Here, we summarize the field operations for net tows, since the focus of this data package is on providing a detailed inventory of zooplankton samples collected at sea and processed on board. Field operations for net tows are documented in the associated abundance data package [knb-lter-nes.25.1]. Subsequent analyses of these samples, such as zooplankton abundance and stable isotopes, will be detailed in separate, dedicated data packages. Zooplankton abundance data package [knb-lter-nes.25.1] is available and encompasses ongoing data from 2018. An event log data package [knb-lter-nes.20] for transect cruises with raw event log data is available. The metadata file included in this inventory package [nes-lter-zooplankton-tow-metadata-v2] includes all pertinent data that was extracted from and corrected from the electronic event logs.
During dedicated Northeast U.S. Shelf Long-Term Ecological Research (NES-LTER) transect cruises, zooplankton sampling occurs at each standard transect station (L1-L11 and in the vicinity of the Martha’s Vineyard Coastal Observatory, MVCO). Zooplankton is collected with 61-cm diameter bongo nets, one with a 335-µm mesh and the other with a 150-µm mesh. Both nets are 3.2 m long and have similar porosity values— 45% and 46%, respectively. The nets are conically shaped and taper down to a detachable cod end. A General Oceanics mechanical flowmeter is attached to the center of each bongo net mouth. A Star-Oddi DST centi-TD time-depth recorder (TDR) is attached to the bongo frame to record the net’s maximum depth, trajectory during the tow, and water temperature (to be accessed post-deployment). During the tow, the depth of the net is checked by measuring wire angle using a handheld inclinometer. Zooplankton sampling tows are conducted as single oblique (descending and then ascending only once), towing at a speed through the water of 1.5 to 2.0 knots. The target net depth is 5 m off the bottom, or 200 m when bottom depth exceeds 200 m. Winch pay-out and retrieval speeds follow NOAA’s EcoMon protocol, with the exception that our maximum pay-out speed is 30 m/min (e.g. when the target sampling depths are >60 m, pay-out is at 30 m/min and retrieval is at 20 m/min). The bongo frame attaches to the end of the wire with a shackle. Attached to the bottom of the bongo frame is a ~1.5 ft section of chain and a 45 kg weight.
In addition to the bongo nets, a 30-cm-diameter ring net with a mesh size of 20 µm is attached ~2 m above the bongo frame with a book clamp. This ring net is non-quantitative and is only used for size fractionated stable isotope analysis—specifically the 20-100 µm and 100-200 µm fractions.
Deployment on the R/V Endeavor is off starboard side with one of the J-frame’s .322 wires.
During transect cruises with collaborators (e.g., Ocean Observatories Initiative in spring and fall through fall 2022), zooplankton are sampled at each regular transect station (L1-L11). Zooplankton are collected using a 30-cm ring net with a 150-µm mesh (2 m long, 46% porosity). A single vertical tow is conducted after lowering the weight to 1 m above the bottom, then positioning the net’s mouth 3 m above the seafloor, as determined by a CTD or onboard depth sounder. The R/V Armstrong uses a dynamic positioning system to maintain a stationary position during sampling. Winch payout and retrieval speeds are set to 30 m/min. These samples are non-quantitative. Fewer samples are collected on these cruises, resulting in many NAs in the metadata file [nes-lter-zooplankton-tow-metadata-v2] for Ring Net only entries.
- ¾ split into 5% buffered formalin-seawater solution for morphological identification (to Richardson lab at NOAA NEFSC)
- ¼ split in 95% ethanol for DNA metabarcoding (to Rynearson lab at URI)
- ¼ split into 95% ethanol for morphological ID (to Llopiz lab at WHOI)
- ¼ split into 95% ethanol for DNA metabarcoding (to Rynearson lab at URI)
- ¼ split to be size fractionated on board (>1000 µm, 500-1000 µm, and 200-500 µm) for stable isotope analyses. Size fractions are frozen (-20°C) in vials.
- ¼ split for fresh sorting on board and freezing of individual taxa (or the split will be frozen for later sorting of taxa in the lab) for stable isotope analyses—4 dominant taxa of copepods (e.g. Calanus, Centropages, Pseudocalanus, Oithona), as well as appendicularians and chaetognaths.
Upon retrieval, the nets are rinsed on the outside with a seawater hose to ensure that all plankton are collected in the cod ends. The cod ends are then carefully removed only after the water level is below the cod end opening, while positioned within a labeled and appropriate bucket. Labeled cod ends are then carried in their corresponding labeled buckets (distinguishing between the 335-µm and 150-µm mesh nets) into the wet lab for processing. In the wet laboratory, samples are split using a Folsom plankton splitter, and aliquots are processed according to established sample allocation. Aliquots are preserved in a 5% buffered formalin-seawater solution, 95% ethanol, or frozen, depending on the purpose of the sample.
- Record flowmeter readings for each net.
- Rinse the nets thoroughly from the outside, starting at the top and working down to the cod end.
- If there are a few large organisms in the sample (e.g. jellies), remove them prior to splitting and preserve them separately in ethanol. Use the large (~5 mm) sieve if needed. Note the removal and preservation of organisms in the Bongo/Ring Event Log, specifying the net from which they were extracted, and lab their jar accordingly.
- Rinse all sieves and any equipment with sample residue between each sample.
- Rinse all gear with freshwater upon completion of sampling for the cruise.
- Split the sample with a Folsom plankton splitter.
- Put 3/4 of the 335 µm sample into a glass quart jar with formalin to a 12% formalin/seawater mix, buffer with 1:1 disodium phosphate:monosodium phosphate.
- Drain and gather the remaining ¼ split onto a ≤335-µm-mesh sieve and preserve in 95% ethanol using a glass pint jar. Jar should be at least 2/3’s full of ethanol and 1/3 or less of plankton.
- Ensure each sample is appropriately labeled both internally and externally.
- Store samples separately.
- Split the sample with a Folsom plankton splitter.
- Preserve two ¼ splits in 95% ethanol by draining and collecting each on a 150 µm mesh sieve and transferring to a plastic pint jar using a squirt bottle of ethanol. One sample is designated for the Llopiz lab, and the other for the Rynearson lab.
- One of the remaining ¼ splits gets size fractionated on 1000 µm, 500 µm, and 200 µm sieves, and what is collected on each sieve gets drained and transferred to a cryovial and then frozen at -20°C.
- The other ¼ split will be drained and frozen at maximum -20°C.
- Ensure each sample is appropriately labeled both internally and externally.
- Place ethanol-preserved splits in their appropriate boxes (Llopiz and Rynearson labs)
- This sample is non-quantitative. Target a well-mixed sub-sample.
- Size fractions 100-200 µm and 20-100-µm are retained, drained and transferred to a cryovial and then frozen at -20°C.
Zooplankton samples for stable isotope analysis originate from the 150 µm mesh bongo net and 20 µm ring net (no size-fractionated zooplankton collected from 335 µm). First, samples are split using a Folsom splitter and then separated by size using sieves of varying mesh sizes (metal sieves for 1000 µm, 500 µm, and 200 µm; and PVC fitted with mesh for the 100 µm and 20 µm sieves). During this process, samples are rinsed with filtered seawater only (not ethanol) to avoid contamination. A quarter (¼) of the sample collected with the 150 µm mesh net is apportioned for the >1000 µm, 500-1000 µm, and 200-500 µm size-fractionated samples. The entire sample collected using the 20 µm ring net is apportioned/allocated to the 20-100 µm and 100-200 µm size fractions. Size fractions are drained, material transferred to cryovials and frozen in -20°C freezer.
Size fractions:
| Net | Sieve | Fraction |
|---|---|---|
| 150-µm bongo | 1000 µm | > 1000 µm |
| 150-µm bongo | 500 µm | 500-1000 µm |
| 150-µm bongo | 200 µm | 200-500 µm |
| 20-µm ring | 100 µm | 100-200 µm |
| 20-µm ring | 20 µm | 20-100 µm |
- Drain and replace the ethanol of ethanol-preserved samples. Note the date changed on the external jar labels and on the Bongo Event Log sheet.
- Test the pH of formalin samples and add additional buffer if the pH is <7.
- Ensure that samples are promptly delivered to their respective labs.
- Advise whether any samples require additional ethanol changes.
Sieve the sample using only seawater and transfer it directly into the jar for preservation. The entire sample will go into a single jar with 95% ethanol. Ethanol will be changed 24hrs after initial preservation.
- Drain the contents into a 150-µm sieve and use pre-filtered seawater to rinse the cod end. Make sure the mesh on the cod end is rinsed well.
- If there are a few large organisms in the sample (e.g. gelatinous or fish), remove them and preserve them separately in ethanol. Use the large (~5 mm) sieve if needed.
- Transfer the entire sample to a plastic 16 oz or 4 oz jar, depending on sample density.
- Ensure each sample is appropriately labeled both internally and externally.
- Rinse all sieves and anything with sample residue with freshwater between each station.
- Rinse all gear with freshwater upon completion of sampling for the cruise.
- Drain and replace the ethanol of ethanol-preserved samples. Note the date changed on the external jar labels and on the Ring Net Event Log sheet.
Data collected with the Star-Oddi DST centi-TD time-depth recorder (TDR) miniature temperature and pressure (depth) data logger is included in this package. The TDR is set up to collect data every second during the bongo nets descent and ascent. Sometimes the TDR had a depth offset, these values were corrected prior to data publishing, so the TDR depth reported here is correct. In some instances, the sampling depth (net maximum depth) was not recorded by the time-depth recorder (TDR) or the CTD attached to the bongo wire. When this occurred during oblique bongo tows, we estimated the maximum tow depth (in meters, necessary for calculating depth integrated meters squared abundance values) using the following equation based on the straight cosine law: Z = L * cos(α)
Where:
- Z = estimated maximum tow depth (in meters),
- L = length of wire paid out (in meters),
- Α = wire angle (in radians)
For vertical tows, where no wire angle measurement is available, the net depth was assumed to be equal to the maximum wire paid out. When TDR, wire out, and wire angle information were unavailable, the target net depth was used as the net sampling depth.
A secondary flag with comments provides information about when this estimation was used. The primary flag for these cases, where TDR/CTD depths are unavailable, is set to 3 (more on data flags below).
The sampling depth is found in the net_max_depth column of the metadata file.
The volume of water filtered was calculated using the following equation: V = (R)(Ff)(A) Where:
- V = calculated volume of water filtered (in meters cubed)
- R = number of flowmeter revolutions during tow
- Ff = factory calibration factor (in meters per revolution) = 0.26873
- A = area of net mouth (in meters squared) = 0.2922
When flowmeters failed, the following equation was used to calculate volume of water filtered:
V = (A)(T)(S)
Where:
- V = calculated volume of water filtered (in meters cubed)
- A = area of net mouth (in meters squared) = 0.2922
- T = duration of tow (in seconds)
- S = ship speed during tow (in meters per second)
These values are recorded in the vol_filtered_m3_335 and vol_filtered_m3_150 columns of the metadata file. The comments indicate whether ship speed and tow duration were used instead of flowmeter readings to calculate the net volume sampled.
Standard haul factors are provided to make multiple tows comparable. These were calculated using:
For 10 meters squared:
H = (Z * 10) / volume of water sampled (meters cubed)
Where:
- H = area (10 meters squared)
- Z = Maximum tow depth (in meters) [net_max_depth_m column]
For 100 meters cubed:
H = 100 / volume of water sampled (meters cubed)
Where:
- H = volume (100 meters cubed)
Post-cruise sample allocation and storage:
- Morphological ID (MorphID): Ethanol
- Stable isotope analysis on size fractions (SIA): Frozen
- Taxa-picking: Frozen
- Samples are examined for proper preservation and ethanol is changed if needed. Date of change is recorded on the jar.
- A layer of electrical tape is wrapped around the opening of the jar to prevent evaporation.
- Jars are placed in cardboard boxes organized roughly by cruise and date.
- Boxes are labeled with the cruise IDs and MVCO dates if applicable.
- Boxes of samples are stored in Llopiz lab: Redfield 226 with ethanol safety tag on the outside of the box.
- Samples are transferred to BioSpecs in the Llopiz storage area.
- In addition to the writing on the box for short term storage, sticker labels available from Phil Alatalo at WHOI are affixed to the boxes and the accompanying data sheet online is filled out.
- Metabarcoding (DNA) 150 µm mesh net: Ethanol
- Metabarcoding (DNA) 335 µm mesh net: Ethanol
- Samples are checked a) for proper, legible labeling and b) to ensure ethanol was changed after the initial fixation. If needed, labels are improved, and ethanol is changed where not yet done.
- Samples are stored in labeled cardboard boxes in drawers in the Rynearson lab.
- Morphological ID (MorphID): Formalin
- In plankton lab on NEFSC Narragansett campus in Narragansett, RI.
- Samples are shipped to Poland a maximum of 3 times a year. These samples are processed following MARMAP NEFSC Sorting Protocols 5.3.1 ECOSYSTEM MONITORING - ZOOPLANKTON.
Ichthyoplankton:
- Vial of cephalopod paralarvae (not ID’ed to species)
- Vial of fish eggs (not ID’ed to species)
- Vial (1) of larval fish for each taxon
Zooplankton:
- Quarter aliquot of the sample
- Vial of individuals for each taxon from the 400+ aliquot of individuals. In some cases taxa are combined in the vials.
These samples are archived at the Narragansett Lab with samples dating back to the 1970s.
Occasionally, the bongo net hits the sea floor during a tow, resulting in missed samples when there is no time to redeploy. Whenever possible, nets are rinsed and redeployed. In some cases, the sand and/or mud is sieved, and samples preserved. These occurrences are documented in the comment and data flags sections of the inventory data package.
Bongo and ring net samples were not collected at the following cruises (stations), primarily due to time constraints, bad weather, or instances where the bongo hit the bottom without time for a redo: EN608 (L8), EN644 (L8), EN655 (L8, L11, MVCO), AR32 (L2, L4, L8, L9, MVCO), AR63 (L1, L3, L6, L8, L9, L10, L11, MVCO), AR38 (L1, L5, L8, MVCO), EN661 (L6, L7, L8, L9), AT46 (L10), and EN712 (L6). EN627 u11c was an opportunistic sample.
The following cruises only used a 30-cm ring net (no bongo net) with a 150 µm mesh mainly for MorphID samples (for Llopiz lab): AR31A, AR39B, AR34B, AR31A, AR28B, AR61B, AR66B. These samples were all non-quantitative vertical tows (no oblique). In the inventory data, these cruises have “NA” for most of the sample inventory collection since they were not part of the protocol yet. These were cruises with OOI collaborators. Upcoming cruises will continue using the bongo net and its associated protocol for oblique tows to obtain quantitative zooplankton data (i.e., abundances). For cruises HRS2303 and AR77, only ship speed over ground is available, with no data on speed through water. Ship speed for cruise EN720 was not yet available at the time of package assembly, so an estimated value of 2 knots was used.
Data quality flags were assigned following the Ocean Best Practices UNESCO 2013 IOC 54:V3 IODE Quality Flag Standard [Available here: https://www.iode.org/index.php?option=com_oe&task=viewDocumentRecord&docID=10762]:
| Flag | Description |
|---|---|
| 1 | Good (passed documented required QC tests) |
| 2 | Not evaluated, not available, or unknown (no QC test performed or information on quality is not available) |
| 3 | Questionable/suspect or of high interest |
| 4 | Bad (failed critical documented QC test(s) or as assigned by the data provider) |
Advanced Flagging Scheme:
In addition to the primary flags, the Ocean Best Practices UNESCO 2013 IOC 54:V3 standard also describes an advanced flagging scheme with more detailed commentary, helping to clarify and provide additional context. In this data set, this scheme includes comments such as:
- Depth recorder (TDR) data not available. Net max depth was calculated based on wire information (cosine law).
- Target depth used for net max depth due to unavailable TDR data and wire information.
- Target depth used for net max depth due to unavailable wire data for Ring Net.
To ensure the accuracy of the entries for physical samples from zooplankton net tows, data was validated using R. This involved checking for ranges of values and unique strings in each column. All coordinates were plotted on a map to ensure that the values were within reasonable ranges. Errors in manually written latitude and longitude were fixed based on the ship’s event log entries. Entries were cross-referenced with the scanned Bongo/Ring Event Log sheets and the event log for each cruise. Missing values were identified, and NAs assigned. Documentation is available at https://github.com/cabanelas/nes\_lter\_zooplankton\_inventory\_v2.
Data package assembly with metadata templates was completed in R, with documentation available at https://github.com/WHOIGit/nes-lter-zooplankton-transect-inventory.
This is the second version of the NES-LTER zooplankton inventory data package [knb-lter-nes24.2]. The previous data package [knb-lter-nes24.1] included sample inventory data for 16 cruises; version two adds 11 for a total of 27 cruises. New in this version is the tow metadata table. Methods were edited and updated. Some column names and descriptions were updated.
Cruises in v1: EN608, EN617, EN627, EN644, EN649, EN655, AR28B, AR31A, AR32, AR34B, AR38, AR39B, AR61B, AR63, EN657, and EN661. New cruises in v2: EN668, AT46, EN687, EN695, HRS2303, EN706, AR77, EN712, AR66B, EN715, and EN720 .
Other related packages include event log data package [knb-lter-nes.20.1] and zooplankton abundance data package [knb-lter-nes.25.1] for transect cruises.