Pilot 1.2 in vivo NiCl2 x Botryllus schlosseri Exposure

study plans
nickel chloride
Author

Celeste Valdivia

Published

October 30, 2023

library(knitr)
library(tidyverse)
── Attaching core tidyverse packages ──────────────────────── tidyverse 2.0.0 ──
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✔ forcats   1.0.0     ✔ stringr   1.5.0
✔ ggplot2   3.4.2     ✔ tibble    3.2.1
✔ lubridate 1.9.2     ✔ tidyr     1.3.0
✔ purrr     1.0.1     
── Conflicts ────────────────────────────────────────── tidyverse_conflicts() ──
✖ dplyr::filter() masks stats::filter()
✖ dplyr::lag()    masks stats::lag()
ℹ Use the conflicted package (<http://conflicted.r-lib.org/>) to force all conflicts to become errors
library(tidyr)
library(dplyr)
library(hrbrthemes)
library(ggplot2)
knitr::opts_chunk$set(echo = TRUE)

Experimental Planning

Animal Handling Prior to Exposure

Divided animals into separate system, 3 days prior to start of exposure, 8 days acclimated to the recirculating system.

Randomize Treatment and Plate Assignments

Here we will have R randomly assign each animal to a treatment and each treatment/animal to a different well on the plate. The idea here is to randomize placements as there may be variations in the well design.

set.seed(3445)
plate_number_12_well <- c(rep(1, times = 12))
well_placements <- c("A1", "A2", "A3", "A4", "B1", "B2", "B3", "B4", "C1", "C3", "C2", "C4")
conditions <- c("Control R1", "Control R2", "Control R3", "Control R4", "5 mg/L R1", "5 mg/L R2", "5 mg/L R3", "5 mg/L R4", "45 mg/L R1", "45 mg/L R2", "45 mg/L R3", "45 mg/L R4")
animal_id <- c("S246C001", "S228C001", "S215C001", "S247C001", "S224C001", "S218C001", "S248C001", "S212C001", "S209C001", "S207C001", "S200C001", "TBD")
well_assignments_exp1 <- data.frame(
  Plate_Number = sample(plate_number_12_well),
  Well_Placements = sample(well_placements),
  Conditions = sample(conditions),
  animal_ID = sample(animal_id)
)
print(well_assignments_exp1)
   Plate_Number Well_Placements Conditions animal_ID
1             1              B4  5 mg/L R4       TBD
2             1              B2  5 mg/L R3  S246C001
3             1              A3 Control R1  S209C001
4             1              A1  5 mg/L R1  S200C001
5             1              A2 45 mg/L R1  S218C001
6             1              C3 Control R2  S228C001
7             1              C4 Control R3  S247C001
8             1              B3 45 mg/L R2  S215C001
9             1              C2 45 mg/L R3  S224C001
10            1              A4 Control R4  S212C001
11            1              C1  5 mg/L R2  S248C001
12            1              B1 45 mg/L R4  S207C001
#save assignments in csv for downstream use as metadata
write.csv(well_assignments_exp1, file = "well_assignments.csv")

plate assignments

Nickel Chloride Dilutions

Review resources prior to handling nickel chloride:

All handling of stock solution and treatments to be done in a chemical fume hood. Once spiked water has been distributed, add animals, parafilm jars, place in secondary container, and move to environmental chamber.

Required Volume of Artificial Seawater

Calculations for spiking artificial seawater. 225 mL of seawater required to mostly submerge glass slide in wide mouth 16 oz Kerr mason jar.

Considerations:

  • 4 replicates

  • 225 mL per jar

  • 3 conditions

  • Account for 10% loss of water during transferring

\[(4*225 * 3) + (0.1*(4*225*3)) = 2,970\]

Need 2,970 mL of artificial seawater total for exposure, particularly, 990 mL of artificial seawater per condition.

Calculations

Stock solution of NiCl2 made by undergraduate Gina Jones on 02/08/23.

  • NiCl2

  • Suspended in MilliQ water (0 PPT)

Low Dose: 5 mg/L

\[ C_1*V_1 = C_2*V_2\] \[ 1 g/L * V_1 = 0.005g/L*0.99L\] \[V_1 = 0.00495 L = 4.95 mL\] Spike 990 mL of aerated, artificial seawater (16 C) with 4.95 mL of stock NiCl2 solution. Change in salinity negligible (+/- 1 PPT).

High Dose: 45 mg/L

\[ C_1*V_1 = C_2*V_2\] \[ 1 g/L * V_1 = 0.045g/L*0.99L\] \[V_1 = 0.04455 L = 44.55 mL\] Spike 990 mL of aerated, artificial seawater (16 C) with 44.55 mL of stock NiCl2 solution. Change in salinity negligible (+/- 1 PPT).

Protocol

  1. Subdivide animals 3 days prior to exposure start.

  2. Make sufficient volume of artificial seawater at appropriate salinity, 25 PPT, to match recirculating seawater system parameters. In this case for a 3 x 4 exposure at 250 mL, will require about 7 L of artificial seawater.

  • 30.8 g / L of Red Sea Salt
  1. Rinse all glassware to be used during exposure as follows:

  • Triplicate rinse with lab detergent

  • Triplicate rinse with tap water

  • Triplicate rinse with DI water

  1. Add lab bench paper to environmental chambers top rack to reduce light intensity. Include 4 slits in bench paper to allow for air flow.

  2. Add 1000 mL of artificial seawater to three 1 L beakers and place in environmental chamber (set to ambient 23 C to achieve 16 C for water) with gentle aeration (targeting ~ 67 % DO) in preparation the night before exposure.

Day of Exposure Commencement

  1. Take 10 mL from each 1 L beaker and document temperature, DO, pH, salinity, and ammonia of artificial seawater prior to spiking the water.

  2. In a chemical fume hood add the appropriate volume to achieve desired concentration of nickel chloride concentration to each of the 1 L beakers using a 25 mL or 50 mL serological pipette. Be sure to homogenize solution well with the serological pipette. Distribute 225 mL of each stock to the appropriate mason jar or beaker using designated serological pipette.

  3. Transfer animals to a portable container with artificial seawater from the recirculating system. Image the animals using the Excelis stereomicroscope. Use a razor blade to clean around the animal and to clean glass slide of excess algae and debris.

  • Document stage, zooid number, cumulative system number.

  1. Distribute animals to appropriate jar in fume hood.

  2. Parafilm jar and place in secondary container.

  3. Transfer secondary container with jars to second rack of environmental chamber.

  4. Leave animals for 24 hours.

Day of Dissections

The following steps should be conducted in a chemical fume hood and iterated through one beaker at a time to avoid desiccation stress:

  1. Remove 100% of the exposure water from beaker and dispose of NiCl2 dosed artificial seawater in the appropriately marked hazardous waste container.

  2. With the animal and glass slide still in the beaker, quadruplicate rinse the glass slide and animal with deionized water.

  3. Glass slide may now be handled with nitrile gloves and should be quadruplicate rinsed with artificial seawater over exposure beaker.

  4. Transfer glass slide and animal to a fresh 16 oz mason jar containing 200 mL of 15 C artificial seawater.

  5. Dispose of excess liquid in exposure beaker in appropriately labeled hazardous waste container.

You may now work outside the chemical fumehood. Note there may be residual NiCl2 in animal so continue to wear the appropriate PPE and work carefully.

  1. Using a stainless steel razor blade, remove one of the two cumulative systems on the glass slide. Use forceps to transfer cumulative system to appropriately labeled snap cap 1.5 mL eppendorf PCR safe tube containing 500 uL of RNAProtect by Qiagen.

  2. Proceed with dissection of the primary buds or zooids of the second cumulative system on the slide. Refer to epithelial isolation protocol

  • However, avoid using any ethanol in this procedure.

  1. Once tissue has been removed, take cell strainer and rinse it with 30 mL ASW-PSA.

  2. Place tissue in aliquot of TCM and immediately go to seed tissue in biosaftey cabinet. Mark time of seeding.

  3. No replacement of media over course of trial.

  4. Transfer samples containing cumulative system suspended in RNAProtect to -20 C freezer overnight. The following day, transfer samples to -80 C freezer for long term storage.