TITLE: Labelling kelps with 13C and 15N for isotope tracing or enrichment experiments
AUTHORS: Anton Kuech, Ursula Witte, Inka Bartsch

[DESCRIPTION]
Isotope tracing experiments can be used to trace organic material flow through the ecosystem by artificially adding labelled biomass into a system. The advantage of this process is the direct control of carbon and nitrogen addition to the system for measuring uptake rates by consumers which can substantially reduce the uncertainties associated with food web models. This protocol details the steps involved in successfully culturing and isotopically enriching (13C & 15N) juvenile sporophytes of two common North Atlantic kelp species (Laminariales): Saccharina latissima and Laminaria digitata. A first-order successful isotopic enrichment study of S. latissima, as well as the first inclusion of 15N enrichment for L. digitata, is detailed. This protocol provides a comprehensive description of the stable isotope enrichment process in two kelp species, potentially serving as a foundation for its application in other macroalgal taxa.

[BEFORE_START]
The generation of juvenile sporophyte material for isotopic labelling can be achieved through various methods, such as sampling from the field or growing sporophytes from spores or vegetative stock gametophytes. For details of our experiment see PLOS ONE article. Juvenile kelp sporophytes for labelling purposes have to be cultured in a way, which ensures good growth to produce actively growing healthy material. The following steps are tailored to the two species cultivated for this experiment and may require adaptation based on the available equipment and sporophyte physiology.

[STEPS]
SECTION: Culture set-up (pre-labelling)
1. Sterile culture bottles (glass or plexiglass) of sufficient size are used for cultivation. In our case, sporophytes of Laminaria digitata and Saccharina latissima were cultured in 5 L DURAN glass bottles with the nutrient addition of 100 mL (half concentration) Provasoli Enriched Seawater (PES) in 10 L fresh seawater stored in temperature-controlled laboratories at 10 °C. Medium was changed weekly and continuously bubbled with dry air via tubes. Irradiance should be set to around 40-50 µmol photons m-2 s-1 in long-day conditions (16 h light : 8 h dark; 16:8 LD). For cultivation of sporophytes, clean and pasteurised seawater is needed, either from natural fully marine sources or artificial seawater at approximately 30-33 PSU. Here, seawater was filtered through a 5" Polypropylene Yarn Water Filter and was pasteurised for 240 min at 99 °C with a combi steamer (PALUX, Germany).
 

 

 

 

 

SECTION: Labelling of kelps
9. Incubation of sporophytes for labelling
Selected kelp sporophytes are stored in Kautex bottles, filled with the pasteurized seawater and labelling solution to the top, tightly closed and placed on rotating or tilting shakers for 9 days. Kautex bottles lie on their sides to allow full illumination. Make sure that there are no air bubbles in the bottle. We used Orbitron rotating shakers at  . Irradiance set to 50 µmol photons m-2 s-1 in a 16 : 8 LD cycle.
 

 

 

 



SECTION: Labelling of kelps
8. Separate sporophytes of the two species by cutting them at the stipe just above the holdfast using nail scissors as detailed in step #4.   
SECTION: Labelling of kelps
7. Labelling conditions
The nutrient concentrations used in von Stosch (VS) were adjusted to represent the same concentration that the macroalgae would receive in the full PES mixture (200 mL PES in 10 L pasteurized seawater). This concentration is considered the ideal medium for growth and is also in accordance with the concentrations used by Braeckman et al. (2019) and Rossi et al. (2013). Exact concentrations of chemicals added separately were 0.01546 millimolar (mM)   ,  0.55457 millimolar (mM)   and 2.35294 millimolar (mM)   . Remaining nutrients and vitamins were added according to the von Stosch (VS) enrichment medium modified to exclude phosphate and nitrate  . 

     Chemical   compound      CAS   number      Concentration   (μmol/L) [as in 1 PES]        Sodium glycerophosphate   – hydrate      55073-41-1      15.46 μmol/L        Sodium   nitrate-15N - Na15NO3      31432-45-8      554.57   μmol/L        Sodium   bicarbonate-13C - NaH13CO3      87081-58-1      2352.94   μmol/L   

 

 

 

SECTION: Harvesting biomass
10. Remove sporophytes from a single Kautex bottle.
SECTION: Harvesting biomass
11. Take picture and wet weight of sporophytes using a macro photography camera or similar.
SECTION: Harvesting biomass
12. Separation of growing from non-growing blade parts
After the incubation period, newly formed blade parts of sporophytes normally become visible by a widening at the basis (see image below). We expected that the basal blade part that was formed during incubation would have a higher labelling result than the distal blade part. This assumption was supported by our results (see PLOS ONE article for details). Thus, if you cut sporophytes at the intersection between the estimated new growth (highly labelled) and prior biomass (slightly labelled), you separate material with a differential labelling result. 
 

 

SECTION: Determining growth rates
2. Macroalgal growth rates should be determined in simulated labelling conditions prior to labelling start to ensure the material is growing well. Otherwise, successful incorporation of labelled compounds is not guaranteed. Wet weight (mg) and surface area (cm2) are the main variables that should be measured at regular intervals (e.g. every 4 days), but blade length can also be used as a variable. In addition, pH measurements can be taken to monitor the acidity of the media.
 

SECTION: Determining growth rates
3. Labelling medium
The simulated medium for labelling consists of von Stosch (VS) medium (in contrast to PES, which is used during early cultivation – see Note below) modified to exclude phosphate and nitrate. Phosphate (0.01546 millimolar (mM)    C3H17Na2O11P), nitrate (0.54908 millimolar (mM)   NaNO3) and bicarbonate (2.32545 millimolar (mM)   NaHCO₃) were added individually in the same concentrations as in PES (table below). The full concentration is 200 mL VS for 10 L fresh seawater.

     Chemical   compound      CAS number      Concentration   (μmol/L) [as in 1 PES]        Sodium   glycerophosphate – hydrate      55073-41-1      15.46   μmol/L        Sodium   Nitrate       7631-99-4      549.08   μmol/L        Sodium   bicarbonate       144-55-8      4650.9   μmol/L   
 

 

 

SECTION: Determining growth rates
4. Sporophyte preparation for labelling
Separate sporophytes of the two species by cutting them at the stipe just above the holdfast using dissecting scissors. This enables single unidirectional growth as compared to an unequal distribution of growth between blade and holdfast. Select sporophytes of a good growth stage (e.g. widening of blade for S. latissima) and avoid individuals with white spots or other noticeable damaged areas. The blade should have an even brown colour.

 

 

SECTION: Determining growth rates
5. Example incubation of sporophytes for determining growth rates
Either two sporophytes of Saccharina latissima or four sporophytes of Laminaria digitata were placed into a single 2 L Kautex bottle (ensure sufficient replication). Wet weight (mg), surface area (cm2) and pH were measured in 4-day intervals for a 14-day period.
 

 

 

SECTION: Labelling of kelps
6. Pre-labelling start conditions
A few days prior to labelling start, full PES (200 mL PES in 10 L pasteurized seawater) should be added and irradiance should be set to 50 µmol photons m-2 s-1 to induce optimum growth rates (16 h light : 8 h dark per 24-hour period).
 

SECTION: Determining labelling uptake
19. Analysis of samples for 13C and 15N isotopes. For details of the results for this experiment see PLOS ONE article.
 

SECTION: Determining labelling uptake
18. Random sub-samples of the powder, such as 4 replicates per species and distal/basal blade part, are weighed into aluminium cups (approximately 1 mg per sample), encapsulated and added onto a 96 well plate (can also be stored in a desiccator filled with silica gel for shorter periods).
 

 

SECTION: Determining labelling uptake
17. Freeze-dried material should ideally be made into powder, best with a ball mill. Remove sporophytes from foil and transfer into the metal tubes from the ball mill. Ball mill set to 3 min at 25 Hz. Afterwards, store the powder in combusted glass vials (previously combusted at 500 °C overnight). 

 

 

 

SECTION: Harvesting biomass
16. Storage of freeze-dried biomass in zip lock bags in freezer at -20 °C (or -80 °C). Material can also be stored in a desiccator filled with silica gel for shorter periods.
SECTION: Harvesting biomass
15. Freeze-drying of samples for 48 hours.
 

SECTION: Harvesting biomass
14. Shock freezing of samples
Sporophytes wrapped in punctured aluminium foil (highly/slightly labelled separate) and either (a) dipped in   until bubbling of liquid stops or (b) stored for a minimum of 24 hours at -80 °C prior to freeze-drying.
SECTION: Harvesting biomass
13. Dip sporophytes briefly in deionized (DI) water to remove residual saltwater and dry material with clean tissue paper.