Nov 14, 2024
Version 2
The Shell Seeker: Oyster Larvae Sampling with the PlanktoScope V.2
This protocol is a draft, published without a DOI.
- 1Louisiana Universities Marine Consortium
Protocol Citation: Marco Milton, Kendall Cosper, Kevin Du Clos 2024. The Shell Seeker: Oyster Larvae Sampling with the PlanktoScope. Protocol exchange https://protocols.io/view/the-shell-seeker-oyster-larvae-sampling-with-the-p-drq355ynVersion created by Kevin Du Clos
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 07, 2024
Last Modified: November 14, 2024
Protocol Integer ID: 112123
Keywords: PlanktoScope, oyster, larvae, imaging, EcoTaxa
Funders Acknowledgement:
Louisiana Sea Grant UROP Program
Grant ID: R/OA-27
Abstract
This is protocol for sampling oyster larvae using a battery-powered pump and the PlanktoScope flow through imaging system. Oyster reefs can only grow when larval recruitment exceeds adult mortality, so quantifying larval abundances is crucial for protecting and restoring of reefs. Oyster larvae are difficult to study in the field, however, due to their low and patchy abundances, so field measurements are rare. We developed this protocol to facilitate rapid and repeatable field measurements of oyster larval abundance.
Image Attribution
Kevin Du Clos
Guidelines
The EcoTaxa project linked below provides identified images of bivalve larvae some other plankton that could be misidentified as bivalve larvae:
Materials
Field Equipment
- Small bucket
- Battery powered pump (Ryobi ONE+ HP 18V 1/4 hp Cordless Battery Powered Transfer Pump or equivalent)
- Carboy (20 liters)
- Squeeze bottle
- Thermometer
Lab Equipment
- 35 µm nylon mesh filter
- Graduated cylinder (100 ml)
- Refractometer
- Glass flasks and beakers
PlanktoScope
Available from https://www.fairscope.com/
Microscope
- 1-ml micropipettor
- Sedgewick rafter counting slide with cover slips
Before start
If you plan to process your data with EcoTaxa, you will need to sign up for an account and wait for verification before creating your project (https://ecotaxa.obs-vlfr.fr/).
Field Collection with Battery-Powered Pump
Field Collection with Battery-Powered Pump
Metadata collection
Collect water from the sample site using a small bucket. Record the collection time (in UTC) and temperature of the water collected in the bucket. Collect another water sample in a squeeze bottle for salinity measurements and concentration in the lab.
Sample collection
Collect 20 liters of seawater in a carboy with a battery-powered pump on low speed (see Figure 1). We collect water at approximately 1 meter below the surface.
Figure 1: Field Materials: (1) 20-liter carboy (2) battery powered pump (3) small bucket (4) squeeze bottle (5) thermometer.
Note
To maintain a consistent sampling depth, mark the inlet hose with tape at a measured distance (1 m) from the inlet and keep the tape close to the water surface while sampling.
Lab Processing
Lab Processing
Salinity measurement
Use a refractometer to measure the salinity of the water in the squeeze bottle (4) collected in step 2.
Concentrate the sample
Run the water collected in the carboy (1) through a 35 µm nylon mesh filter, pouring off the concentrate and rinsing the filter with the squeeze bottle (4) into the beaker (3) as needed.
Re-filter the collected sample to reach a volume less than 100 ml if possible. If the filtered volume was less than 100 ml, use the squeeze bottle (2) to bring the volume up to 100 ml in the graduated cylinder (4). If the volume was more than 100 ml, use a larger graduated cylinder to measure the final volume.
Note
The final concentrated volume will be entered as metadata when processing with the PlanktoScope and will be used to calculate abundances.
Transfer the sample
To improve PlanktoScope processing, let the water in the graduated cylinder (4) sit for 2 minutes to allow some of the sediment to settle out. After this settling period, transfer the sample to a flask (5) (leaving behind the sediment).
Note
Tip: Removing sediment helps to improve PlanktoScope processing. Keep the settling time consistent to avoid introducing processing error.
Figure 2: Sample concentration materials: (1) carboy from step 2 (2) squeeze bottle from step 1 (3) beaker (4) 100-ml graduated cylinder (5) 200-ml flask (6) beaker (7) 35 µm nylon mesh.
Quantify the sample
Quantify the sample using the PlanktoScope (10 1-ml acquisitions) and a Sedgewick rafter (3 1-ml samples).
See below for microscope and PlanktoScope details.
Note
Tip: The PlanktoScope protocol includes a pre-filter step with a 200 µ m mesh.
We skip this step to avoid losing larvae.
Microscope Counts
Microscope Counts
Transfer sample
Transfer 1 ml of sample to a Sedgewick rafter counting slide with a micropipette and apply the cover slip.
Perform Count
Count larvae across the entire slide in a zig-zag pattern using a compound microscope with a 10x objective.
Figure 3: Oyster larvae though the microscope
Note
Tip: Oyster larvae can be tricky to identify. Shape is important when identifying the larvae! Look for a letter D-shape or a football shape. See the Guidelines section for a link to a guide with identification advice.
Repeat
Obtain at least 3 replicate counts by repeating steps 7 and 8.
PlanktoScope Processing
PlanktoScope Processing
Set up your PlanktoScope
Assemble and turn on your PlanktoScope (refer to the PlanktoScope protocol linked at the end of this section for more detailed instructions).
Connect to the PlanktoScope
Connect to the PlanktoScope's Wi-Fi network with your computer and use an internet browser to access the dashboard (http://192.168.4.1:1880/ui/).
Setting the Date and Time
Set the PlanktoScope's internal date and time by navigating to the System Monitoring page and pressing the button labeled "CHANGE INSTRUMENT'S TIME TO MATCH WEB BROWSER'S TIME".
Figure 4: System Monitoring Metrics display
Note
This feature first became available with PlanktoScope software release v2024.0.0-beta.0.
Enter Metadata
On the Sample page fill out the metadata as shown below. Use "Single location (with net or bucket)" for the Sampling gear. Use metadata specific to the collected sample.
Figure 5: Metadata setup display
Load sample
Load at least 10 ml of your sample from step 5.
Optic configuration
Turn the LED light on, pump a small volume through and flow cell, and check the focus.
Fluidic Acquisition
Set an acquisition ID, starting at 1 and increasing by 1 for each additional acquisition.
Set the number of images per acquisition (100 is recommended).
Set volume to pump and the flow rate. We use the default settings.
Note
We recommend 10 acquisitions of 100 images to obtain sufficient larvae images for quantification at low larval abundances.
Segmentation
Select the "Yes" option for EcoTaxa.
Check the boxes for each acquisition and start segmentation.
Export Images
On the Gallery page go to Export > EcoTaxa folder and select the files to export. Export the selected files using the download button (files can also be downloaded over FTP).
Note
For more details refer to the PlanktoScope protocol: https://www.protocols.io/view/planktoscope-protocol-for-plankton-imaging-bp2l6bq3zgqe/v3.
EcoTaxa
EcoTaxa
Create a Project
Create a new project at https://ecotaxa.obs-vlfr.fr/. A separate project for each sampling date is recommended. Fill out the relevant metadata to begin.
Figure 6: Display of EcoTaxa project setup
Import Images
Import all images collected by the PlanktoScope.
Train and Predict
Train and predict the identification software by selecting "Train and Predict classifications" in the "Project" drop down menu in the upper left corner. This will obtain an initial larvae count and begin the identification process.
Note
The EcoTaxa project linked below provides identified images of bivalve larvae some other plankton that could be misidentified as bivalve larvae:
Identify larvae manually
Review and classify images. Images can be classified by dragging them to the appropriate class on the left side of the screen or typing the start of the name in the box at the top of the taxonomy list and selecting the correct classification from the auto-completed list.
Figure 7: Larva characteristics
Note
Tip: Refer to the Guidelines section for more information on identification.
Repeat the previous two steps until all larvae are identified
Export summary data
Export data for analysis by selecting Export from the Project menu and choosing the Summary Export option. Selecting Acquisition for the Computations aggregation level option will facilitate the abundance calculations described below (Step 26).
Figure 8: Summary export display
Note
Tip: Refer to the official PlanktoScope protocol for a more detailed description of processing with EcoTaxa:
Calculations
Calculations
Calculating abundances (microscope counts)
Use the equation below to calculate field abundances from microscope counts:
The volumes used in the protocol are included in parentheses. These terms simplify to 5, so the field abundance in larvae per liter can be calculated by multiplying the mean of the microscope counts by 5.
Calculating abundances (PlanktoScope/EcoTaxa)
Use the equation below to calculate field abundances from EcoTaxa projects:
Larvae images is the number of validated bivalve larvae images in a project. Acquisitions is the number of acquisitions imported into the EcoTaxa project. This can be determined by exporting the data from EcoTaxa using the Summary export option, selecting By acquisition for the Computations aggregation level, then counting the number of acquisitions in the exported CSV file.
The imaged volume/acquisition is the volume of each PlanktoScope acquistion; the inverse of this number will be used in the calculation. This number is based on the PlanktoScope calibration. It can be found in the Acquisition details section in EcoTaxa (imaged_volume).
Protocol references
Cowen, R. K. and Guigand, C. M. 2008. In situ ichthyoplankton imaging system (ISIIS): system
design and preliminary results. Limnology and Oceanography: Methods. 6:126--132.
Chanley, P. 1971. Aids for identification of bivalve larvae of Virginia. Malacologia. 11:45--119.
Garland, E. D. and Zimmer, C. A. 2002. Techniques for the identification of bivalve larvae.
Marine Ecology Progress Series. 225:299--310.
Goodwin, J. D., North, E. W., and Kennedy, V. S. 2016. Identification of eastern oyster
Crassostrea virginica larvae using polarized light microscopy in a mesohaline region of
Chesapeake Bay. Journal of Shellfish Research. 35:157--168
Hendriks IE, Van Duren LA, Herman PM. 2005. Image analysis techniques: A tool for the identification of bivalve larvae?. Journal of Sea Research. 54(2):151-62.
Kim, C.-K., Park, K., Powers, S. P., Graham, W. M., and Bayha, K. M. 2010. Oyster larval transport in coastal Alabama: Dominance of physical transport over biological behavior in a shallow estuary. Journal of Geophysical Research: Oceans. 115.
Losee E. 1979. Relationship between larval and spat growth rates in the oyster (Crassostrea virginica). Aquaculture. 16(2):123-6.
Poirier LA, Clements JC, Davidson JD, Miron G, Davidson J, Comeau LA. 2019. Sink before you settle: Settlement behaviour of Eastern oyster (Crassostrea virginica) larvae on artificial spat collectors and natural substrate. Aquaculture reports. 13:100181.
Zu Ermgassen, P. S., Spalding, M. D., Grizzle, R. E., and Brumbaugh, R. D. 2013. Quantifying
the loss of a marine ecosystem service: filtration by the eastern oyster in US estuaries. Estuaries
and Coasts. 36:36--43.