Halosphaera
Sampling Protocol
Background
Marine phytoplankton produce almost half of our planet’s oxygen through photosynthesis, and most of this productivity occurs in estuarine and upwelling regions (Field et al., 1998). Recent literature suggests that a changing climate has a strong correlation with changes in phytoplankton phenology (Kahru & Elmgren, 2014; Winder & Schindler, 2004), and cataloging their blooms remains an important metric for showing the effects of such a big problem.
The Salish Sea is a region in the North Pacific fed by several major rivers, and several smaller waterways. This region supports a wide range of marine organisms – some of which are the phytoplankton, Halosphaera. Halosphaera are unicellular green marine algae in the group Prasinophyceae that lack detailed description of their life cycle and ecological function. Historically, they have not been well studied and most literature about them was published from the 60’s - 80’s. This indicates that a newer more robust study could give us insight into a genus we know little about.
Halosphaera are uniquely found during the winter months in the Salish Sea (pers. obs. Dube & Kodner), whereas in warmer open waters they have only been reported during the summer months (Jenkinson, 1986; Wiebe et al., 1974). Halosphaera have two major life stages, a motile flagellated stage and a nonmotile lipid-like phycomated stage. During these phases they undergo significant size transformation, from approximately 10 µm for the flagellated stage to about 400 µm for the phycomated stage (Parke & Hartog-Adams, 1965). The phycomates are like balloons and they float passively to the water surface, while the flagellates can actively move around.[insert part about sinking phycomates determining where they are when they release flagellates] Some of the lingering questions we have about them are:
- Where are they during the summer months?
- What part of the water column are the flagellates found?
- Why are they only found in the Salish Sea during the winter months?
To help answer these questions we will need to collect and analyze Halosphaera both spatially and temporally.
Because Halosphaera have been suggested to be most abundant during the months of (pers. obs. Dube & Kodner), using microscopy to capture both life phase images through size fractionation will quickly give us important details about this organism (i.e. what size, abundance of phycomates to flagellates per sample, etc…) within the Salish Sea. Sampling data, microscopy, and culturing data will then be used to tell a story about Halosphaera’s life cycle.
During the late 60s, three distinct species of Halosphaera were described through morphological observations and microscopy; H. viridis, H. minor, and H. russellii (Manton et al., 1963; Parke & Hartog-Adams, 1965). A more recent study on the environmental DNA (eDNA) analysis of picoplankton suggests that Prasinophyceae has a much higher genetic diversity than previously thought (Viprey et al., 2008). Another recent study using morphological data on collections in the Salish Sea, suggests that the species of Halosphaera in this region is, in fact, a different species entirely than those previously described (Kodner, 2007) . Finally, advances in genetic analyses demonstrate the need for a more up to date investigation on this algal group [find a good paper on eDNA]. By implementing both PCR and amplicon sequencing analyses, we should be able to determine which species of Halosphaera are in the Salish Sea along with what other organisms are present.
Fig. 1: (a) Flagellated phase for H. minor, suggested to be the most similar to what we are seeing in the Salish Sea (Parke and Hartog-Adams 1965; Kodner 2007). (b) Immature phycomate. (c) Formation of rosettes that will become motile flagellates. (d) Intermediate stage of Halosphaera sp., each collected from Taylor Dock, Bellingham, WA in January 2023.
This protocol goes into detail about how we will investigate each of these areas. We plan to begin sampling during the week of October 15th, 2023 and continue until the cells can no longer be found.
Greater Impact
The exterior of the phycomate is thought to be made up of an unknown recalcitrant biopolymer, which can be fossilized in marine sediments (Cohen et al., 2009). This makes Halosphaera a potential model for the most ancient eukaryotic microfossils, and will allow us to study a “living fossil”. Investigating Halosphaera sp. biology in our modern ocean is important for algal evolution and adaptability, since it has survived extreme environmental change over time. This preliminary work will help set the stage for this research to be investigated in the future.
Goals
The goal of this research is to describe Halosphaera in the Salish Sea. This project aims to build the foundation necessary for answering more complex questions about this marine phytoplankton. Our hope is that this project can be the first step in quantifying interactions that exist between this marine phytoplankton and other organisms, describing its journey through space and time, and figuring out where it goes when it is gone.
Take surface & depth plankton tows from October 15th - March 31st for microscopy.
Use FlowCam to quickly determine the size and abundance of phycomates during a portion of their bloom.
Through DNA analyses, determine which species of Halosphaera are in the Salish Sea & how abundant they are in comparison to other organisms.
Research Questions
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What species of Halosphaera are in the Salish Sea and can we describe their life cycle?
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1. How long is the phycomate bloom in the Salish Sea?
When do we first start seeing phycomata in the water (aka: when does the bloom begin)?
When does the bloom end?
2. Can we find flagellated cells in the Salish Sea?
What is their duration?
Are there patterns in time when they are most abundant?
3. Can we find Halosphaera in multiple locations?
Are there locations in the Salish Sea that you are more likely to find them?
Are flagellates found throughout the water column?
4. Are Halosphaera found more abundantly at the chlorophyll max?
Are these depth samples dominated by phycomates or flagellates?
Is there a depth that you can find most often?
5. What size are the phycomates throughout their life cycle?
At what days in the month are phycomates the largest?
Are there periods of the month where flagellates are the most abundant?
6. What species of Halosphaera are in the Salish Sea?
Is this species different from others observed and can we name it?
Are flagellates present when phycomates are not?
Are Halosphaera actually the most abundant green algae in the winter months within the Salish Sea?
Sampling Design
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I. Temporal
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Background
Parke & Hartog-Adams noted that pycomated cells were abundant in surface waters on or around the new and full moon (Parke et al., 1978) . A preliminary investigation from January - March of 2023 support this, with data shown below:
haloJulianData <- read.csv("haloJulian23.csv")
ggplot(haloJulianData, aes(Julian, Cells, color=Cells))+
geom_point(show.legend = F, na.rm=T)+
labs(title="Lunar periodicity is possible", y="Cell #/tow", x="Julian")+ #all -1 values are days that I did not sample
geom_vline(xintercept = 24, color="#63acac")+
geom_vline(xintercept = 9, color="plum")+
geom_vline(xintercept = 38, color="plum")+
geom_hline(yintercept = 0, color="gray")+
geom_vline(xintercept = 0, color="gray")
Fig. 2: Number of cells counted per tow on the y-axis with time on the x-axis (Day 1 began on January 13th, 2023).
The plum colored vertical lines in Figure 1 show when the new moon occurred, and the blue vertical line shows when the full moon occurred. The sample with 55 cells was collected 4 days before the full moon. Unfortunately there was only one full moon event during this last sampling season - so the goal of this year is to collect more information surrounding the full moon. Sampling dates will be created following the lunar calendar and NOAA’s tide chart.
New Moon Schedule 23-24: Nov 13, Dec 12, Jan 11, Feb 9, Mar 10
Full Moon Schedule 23-24: Nov 27, Dec 26, Jan 25, Feb 24, Mar 25
Taylor Dock: We will collect samples 2x/week beginning on October 15th, 2024. Sampling will increase to 3-4 times weekly on November 15th, 2024. Sampling will increase to 1 time per day on January 15th, 2024. During initial counts taken in January 2023 we noticed that we had peak phycomates on February 1st.
Boat: Samples will occur tentatively in hopes that they will occur on or around:
- Wednesday November 1st with Dr. Ponton.
- Friday December 8th. Monday January 8th or Tuesday January 23rd.
- Wednesday February 7th or Friday February 23rd.
If phycomates are still abundant in the water column in the beginning of March we will discuss a possible boat trip during that time.
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II. Spatial
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Background:
Sampling taken on the R.V. Magister on February 2, 2023 included sampling at sites 2 - 4 (Fig. 3). Surface tows were not collected on this trip. Another trip on March 8, 2023 went only to site 3. A surface tow was collected at both site 3 and site 1 that day. Site 3 had 15 cells at the surface and site 1 had 3 cells. These trips provided the following CTD data:
NSICTDMetadata <- read.ctd("NSI001NTS.cnv")
NSICTDtibble <- NSICTDMetadata@data%>%
as_tibble()
# makes a long table: gtsummary::tbl_summary(NSICTD)
#NSICTD %>%
# select(pressure, depth, temperature, conductivity, salinity, oxygen2, par, fluorescence) %>%
#tells number of rows
#data.table(options = list(pageLength = 3))
head(NSICTDtibble)%>%
gt |>
opt_stylize(style=5, color="cyan") |>
opt_table_font(google_font("Muli")) |>
tab_caption(caption = md("**Table 1:** showing the different parameters collected during boat sampling. This particular data is from North Smith Island on February 2, 2023."))|>
tab_options(table.font.size = 9)| scan | timeS | soundSpeed | pressure | depth | temperature | conductivity | salinity | theta | oxygen | oxygen2 | beamAttenuation | v4 | par | fluorescence | density | sigmaTheta | flag |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 0.00 | 1429.3 | 0.075 | 0.074 | 5.6885 | 0.069897 | 0.0512 | 5.6885 | 11.66599 | 364.570 | 0.0114 | 0.1856 | 390.40 | 0.0600 | 999.9934 | -0.0069 | 0 |
| 2 | 0.25 | 1429.3 | 0.075 | 0.075 | 5.6886 | 0.069841 | 0.0511 | 5.6886 | 11.66979 | 364.689 | 0.0111 | 0.1853 | 387.32 | 0.0346 | 999.9934 | -0.0070 | 0 |
| 3 | 0.50 | 1429.3 | 0.075 | 0.074 | 5.6885 | 0.069812 | 0.0511 | 5.6885 | 11.67413 | 364.824 | 0.0105 | 0.1787 | 384.34 | 0.0173 | 999.9934 | -0.0070 | 0 |
| 4 | 0.75 | 1429.3 | 0.075 | 0.074 | 5.6884 | 0.069841 | 0.0511 | 5.6884 | 11.67596 | 364.881 | 0.0110 | 0.1789 | 383.19 | 0.0726 | 999.9934 | -0.0070 | 0 |
| 5 | 1.00 | 1429.3 | 0.074 | 0.074 | 5.6884 | 0.069812 | 0.0511 | 5.6884 | 11.67243 | 364.771 | 0.0115 | 0.1789 | 380.18 | 0.0565 | 999.9934 | -0.0070 | 0 |
| 6 | 1.25 | 1429.3 | 0.075 | 0.074 | 5.6883 | 0.069784 | 0.0511 | 5.6883 | 11.66670 | 364.592 | 0.0116 | 0.1792 | 379.57 | 0.0496 | 999.9934 | -0.0070 | 0 |
Correlation coefficients will be used on this data to help
create an association network alongside our eDNA samples (Han et al., 2022 ). The locations used in our
pilot study are being discussed on Thursday, November 2, 2023 with
Dr. Sam Kastner. Boat sites will be updated following that
discussion.
Boat Sites:
Near Smith Island (NSI)
Near Burrows Bay (BuB)
In the Strait of Juan de Fuca (SFJ001)
Fig. 3: Previous sample sites from our pilot study January - March, 2023 taken at multiple locations within the Salish Sea. We took two trips on the R.V. Magister; on February 2nd and March 8th. The February 2nd trip traveled to sites 2-4, the March 8th trip only traveled to site 3, a surface tow was also collected at Taylor Dock and aboard the Magister. The Taylor Dock sample had only 3 cells and the R.V. Magister sample had 15 cells. (1) Taylor Dock, (2) Burrow’s Bay, (3) Smith Island, (4) Strait of Juan de Fuca 001.
Tow Type:
Surface tows will be collected at each site. Conductivity, Temperature, and Depth (CTD) Device will be used to collect water samples in the water column. Using the monitor on the R.V. Magister – the chlorophyll max can be detected for chlorophyll maximum beneath the water’s surface. This will help in determining which depths to collect samples. Literature suggests that Halosphaera can be found in greater abundance at depth (Jenkinson, 1986) . These profiles will also help us determine if flagellates are present.
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III. Measurements
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Dock:
Fresh Tow: samples will be collected using 20 micron mesh collection net. Tows will be taken over the edge of Taylor Dock starting at the boat ramp and walking north about 450 feet. Sample will then be pulled up and poured into a 20 oz. Nalgene bottle. The net will be placed back into the water at that point and sample will be collected walking back about 450 feet toward the boat ramp. Sample will be pulled up and poured into the same Nalgene bottle.
Preserved Tow : samples will be taken just like fresh tow, but this tow will be used to collect suspension after sample settles. This suspended material will be preserved in gluteraldehyde. The bottom of the sample will also be stored in gluteraldehyde to determine if flagellates are present.
DNA: samples will be taken off Taylor Dock’s edge by adding about 150 mL of water to the sample, and pulling that water through a micropore filter. The micropore filter will collect DNA which will be placed into RNAlater and frozed for future eDNA analysis.
Boat:
Three Samples: 3 water samples using CTD will be collected during vertical cast. These vertical tows will allow us to record conductivity, temperature, and chlorophyll max at different sites in Salish Sea, as well as collect cells at different depths.
1 of those samples will be collected at depth for live observations.
1 sample will be collected at depth for eDNA.
1 sample will be collected at depth for preservation.
Three Surface Tows: 3 surface tow samples from the boat will be collected by tying 20 micron plankton net to the boat and sampling the surface water while the boat turns 360°.
1 sample will be collected at the surface for live observations.
1 sample will be collected for at surface for eDNA.
1 sample will be collected at surface for preservation.
FlowCam:
In Mid January we expect to use FlowCam to quanititatively analyze water samples. This instrument will allow us to measure the abundance of Halosphaera in phycoma/mL. Using size fractionation we should also be able to duplicate those measures with flagellates. The FlowCam can measure other parameters in the samples concurrently, so this data will be useful in determining other factors involved in the transition from phycomate to flagellate.
Equipment
- Plankton tow net (currently: ~20 micron net mesh so it will not catch all flagellates)
- PVC Pipe
- Rope
- Tape
- Marker
- Nalgene bottle 500 ml (2)
- 500 ml Nalgene plastic vacuum flask
- plastic tube attachment
- Handheld pressure pump
- Membrane disk filters 0.2 micrometer with 47 mm diameter
- Tweezers (2)
- Alcohol wipes
- RNALater
- Plastic pipettes
- Micro-centrifuge tubes 0.5 ml
- Glutaraldehyde
- plastic pipette
- Nalgene bottle 500 ml
- Flow cytometer
- Nate’s fast vacuum pump (for boat DNA samples)
- Nikon SMZ745T Dissection scope
- EVOS Digital Inverted XL Core AMEX1200
- FlowCam 8000
- Refrigerator
- Freezer
- ZymoBIOMICS DNA Miniprep Kit
- Pipette 0.1 micrometer (for picking cells)
Sampling Procedure
Collections
There will be at least 75 samples total during this collection season.
An 16 oz Nalgene of water will be collected at the surface, in case there are flagellates being missed. This sample can be settled and observed under the EVOS Digital Inverted XL Core AMEX1200.
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I. Dock Tow
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Ensure plankton net is securely attached to PVC rope using a fisherman’s knot. Ensure that the plankton basket is properly attached to net. Lower net over the edge of Taylor Dock at location labelled START on map. Drag net through water about 450 feet to location labelled END on map. Pour this sample into your Nalgene and return to the starting point while continuing a plankton tow. Pour sample into the same Nalgene. This will be one sample. Repeat again for a second sample. This sample will be the preserved sample. Preserved samples will be left in the refrigerator for up to 24 hours. At this time, material will be assessed under the EVOS Digital Inverted XL Core AMEX1200 microscope, if flagellates are present then the sample will be preserved with Glutaraldehyde for later inspection. [eDNA protocol], [8 oz nalgene protocol]
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II. Boat Tow & CTD Cast
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Get vacuum flask prepared with filter in place and sampling vials labelled prior to getting to the site. Once at the site the CTD will be lowered while inspecting the monitor for the chlorophyll maximum. The CTD should fire three bottles on the upcast at that chlorophyll maximum. Once the CTD is brought back up to the surface, water samples will be collected in Nalgene bottles. One of which will be pulled through the vacuum flask right away for eDNA analysis. This sample will be poured into the top of 500 ml flask, and the electric pump will pull the water over the filter. Once the water has emptied from the top chamber – the filter will be placed in a 0.5 ml micro-centrifuge tube and RNALater will be added to the top. The two live samples will be placed into the refrigerator and the DNA sample will be placed into the freezer.
Field Notes
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Field notes will be taken inside of a Rite in the Rain notebook. This information should include:
Date
Location of sample (gps coordinates when possible)
Time
Ebb or flood
Current tidal height (ft)
Moon age (how far away from a NM or FM is it)
Documentation
All samples should be labelled with:
- Date
- Site
- Taylor Dock (TD)
- Friday Harbor (FH)
- Burrows Bay (BuB)
- North Smith Island (NSI)
- Strait of Juan de Fuca (SFJ)
- Colville Island (COI)
- Initials
All field notes will go into shared Google Sheet that can then be converted into .csv file for further analysis later. Cell counts will be added in as microscopy takes place. Photographs will be placed into Google Drive folder with the date and the cell count number. All other files will go into this folder, which will be publicly available at the end of this project.
Sample Storage
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Live tows should be picked within 4 days of sampling and stored under low light at 12°C. DNA samples will be stored in the freezer. Preserved samples will be stored in the refrigerator under low light at 12°C. Once live tows have been picked and photographed, they can be disposed of down the sink. Glutaraldehyde samples can be disposed of down the drain with lots of water.
Sampling Analysis
Microscopy
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Nikon SMZ745T Radioscope
This microscope will be used to take images of phycomates and to pick Halosphaera from live samples.
EVOS Digital Inverted XL Core AMEX1200
This microscope will be used to pick through for imagery of flagellates. I am hoping there is a measurement tool on this instrument so that I can also take measurements of these cells.
This microscope can also be used to pick through preserved samples.
FlowCam 8000
This instrument will be used on all samples during January 15 - February 15. It will take imagery with multiple parameters. We will fractionate our samples by size, which will allow us to get measurements on both the phycomates in a sample and the flagellates.
FlowCam can image up to 64 different cell characteristics (i.e. cell texture, shape, color, size). These will be incredibly useful for quantitative analysis of their life cycle and description.
Micro-computed Tomography
I plan on testing this instrument to see if it can take nice imagery of our algal cells for displaying life cycle. I have seen xray imagery that it took of a flower and I think it could be very useful.
Scanning Electron Microscopy (SEM)
This instrument will be added to this protocol in the event that we have time. This will be used to determine if Halosphaera fossils are present in the sediment of Bellingham Bay. These sediment cores are already sampled and just need analyzed.
DNA
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ZymoBIOMICS DNA Miniprep Kit
This kit will be used for metagenome analysis. It can help us determine if Halospahera is present as a flagellate, even when we are not finding phycomates. This will help us better understand its life cycle and figure out who else is sharing the water column with them.
Data
Storage
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All data will be stored on a Google Drive, which will be downloaded into Microsoft Teams once it is finished. This data will also be stored in a repository on Github so that people can access it publicly.
Analysis
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Nikon SMZ745T Radioscope will be completed on live tows to count out the cell number. These cell numbers will be used to figure out when phycomates are the most abundant in Bellingham Bay throughout the month.
EVOS Digital Inverted XL Core AMEX1200 microscopy will be completed on live tows at multiple magnification. All photos for microscopy should include this nomenclature upon saving:
23-NSI_009-030823-settle-jb Where 23 is the year, NSI is the location quick code, 009 is the depth (s for surface), 030823 is the date of collection, settle is the type of sample you are viewing, and jb are my initials.
These photos will be analyzed using 10x-40x objectives. 4/10 and 20/40 phase filter (PH) helps refract light, so this is necessary when looking for flagellates. BF is bright field and can be used to view phycomates. These images could possibly be used to determine the size of cells throughout the month. Abundance might be harder, but is still possible through cell settling.
We will work with Savannah Judge from FlowCam to figure out the best setup for the FlowCam 8000. The FlowCam captures images rapidly as water is being pumped through a flowcell. We will use Autoimage setup on the instrument coupled with machine learning to pick through samples quickly. These phytoplankton images will be used to determine volume and abundance in each sample. Other characteristics will also be stored to determine patterns in phycomate size ranges throughout the month color, and rosette number.
References
Calendar
Calendar code is currently being worked on. Coming soon: all dates we plan to sample, lunar calendar, etc…
calendarHalo <- read.csv("CalendarHalo.csv")
calendar(calendarHalo,
view = c("month", "week", "day"),
navigation=T,
isReadOnly = T,
useDetailPopup=T,
useCreationPopup=T,
width = 600,
height = 400,
defaultDate = Sys.Date()) %>%
cal_month_options(
startDayOfWeek = 1,
daynames = c("S", "M", "T", "W", "Th", "F", "S"),
visibleEventCount = T
)