-culture-collection.org/rcc-strain-details/4401
Methods
Vaulot, D., Gall, F., Le Marie, D., Guillou, L., & Partensky, F. (2004). The Roscoff Culture Collection (RCC): a collection dedicated to marine picoplankton. Nova Hedwigia, 79(1–2), 49–70. https://doi.org/10.1127/0029-5035/2004/0079-0049
Methods

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Parameters

ParameterDescriptionUnits
Sample_name

Unique name specifying treatment, incubation time, and replicate

unitless
Treatment

Infected, healthy host, or f/2 filtrate

unitless
Time

Hours of incubation post filtrate added

hours
Replicate

Treatment bottle replicate

unitless
Date

Date of sample collection

unitless
Cell_concentration_hbact

Concentration of bulk heterotrophic bacteria

Cells per ml (cells mL-1)


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Instruments

Dataset-specific Instrument Name
Guava easyCyte HT flow cytometer (Millipore)
Generic Instrument Name
Flow Cytometer
Dataset-specific Description
Guava easyCyte HT flow cytometer (Millipore) to measure cell counts of bulk heterotrophic bacteria. 
Generic Instrument Description
Flow cytometers (FC or FCM) are automated instruments that quantitate properties of single cells, one cell at a time. They can measure cell size, cell granularity, the amounts of cell components such as total DNA, newly synthesized DNA, gene expression as the amount messenger RNA for a particular gene, amounts of specific surface receptors, amounts of intracellular proteins, or transient signalling events in living cells. (from: http://www.bio.umass.edu/micro/immunology/facs542/facswhat.htm)


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Project Information

Characterizing plankton parasite-host metabolites and the response of heterotrophic bacteria (Plankton parasite-host metabolites)


Nearly half of annual primary production in the ocean is released by phytoplankton as labile metabolites, making up a large portion of dissolved organic carbon (DOC) that can fuel microbial food webs and mediate cell signaling and species interactions. Phytoplankton mortality is a major source of labile DOC, and yet, the contribution of different sources of mortality to carbon cycling remains unclear. This is especially true for parasitism. In recent genomics surveys, parasitic protists have been found to be widespread in the ocean and well-connected to potential phytoplankton hosts via network analysis. The family Amoebophryaceae (Syndiniales) are one of the ubiquitous and phylogenetically diverse groups of protist parasites, known to infect a range of hosts and drive shifts in microbial diversity and bloom phenology. Though not well quantified, these parasites are thought to have a similar impact on carbon cycling as viruses, with rapid host infection (2-3 d) and a rerouting of particulate carbon to labile DOC. In this project, we performed culture-based experiments and used untargeted metabolomics to profile labile DOC released over an infection cycle. Phytoplankton cultures included two strains of Amoebophrya sp. that infect the same dinoflagellate host (Scrippsiella acuminata), allowing us to explore new insights into strain-level infection dynamics. Separate incubation experiments were also conducted to expose parasite-derived filtrate (collected fresh from cultures after host lysis) to natural bacterial communities. Samples were collected for metabarcoding and metatranscriptomics at discrete time points to characterize changes in bacterial community assembly and gene expression in the presence of labile DOC. This work aims to provide new perspectives on plankton parasitism and its role in biogeochemical cycling, which can inform future research directions in microbial parasite-host systems and lead to more accurate food web and ecosystem models in a changing ocean environment.



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Program Information

Center for Chemical Currencies of a Microbial Planet (C-CoMP)


Coverage: North Atlantic, BATS, global/other


Functions carried out by microscopic inhabitants of the surface ocean affect every aspect of life on our planet, regardless of distance from the coast. Ocean phytoplankton are responsible for half of the photosynthesis on Earth, the first step in a complex system that annually withdraws 50 billion metric tons of carbon from the atmosphere to sustain their growth. Of this, 25 billion metric tons participate in a rapid cycle in which biologically reactive material is released into seawater and converted back into carbon dioxide by marine bacteria within hours to days. The chemical-microbe network at the heart of this fast cycle remains poorly constrained; consequently, its primary currencies and controls remain elusive; its sensitivities to changing ocean conditions are unknown; and its responses to future climate scenarios are not predictable. The Center for Chemical Currencies of a Microbial Planet (C-CoMP) integrates research, education and knowledge transfer activities to develop a mechanistic understanding of surface ocean carbon flux within the context of a changing ocean and through increased participation in ocean sciences. C-CoMP supports science teams that merge biology, chemistry, modeling, and informatics to close long-standing knowledge gaps in the identities and dynamics of organic molecules that serve as the currencies of elemental transfer between the ocean and atmosphere. C-CoMP fosters education, outreach, and knowledge transfer activities that engage students of all ages, broaden participation in the next generation of ocean scientists, and extend novel open-science approaches into complementary academic and industrial communities. The Center framework is critical to this mission, uniquely facilitating an open exchange of experimental and computational science, methodological and conceptual challenges, and collaborations that establish integrated science and education partnerships. With expanded participation in ocean science research and ocean literacy across the US society, the next generation of ocean scientists will better reflect the diverse US population.

Climate-carbon feedbacks on the marine carbon reservoir are major uncertainties for future climate projections, and the trajectory and rate of ocean changes depend directly on microbial responses to temperature increases, ocean acidification, and other perturbations driven by climate change. C-CoMP research closes an urgent knowledge gap in the mechanisms driving carbon flow between ocean and atmosphere, with global implications for predictive climate models. The Center supports interdisciplinary science teams following open and reproducible science practices to address: (1) the chemical currencies of surface ocean carbon flux; (2) the structure and regulation of the chemical-microbe network that mediates this flux; and (3) sensitivity of the network and its feedbacks on climate. C-CoMP leverages emerging tools and technologies to tackle critical challenges in these themes, in synergy with existing ocean programs and consistent with NSF’s Big Ideas. C-CoMP education and outreach activities seek to overcome barriers to ocean literacy and diversify participation in ocean research. The Center is developing (1) initiatives to expand ocean literacy in K-12 and the broader public, (2) ocean sciences undergraduate curricula and research opportunities that provide multiple entry points into research experiences, (3) post-baccalaureate programs to transition undergraduates into graduate education and careers in ocean science, and (4) interdisciplinary graduate student and postdoctoral programs that prepare the next generation of ocean scientists. The C-CoMP team includes education faculty who evaluate the impacts of education and outreach activities and export successful STEM initiatives to the education community. C-CoMP is revolutionizing the technologies for studying chemical transformations in microbial systems to build understanding of the outsized impact of microbes on elemental cycles. Open science, cross-disciplinary collaborations, community engagement, and inclusive practices foster strategic advances in critical science problems and STEM initiatives. C-CoMP science, education, and knowledge-transfer themes are efficiently addressed through a sustained network of scientists addressing critical research challenges while broadening the workforce that will tackle multi-disciplinary problems with academic, industrial and policy partners.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

The Program's Data Management Plan (DMP) is available as a PDF document.



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Funding

Funding SourceAward
NSF Division of Ocean Sciences (NSF OCE)

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