Background

The Smurf Assay (SA) is based on the Drosophila feeding assay described in (Wong et al., 2009). The assay assesses food intake by the co-ingestion of a blue dye, which is not absorbed by the digestive tract, thereby allowing direct quantification. For the SA it is essential that this specific blue dye does not pass an intact intestinal barrier since its readout is a whole body coloration (here blue). This property allows direct in vivo assessment of gut permeability, which has been shown to increase with age (Rera et al., 2011 and 2012).

As recently discussed in Rera et al. (2018), the Smurf Assay is not only a simple way to assess intestinal permeability in vivo, but also an elegant way to assess the physiological age of individuals in a broad range of organisms. As such, it allows novel approaches to study the various events occurring in aging individuals (Tricoire and Rera, 2015; Rera et al., 2018).

In recent years, we have received numerous comments and questions about the initial protocol, leading us to develop the present extended protocol.

Specifics of the dye used: The dye typically used is FD&C blue dye #1, but we have also validated the use of red #40 and fluorescein (Rera et al., 2011 and 2012). We adapted the use of the very same blue #1 to zebrafish (Dambroise et al., 2016) and killifish (Rera et al., 2018) but found it is easier to use fluorescein with nematodes although the same blue #1 can also be used as demonstrated in (Gelino et al., 2016). The dye is non-toxic and does not decrease the lifespan of individuals when exposed during their whole life (Figure 1A). Moreover, no reduction in longevity is detected even when the gut becomes permeable and the dye diffuses into the body, contrary to what was recently suggested in Clark et al. (2015). We confirmed this by placing newly identified Smurfs on normal non-dyed media, and this did not lead to a longer lifespan (Figure 1B).


Figure 1. The blue dye #1 is not toxic neither for non-Smurfs or Smurfs. A. The longevity curve of 1,146 individual female flies maintained on blue medium overlaps the longevity curves of 295 female flies maintained on standard medium by groups of 28-32 individuals (longevity data from Tricoire and Rera, 2015). B. The longevity curve of 173 individual female flies maintained on blue dye for their whole lives overlaps the longevity curve of 172 individual female flies transferred back to standard medium when they became Smurf. This confirms that Smurfs do not die prematurely because the dye gains toxic properties when it diffuses through the gut.

Catching Smurfs
Although we initially described Smurfness as a well-marked, almost binary phenotype (Rera et al., 2011 and 2012; Tricoire and Rera, 2015), Smurfness is, as most phenotypes are, continuous (Figures 2A-2D, Clark et al., 2015). Thus, it is important to understand that the lighter the Smurf is, the greater the chance of misidentifying Smurf individuals. Indeed, the major part of uncertainly identified Smurfs appears in the few days preceding clear mortality acceleration in the population (Figure 3). The continuous nature of the Smurf phenotype can have two main causes. First, the dye might take some time to diffuse through limited gut permeability, thus generating a determined relationship between Smurfness and the level of gut permeability. Second, there can be biological (environmental and/or genetic factors) that can cause variation in the phenotype observed.

Moreover, there can be observer bias, attributable to the experimenter who is sorting and classifying individuals. We noticed that the earlier in the lifespan, and the fewer Smurfs are present in the group, the more likely an observer is to classify individuals as Smurfs, despite subsequently being scored as non-Smurf. The latter is probably inherent in the way we distinguish individuals based on their surrounding individuals, and hence the more Smurf individuals are present, the more stringent we are on their identification. To circumvent this, single individuals could be photographed for independent verification. In practice, however, it is difficult to both sort large numbers of flies and photograph every individual for subsequent blue hue quantification. This problem is less relevant to larger organisms.


Figure 2. The Smurf phenotype is not binary but rather continuous. A. The continuous aspect of the Smurf phenotype was previously described in Clark et al. (2015), but we noticed much more subtle shades of blue in our experimental conditions. B. Nevertheless, only the two categories of Smurfs and non-Smurfs showed significant blue hue difference on all body parts (n = 31 female from the drsGFP genotype, n_ns = 16, n_? = 4, n_ls = 4 and n_s = 7)–a subsequent experiment with larger n was conducted and showed significant differences only between Smurfs and non-Smurfs (not shown). C. The continuous Smurfness distribution is not just due to the Smurf (blue dye based) assay but is also observable in the drsGFP individuals by D. measuring GFP intensity in Smurfs (n = 33) and non-Smurfs (n = 130). The drosomycin promoter-driven GFP expression has been shown to be a surrogate of Smurfness in Rera et al. (2012). Mated 35-40 days old female Drosophila.


Figure 3. All individuals eventually become Smurf prior to death. Estimating Smurf survival time requires taking into account individuals from different moments of the survival experiment to prevent misestimation. A. All 1,146 individual female flies became Smurf prior to death and survived for various duration in that state. B. The uncertainty on the Smurf status has the strongest effect on the youngest identified Smurfs. Restricting Smurf studies to that period is thus at high risk of misestimating their remaining lifespan. We recommend to study them close to the T₅₀ of the population. Original data from (Tricoire and Rera, 2015). C. Proportion of the three different types of living Smurfs at various percent survival in the population. The largest population of uncertain Smurf individuals is restricted to the first few days of Smurf apparition in the population.

Other experimental considerations
The duration of exposure to the dye does not affect survival nor the Smurf Increase Rate (SIR). For ease we now use overnight feeding on the blue dye. The fly population density is of critical importance: we observed that at a too high density, individuals tend to get covered with blue faeces. Although easy to rinse with water (the addition of some ethanol can help immersion) to discern ‘false’ Smurfs from legitimate ones, we do not recommend more than 30 individuals per vial for overnight exposure. For continued exposure to the dye, lower numbers should be considered. Moreover, when learning how to distinguish smurfs we recommend washing flies to confirm the phenotype. These considerations could be especially important as behaviour and the quantity of feaces produced can differ between genetic backgrounds and experimental conditions.

We received a significant number of questions regarding ‘the number of Smurfs with time’. As previously stated in (Rera et al., 2011 and 2012; Tricoire and Rera, 2015; Dambroise et al., 2016), it is the Smurf proportion calculated as at a specific age that increases as a function of age, rather than the absolute number of Smurf individuals. The interpretation of this number is similar to that of mortality risk, as it is related to the age-specific risk of an individual in the population to become a Smurf. Note that Smurfs remain in the population for a short time until they die and thus remain in the numerator of the formula above. The Smurf proportion is thus not equal to the risk of becoming a Smurf, but could be calculated as such (Promislow et al., 1999).

Most of the Smurf-related studies we conduct are based on female flies because, as we described in (Rera et al., 2012), they are easier for Smurf identification, principally since their abdomen is larger. In addition, the age-dependent SIR is weaker in males (see Figure S1A in Rera et al., 2012). This might be due to a much shorter remaining lifespan of males when they are in the Smurf state, as we recently observed (unpublished) and/or their smaller body. It is interesting to notice that in zebrafish the sex-specific SIR intensity was inverted (see Figure S1B in Dambroise et al., 2016). Regardless, male Drosophila do undergo the Smurf transition prior to death (Figure 4), contrary to what was recently suggested in Regan et al., 2016.


Figure 4. The Smurf phenotype occurs in male Drosophila melanogaster. Two examples are pictured: A. A smurf male; B. A non-smurf male. 35 days old males.