Materials and Reagents

Parafilm
0.22 µm sterile vacuum filter (Corning^®bottle-top vacuum filter, Corning, catalog number: 430015 )
0.45 µm filter (VWR, catalog number: 514-4127 )
Note: The 0.45 µm filter is for fish.
30 G syringe (BD, catalog number: 324826 )
Narrow plastic vials
22-G Micro-Renathane Implantation tubing (Braintree Scientific, catalog number: MRE025 )
Disposable syringe 0.3 ml BD needle 30 G (Insuline) (BD, catalog numbers: 324826 , 320837 )
Soft sponge of approximately 20 mm in height (such as Jaece Industries, catalog number: L800-D )
Drosophila and fish (any source)
FD&C blue dye #1 (Sigma-Aldrich, catalog number: 861146 , SPS Alfachem Ref: 101-2912 and A & Z Food Additives Brilliant Blue FCF(CAS No. 3844-45-9), FD&C Blue 1, E133)
Fluorescein sodium salt (Sigma-Aldrich, catalog number: F6377-500g )
FD&C Red #40, Allura Red (SPS Alfachem)
Bi-distilled deionized water (ddH₂O)
Hanks’ balanced salt solution (HBSS, Thermo Fisher Scientific, Gibco^TM, catalog number: 14175053 )
Buffered tricaine methanesulfonate (Sigma-Aldrich, catalog number: A5040 ) 164 mg/L in fish tank water
Moldex (VWR, catalog number: 25605.293 )
Blue #1 stock solution (12.8x) (see Recipes)
Dyed media (see Recipes)

Equipment

Magnetic stirrer
5 L glass beaker
2 L glass bottle
LED cool white lighting
White background
Epi-fluorescence microscope Nikon Eclipse 80i for nematode Smurfs (Nikon, model: Eclipse 80i )

Software

ImageJ 1.46j and above
GraphPad Prism 6.01

Procedure

Prepare solutions
See ‘Recipes’ section.
Smurf assay
Notes:
1. All fish work was performed following Animals (Scientific Procedures) Act 1986 and UK Home Office approval (PPL 70/8681).
2. If the blue dye is used at the correct concentration, well dissolved and filtered, no adverse effects are expected from the 30 min incubation period, except some temporary stress and discomfort. After blue-dye incubation, fish should be kept in separate tanks for at least 4 h before joining the main aquarium tanks where water is shared, to ensure all dye is excreted.
3. Any signs of potential adverse effects such as impaired prolonged swimming and feeding should be brought to the attention of the NACWO and/or NVS and any definite or probably adverse effects should lead culling of the animal using an approved procedure performed by a person registered as competent by the certificate/licensed holder (10.1089/zeb.2016.1248).
It is important to standardize the conditions under which Smurfs are examined.
1. Transfer individuals onto SA medium
  1. Drosophila and nematodes: overnight.
  2. Fish: 30 min.
2. Transfer individuals onto fresh normal medium for immediate scoring
  1. Drosophila: if you have to process large numbers of vials, you might want to transfer vials one at a time to prevent individuals from clearing the blue dye before scoring.
  2. Nematodes can be examined directly on the fluorescein medium using an epi-fluorescence microscope.
  3. Fish: rinse them until no blue comes out of the individuals in successive and independent fish tanks (Figure 5).
    
    Figure 5. Identifying Smurfs in zebrafish using the ‘bathing protocol’. A. Successive fish tanks are used to rinse individuals until no further dye is emitted. B. Representative images of Smurf and non-Smurf individuals in WT AB zebrafish.
3. Count individuals that are positive for Smurfness (SA+) as well as those that are negative (SA-).
  The proportion of Smurfs in the population is then
4. Smurf and non-Smurf individuals can then be monitored separately for further analysis.
  An alternative protocol for fish (Raquel Martins and Catarina Henriques): Since penetration of the blue dye through the skin of fish will always be a confounding factor with a dipping protocol, we tested oral administration of the blue dye by following the adult zebrafish gavage protocol published in Collymore et al. (2013). 5 µl of a 5% (m/v) filtered blue dye dissolved in Hanks’ balanced salt solution (HBSS) was administered through oral gavage. Older individuals show the Smurf phenotype whilst we did not observe this in younger individuals (Figure 6C). This method requires training to prevent the experimenter from injuring the gut. It is nevertheless non-lethal, thus allowing further monitoring of individuals post assay.
  
  Figure 6. Gavage allows to identify zebrafish Smurfs. Smurf zebrafish obtained using the ‘gavage protocol’ (A). All six Zebrafish WT AB individuals of 6 and 29 months of age used (B) and respective quantification of the blue hue that leaked through the gut and is now visible on the skin. (C) Quantifications were performed from digital pictures in ImageJ selecting three different parts of the body/fish, avoiding the blue stripes and quantifying the median of pixels per area. All 3 older fish turned bluer than the younger fish (t-test assuming normality and equal variances: P < 0.05 or more conservative: unequal variance t-test or non-parametric Wilcoxon rank test P ≤ 0.10). See Video 1.
  
  Video 1. Detailed zebrafish Smurf gavage protocol
  
  Pre-injection preparation:
  1. Before the gavage procedure, the fish must be fasted for 24 h or at least 4 h prior to gavage, which will empty the intestinal bulb (stomach) contents.
  2. On the flat face, make a cut (10-15 mm deep) in the soft sponge, which will hold the fish for injection.
  3. Then, set the sponge into a 60 mm Petri dish. This will hold water to help maintain sponge moist.
  Anesthesia, Injection and Recovery:
  1. Anaesthetize fish with diluted Tricaine and monitor fish behavior. When fish is quiet, transfer it to the wet sponge (soaked in Tricaine) and move the sponge into a vertical position, as illustrated in the video.
  2. Then, open the zebrafish mouth using the 22-G catheter tubing adjusted to the size of the fish (approximately 2.5 cm), and gently insert the tubing until the tip is past the gills (approximately 1 cm or the length of the tubing). The implantation tubing should not need to be forced. Resistance suggests the tube may be hitting the gill arch or heart. Thus, if there is resistance, gently withdraw, reposition and try again.
  3. When the tube is completely inserted (reaching the middle intestine), inject the material slowly (5 μ of filtered (0.45 μm syringe filter) 5% (w/v) blue #1 diluted in HBSS). To accurately measure 5 μl, pipette 5 μl of the solution onto Parafilm and aspirate with a syringe, avoiding air bubbles. While injecting, make sure that the solution does not exit via the gills or the mouth.
  4. After injecting all the content, remove the fish from the sponge and place into the recovery tank. Fish should be monitored for regurgitation as shown by visualizing the fish actively expelling material from its mouth, or no opercular movement. Fish can be returned to their regular tank, or kept separately in a mating box, once they have recovered.
Blue hue measurements
Using ImageJ freehand selection tool, ROIs are selected on a picture converted to HSB space (command Image → Type → HSB Stack). Then, the hue is measured by the mean gray value (Analyze → Set measurements… → Mean gray value) in the Hue dimension of the picture.

Data analysis

It is possible to consider two distinct approaches for comparing the Smurf proportion in condition A versus condition B.

At one given time-point, comparing the proportion of Smurfs between conditions A and B:
1. Use a two-sided binomial test to estimate the statistical significance of the difference between distribution of SA+ individuals amongst the total population, in condition B compared to the distribution in control condition A. We used this approach in (Rera et al., 2011). Since GraphPad Prism Chi-square is just an approximation, one can use the exact test implemented in Excel or R.
2. Use a Mann-Whitney test on the average proportion of SA+ individuals per vial in condition A and B.
Across multiple time-points, comparing the slope and y-intercept of age-dependent proportion of Smurfs between conditions A and B. We initially described this approach in (Rera et al., 2012) using GraphPad Prism. The calculations follow a method spelled in Chapter 18 of J Zar, Biostatistical Analysis, 2nd edition, Prentice-Hall, 1984. It is equivalent to analysis of covariance (ANCOVA). Similar approaches are available in mixed models, in a binomial model context and should be considered if one wants to correct for experimental confounds, such as batch or other random terms (Gelman et al., 2004).

We recommend the use of method 2 since the time trend ensures a smaller risk of false positives.

Notes

It is crucial to ensure that the bathing solution for fish does not contain clumps of the blue dye powder as they tend to stick to scales, making it harder to distinguish Smurf individuals.
Standardize your Smurf scoring conditions.
Train multiple people to recognize Smurf individuals and cross-check your own scoring with them.
Keep in mind that the genetic background of the individuals you assess for Smurfness can influence its result, so adapt your scoring to it.

Recipes

The FD&C blue dye #1 comes as an extremely volatile dark purple powder. The first step is to put it in an aqueous solution. The maximum concentration we could reach at room temperature is 320 g L^-1. This corresponds to a 12.8x stock solution as the final concentration in the medium is 2.5 g per 100 ml (2.5% w/v).

Blue #1 stock solution (12.8x)
1. Pour 700 ml dH₂O into a 5 L glass beaker
2. Weigh 400 g of the blue #1 powder
3. Gently pour the powder into the water containing beaker
4. Add the magnetic stirring bar
5. Cover the beaker with Parafilm
6. Stir until the powder is totally dissolved (typically a couple of hours)
7. Slowly rinse the walls of the beaker with the remaining 300 ml of dH₂O
8. Stir for 30 min
9. Vacuum filter the blue solution into a sterile bottle
Notes:

For fish: prepare the 1x solution directly in water extracted from the fish tank and filter it.
For fluorescein: use the same procedure as for the blue #1.
The solution is preferentially used immediately or frozen and not stored at room temperature since its dark color does not allow the observation of any potential contamination.

Dyed medium
1. Prepare your standard Drosophila food recipe (Rera et al., 2011; Katzenberger et al., 2015; Tricoire and Rera, 2015; Barekat et al., 2016; Regan et al., 2016) with 7.8% less water
2. After boiling the medium, add the Moldex (VWR)
3. Add 7.8 ml of the Blue #1 stock solution per 100 ml (final) of food
4. Stir thoroughly until the coloration is homogenous
5. Dispense in the narrow vials (minimum 1.25 ml for overnight SA)
Notes:

For nematodes: use the same procedure as for the blue #1. Although the molar mass of fluorescein is 2.9 times less than that of blue #1 at that concentration the fluorescein does not fluoresce. This allows an easy identification of nematode fluorescent Smurfs directly on the medium.
The fluorescein solution is added directly to the agar plate or mixed with the bacteria.

Acknowledgments

This work was supported by the CNRS to M.R. This protocol is adapted from (Rera et al., 2012; Dambroise et al., 2016). M.J.P.S. is supported by a Sir Henry Wellcome and a Sheffield University Vice Chancellor’s Fellowship and the Natural Environment Research Council (N013832). A.W.McC. is supported by the NERC ACCE Doctoral training program. C.M.H is supported by a Sir. Henry Dale Fellowship by the Wellcome Trust & Royal Society and a Sheffield University Vice Chancellor’s Fellowship. R.R.M is supported by a Sheffield University Doctoral Academy PhD Fellowship. The authors declare no conflict of interest nor competing interests.

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