Panagrolaimus superbus tolerates hypoxia within Gallium metal cage: implications for the understanding of the phenomenon of anhydrobiosis

Panagrolaimus superbus, first isolated from Surtsey Island (Iceland) (Sohlenius, 1988), was maintained in the dark, at 20°C, on Nematode Growth Medium (NGM) agar plates and fed with a layer of Escherichia coli (OP50 strain).

Mixed populations of P. superbus (i.e. containing all developmental stages) were subjected to synchronization using the bleaching protocol proposed by Stiernagle (2006). Briefly, these populations were exposed to a bleaching solution (NaOH 1 M and NaClO 40%) for 8 min, disintegrating all the worms, only eggs remained. The reaction was stopped by three consecutive washes with M9 buffer, intercalated with centrifugations at 2,000 × g for 30 sec. All the remaining eggs were deposited on agar NGM plates containing E. coli OP50 and maintained in the dark at 20°C. After 36 hr, plates were rinsed with M9 buffer to collect L2 larvae, which were subjected to the desiccation process.

Nematodes were subjected to desiccation assay according to Shannon et al. (2005). L2 larvae were immobilized on 0.45 µm Supor filter membranes (Sigma Aldrich) by vacuum filtration with a Sartorius funnel (n = 200 worms per membrane), which were stored in 1.5 ml open test tubes and then subjected to sealed chambers under the following conditions: pre-conditioning in 98% relative humidity (RH) for 24 hr over a saturated copper sulphate solution and desiccation in 10% RH for 24 hr on dry silica gel.

Three membranes containing around 600 desiccated L2 larvae were placed in test tube caps containing 100 µl solid Ga 99.99% (Sigma Aldrich). Subsequently, 180 µl liquid Ga 99.99% (Sigma Aldrich), at 50°C, was added to the test tube caps fully covering the membranes (Fig. 1A, B). To check the ability of metal Ga in blocking external moisture, Ga cages containing nematodes were exposed to different levels of relative humidity. Test tube caps were separated into five groups, in the following conditions: negative control 1 (NC1) – membranes with desiccated worms, without Ga, kept at 10% RH; Ga treatment 1 (GT1) – membranes with desiccated worms, covered by Ga and kept at 10% RH; hydrated control (HC1) – membranes with hydrated worms, covered by Ga, kept at 100% RH; negative control (NC2) – membranes with desiccated worms, without Ga, kept at 99% RH; Ga treatment 2 (GT2) – membranes with desiccated worms covered by Ga, kept at 99% RH. All groups were maintained under these same conditions for 7 d, at 20°C in the dark.

Figure 1:

To investigate nematode viability after treatments, we performed worm survival assay using a modified version of Krause et al. (1984) protocol, which was first used for isolated cells but has already been successfully used for nematodes by Evangelista et al. (2017). Briefly, solid Ga blocks (placed within test tube caps) (Fig. 1) were heated at 50°C for 15 min, in order to melt the metal cages. Subsequently, most of the liquid metal was collected from the tube caps by using a micropipette. Finally, we used tweezers to collect the membrane from the residual liquid Ga inside the tube caps. All membranes were deposited in 1.5 ml open test tubes, which remained in 99% RH for 24 hr, for pre-rehydration. Then, one membrane per treatment (n = 200) was transferred to other 1.5 ml test tubes containing 1 ml erythrosine B dye (0.4% w/v in M9 buffer). After 1 hr, samples were washed three times with M9 buffer, to remove the dye excess. Totally and partially dyed worms were scored as dead.

Two membranes per treatment (n = 400) were submerged in 1 ml M9 buffer and the worms were rehydrated. About 100 worms were placed on new NGM plates with E. coli OP50 and maintained at 20°C in the dark. Population growth was measured after 20 d, as well as the survival percentages, and about 100 worms of each treatment were placed on new NGM plates with E. coli OP50 for more 20 d (until day 40). Population growth was determined as follows: [(number of worms − output) × (final survival percentage)] divided by [(number of worms − input) × (initial survival percentage)].

All experiments were performed in biological and technical triplicates with around 100 individuals. Mean values and standard deviations were generated for each experimental group. Shapiro–Wilk’s and Levene’s tests were first performed (P > 0.05) and, then, statistical analyses were performed using one-way ANOVA and Tukey’s post hoc test. Statistical differences were considered significant at P < 0.05.

Desiccated P. superbus survival percentages (Fig. 2A) after 7 d fully immersed in Ga confinements did not show statistically significant differences compared to NC1. The experimental group GT2 (66%) displayed differentially (P < 0.05) higher survival percentages compared to NC2 group (31%), but statistically equivalent to NC1 and GT1 groups (73 and 72%, respectively). Lastly, HC1 group showed no survival (0%) after immersion in Ga for 7 d.

Figure 2:

Likewise, NC1, GT1, and GT2 population growth values (Fig. 2B) did not significantly (P  > 0.05) differ within 40 d (58.8, 55.6, and 63.9-fold at day 40, respectively). Over 40 d, NC2 group showed statistically significant lower population growth (15.3-fold at day 40) compared to all experimental groups. No population growth was observed in group HC1.