Preliminary Trophic Transfer Isotope Experiment

(PRTTIE)

Day 1

Water Collection

  • collect 80L (60L for expt plus 20L for rinsing, etc)2 psu water into four 20L acid washed, DI rinsed carboys
  • transport under low light (black bags, container) to RTC

Experimental Water Prep

  • reverse-filter all water through 75µm mesh sieve to remove copepods and reduce grazing overnight
  • store fractionated water in same 20L carboys overnight in dark environmental chamber set to ambient temp (18°C)

Zooplankton Prep

  • fractionate zooplankton into >200µm (including Pseudodiaptomus) and <200µm (?? Toni, how did you separate Limno?) (fraction containing Limnoithona)
  • dilute bugs as needed, add food (mixed phytoplankton) and put them in CT room set to ambient temp (18°C)
  • determine approximate species/stage composition on a semi-quantitative sample

Day 2

Zooplankton counts -- need 6000 Limnoithona, 500 Pseudodiaptomus in entire 60L

  • siphon off bugs from top third of bucket to get only live/healthy bugs
  • sample to determine density of bugs
  • calculate volume of water from bucket needed to get 6000 Limno and 500 Pseudo total
  • remove this volume from holding bucket and rinse concentrated bugs several times with 35µm filtered water (to reduce food (phyto) added for overnight feeding)
  • hold bugs in large (4L) volume of water while transporting them to seawall tank (where mixing is done)
  • add bugs to 20L carboys to further dilute them until added to 100L carboy

Experimental Setup

  • transfer 60L of water (containing zooplankton) into 100L acid washed Nalgene carboy
  • sample for initial nutrients, NH4 and DIC (Dugdale)
  • add stable isotopes: 13C (NaHCO3 – 51 mg) and 15N (as NH4Cl – 1.63 mg) will be added to 60L to approximate 10% of ambient DIC and NH4 concentrations (assuming 1000µM DIC and 5µM NH4 at station 2)
  • homogenize 60L isotope-spiked, zooplankton-loaded water sample with an acid cleaned paddle
  • dispense mixture into sixteen 4L acid washed cubitainers using an acid washed silicone siphon
  • place cubitainers intoa seawall incubator table under 4 layers of window screening (to reduce light to ca. 15% PAR)
  • immediately remove 3 (?)control (no zooplankton) and experimental (with zooplankton) cubitainers for To sampling in the lab (Bldg 36)

Sample water manipulation and sampling

  • gently pour water from cubitainer through 200µm mesh sieve into 4L acid washed beaker
  • rinse concentrated >200µm fraction(containing Pseudodiaptomus) and transfer to filter cup (using GF/F filtered water, 0.7µm nominal pore size)
  • filter >200µm zooplankton onto combusted (450ºC, 4-6h) GF/F filters and place in cryovials
  • gently pour <200µm-filtered water through 75µm mesh sieve into another 4L acid washed beaker
  • rinse concentrated 75-200µm fraction(containing Limnoithona) and transfer to filter cup
  • filter 75-200µm zooplankton onto combusted GF/F filters and place in cryovials
  • combusted filters containing zooplankton will be dried at 50ºC for 2 days prior to isotope analysis
  • preserve 100mL sample of 200µm- and 75µm-filtered water in 5mL acid Lugols (treat controls in same manner: put through both sieves prior to preservation)
  • give remaining water (>500mL) to Dugdale/Wilkerson/Parker lab for additional sampling
  • three replicate 4L cubitainers will be harvested at each time point (To, T24, T48, T72); control cubitainers will be sampled and returned to incubation tanks

Dugdale lab group analyses:

  • 100mL filtered through 47mm 1µm polycarbonate filter – backwashed onto combusted GF/F filter (“phytoplankton fraction”); filtrate collected in acid washed vacuum flask and filtered onto separate GF/F filter (“bacterial fraction”, 0.7 – 1.0µm). We may use 0.3µm silver filter but having problems with those filters
  • 100ml chlorophyll a
  • 25ml nutrient
  • 25ml NH4
  • 25ml DIC
  • 10ml DOC
  • 10ml bacterial abundance
  • 10ml bacterial production (total bacterial production)

Days 3-5

  • continue daily harvest of cubitainers (same time each day as To)