RESEARCH LETTER

 

Metabarcoding of zooplankton to derive indicators of pelagic ecosystem status

 

 

Jenny A. Huggett^{I, II}; Johan C. Groeneveld^{III, IV}; Sohana R Singh^III; Sandi Willows-Munro^IV; Ashrenee Govender^IV; Riaan Cedras^V; Shaun H.R Deyzel^{VI, VII}

^IOceans and Coastal Research, Department of Forestry, Fisheries and the Environment (DFFE), Cape Town, South Africa
^IIDepartment of Biological Sciences, University of Cape Town, Cape Town, South Africa
^IIIOceanographic Research Institute, Durban, South Africa ⁴School of Life Sciences, University of KwaZulu-Natal, Rietermaritzburg, South Africa
^VDepartment of Biodiversity and Conservation Biology, University of the Western Cape, Cape Town, South Africa
^VISouth African Environmental Observation Network (SAEON), Elwandle Coastal Node, Gqeberha, South Africa
^VIIInstitute for Coastal and Marine Research, Nelson Mandela University, Gqeberha, South Africa

Correspondence

 

 

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ABSTRACT

Zooplankton play a key role in marine food webs and carbon cycling and are useful indicators of climate-related changes and ocean health in pelagic ecosystems. Zooplankton are traditionally identified to species through microscopy, but new molecular techniques have enabled the identification of individual specimens (DNA barcoding) or multiple species in the same sample (DNA metabarcoding). Metabarcoding has been tested and refined using zooplankton collected in South African waters for the first time. Challenges to the implementation of DNA-based methods to measure zooplankton biodiversity easily and routinely include an incomplete DNA barcode reference library, logistical complexity and uptake of the new technology by environmental management agencies. These challenges call for a national effort to intensify zooplankton barcoding initiatives and to effectively engage stakeholders in developing a roadmap towards application of DNA-based methods in marine environmental management.
SIGNIFICANCE: • Metabarcoding has been successfully applied to marine zooplankton for the first time in South Africa, demonstrating its potential as a tool to generate ecosystem indicators during routine ocean observations
• National barcoding efforts must be intensified to provide a comprehensive reference library of zooplankton DNA.
• Effective engagement with stakeholders is required to overcome logistical and policy challenges, and to provide a roadmap towards application of DNA-based methods in marine environmental management.

Keywords: barcoding reference databases, climate change, essential ocean variables, ocean observations, zooplankton biodiversity

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The need for ocean indicators

Recent warming, acidification and deoxygenation associated with climate change have greatly affected physical and chemical conditions in oceans.¹ Altered marine habitats have led to shifts in the distribution and phenology of organisms, with major implications for biological productivity of marine ecosystems.¹ Yet, human reliance on the goods and services provided by oceans continues to grow.² Against a backdrop of climate change and increasing exploitation of marine resources, observations of key ocean indicators are critical to inform policy and support ocean governance and management.^2,3

The importance of ocean observation systems is recognised by the United Nations Decade of Ocean Science for Sustainable Development (2021-2030), with one of the ten Ocean Decade Challenges being to expand the ocean observing system globally.⁴ The Global Ocean Observing System (GOOS) programme has identified a suite of priority physical, biogeochemical and biological ecosystem variables, known as essential ocean variables (EOVs; Supplementary table 1), for routine and sustained observation to assess ocean changes globally, in support of ocean governance.³ A complementary set of essential biodiversity variables (EBVs)⁵, to monitor and reduce biodiversity loss, has been defined by the Group on Earth Observations - Biodiversity Observation Network (Supplementary table 1). Zooplankton biomass and diversity were included as biological EOVs that represent the base of marine food webs.² Here, we report on recent molecular methodology (DNA barcoding and metabarcoding) that allows for the rapid and accurate measurement of marine zooplankton biodiversity and relative abundance, and its potential application as a long-term indicator of pelagic ecosystem status in South African coastal and neritic waters. We comment on both methodological and logistical considerations.

 

Assessing zooplankton biodiversity

Zooplankton play a vital role in the functioning of marine ecosystems. As grazers in pelagic food webs, they provide the main energy pathway from primary producers to higher trophic levels such as fish, squid, and marine mammals. Their excretions fuel the microbial food web and contribute significantly to carbon sequestration via the biological pump.^6,7 Zooplankton are physiologically sensitive to temperature and have short life spans, thus thermal changes are rapidly reflected in their population dynamics.⁸ Because they are not fished commercially, changes in zooplankton communities reflect actual environmental or ecosystem-mediated changes, largely unaffected by exploitation trends.^8,9 Zooplankton are therefore excellent 'sentinels' or indicators of change in pelagic ecosystems, with applications extending to climate, fisheries, invasive species, ecosystem health, definition of pelagic ecoregions, biodiversity and ecosystem assessments.^8,9

Ongoing advancement of DNA barcoding reference data sets (e.g. BOLD, GenBank; Supplementary table 1 ) coupled with recent metabarcoding technologies now makes rapid and accurate processing of taxonomically complex zooplankton samples logistically feasible. This new technology prompted a shift from traditional microscopy methods to genes as a measure of marine diversity.¹⁰ DNA barcodes can distinguish between visually similar or cryptic species, are independent of life stage, and reduce researcher bias through standardisation of reference systems.¹⁰ DNA barcoding has been applied successfully to global biodiversity studies, including the Census of Marine Zooplankton (CMarZ).¹¹

South Africa formally committed to the International Barcode of Life Project (iBOL) in 2011. Barcode reference databases with records of marine zooplankton from South African waters were reviewed recently.¹² Records were proportionally below global levels in nearly all taxa examined and were dominated by species from easily accessible habitats and those with commercially important life phases (i.e. drifting larvae of fish and benthic invertebrates) (Figure 1). Holoplanktonic species were grossly under-represented, despite making up the bulk of zooplankton biomass and with high importance as ecological indicators. The paucity of holoplanktonic barcodes stemmed from too few specialist taxonomists, the so-called 'taxonomie impediment'.¹³

Metabarcoding uses the same reference databases as barcoding but allows for identification of multiple taxa simultaneously from mixed samples by using high-throughput sequencing platforms.¹⁴ Potential applications of metabarcoding are broad-ranging, including revolutionising biodiversity assessments in any environment for which DNA barcode reference databases are available¹⁵, establishing time-series of diversity¹⁶ and developing biotic indices for routine biomonitoring¹⁷. The progression from barcoding of individual specimens to metabarcoding of entire

communities is now well underway in South Africa, with studies published on diatom communities in the St Lucia estuary¹⁸, bacterial communities in waterholes in the Kruger Park¹⁹ and biomonitoring of freshwater macro-invertebrates²⁰, among others.

 

Towards routine biodiversity monitoring

The adoption of molecular approaches such as metabarcoding in marine environmental management requires an iterative 'translational molecular ecology' approach (constant two-way communication between scientists and stakeholders).¹⁷ Recent applications that demonstrate this process internationally include routine monitoring of ichthyoplankton, biosecurity monitoring for non-indigenous species, and ecological status assessments.¹⁷ As part of the translational molecular ecology approach, the development of and adherence to standardised protocols to guarantee data comparison across spatial and temporal scales are crucial.¹⁷

Metabarcoding of marine zooplankton in South Africa has been optimised to follow best-practice protocols set by the international Scientific Committee on Oceanic Research working group MetaZooGene (Supplementary table 1). Optimisation challenges were the design of taxon-specific mini-barcode primers to increase species detection rates²¹, experimental validation of primer cocktails to test their efficiency in detecting rare species²², and comparison of species identities obtained from metabarcoding and microscopy¹². The strategic importance of an expanded DNA barcode reference database for the region is recognised by the Foundational Biodiversity Information Programme of the South African National Research Foundation. This Programme currently funds integrative molecular morphology projects to increase regional reference databases (including for zooplankton), with new records uploaded to open-access databases such as BOLD and GenBank.

Further incorporation of zooplankton biodiversity in ocean observation programmes as biological EOVs faces several logistical challenges in South Africa. Key among these are: access to sea-going vessels and sampling gear, laboratories for sample processing, technical and scientific expertise across various disciplines (genetics, taxonomy, bioinformatics, biodiversity, oceanography) and data management from the point of observation to implementation by users. To evaluate the underlying science, operational costs and benefits, a small-scale pilot project was initiated in 2018, along a single cross-shelf transect at the KwaZulu-Natal Bight Sentinel Site (Supplementary table 1 ; Supplementary figure 1). The pilot project is a collaboration between several academic institutions, which are in turn funded by the Department of Science and Innovation and the National Research Foundation.^12,21,22 Key findings were that costs, coordination and uptake will be the main challenges for a prospective long-term programme.

In addition to a successful 'proof of concept' exploratory project and an improved barcode reference database, progress towards the routine use of metabarcoding in monitoring of the marine environment in South Africa will require a 'translational molecular ecology' process to facilitate its uptake at environmental management and policy levels.¹⁷ The role of national environmental observation agencies, primarily the South African Environmental Observation Network (SAEON) and the Department of Forestry, Fisheries and the Environment (DFFE), in providing operational budgets and coordination, and as long-term custodians of data and indices, will be critical for actionable progress beyond exploratory research. Our Research Letter describes the initial development of a DNA-based method for biodiversity assessments of South African pelagic ecoregions, including enabling conditions for its uptake in marine environmental management.

 

Acknowledgements

J.A.H. acknowledges support from the Scientific Committee on Oceanic Research (SCOR) WG157 MetaZooGene. The South African Institute of Aquatic Biodiversity (SAIAB) is thanked for the use of their research vessel (RV) Phakisa and sampling equipment, and the assistance of the vessel's crew. The KwaZulu-Natal Research and Innovation Sequencing Platform (KRISP) is thanked for undertaking Illumina sequencing of mixed zooplankton samples. The National Research Foundation (NRF) is thanked for doctoral and post-doctoral financial support for A.G. and S.PS. The provision of research platforms (Coastal Crafts, Sentinel Site Instrumentation and Data Management) by the Shallow Marine and Coastal Research Infrastructure (SmCRI) of the Department of Science and Innovation (DSI) hosted by NRF-SAEON Elwandle Node and NRF-SAIAB is acknowledged.

 

Competing interests

We have no competing interests to declare.

 

Authors' contributions

J.A.H.: Conceptualisation, methodology, writing - the initial draft, writing - revisions, project leadership, project management.

J.C.G.: Conceptualisation, methodology, writing - the initial draft, writing - revisions, project leadership, project management. S.P.S.: Methodology, data collection, data analysis, writing - the initial draft. S.W.-M.: Methodology, data analysis, writing - the initial draft, student supervision. A.G.: Methodology, data collection, data analysis, writing -the initial draft. R.C.: Methodology, data collection, data analysis, writing - the initial draft. S.H.PD.: Methodology, data collection, data analysis, writing - the initial draft.

 

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Correspondence:
Jenny Huggett
Email: jenny.huggett@gmail.com

Received: 16 Dec. 2021
Revised: 08 July 2022
Accepted: 30 Aug. 2022
Published: 30 Nov. 2022

 

 

Editor: Sydney Moyo
Funding: South African National Research Foundation