Nematodes have adapted to live in different ecosystems (marine, freshwater, and terrestrial environments) and hosts (as parasites), with a cosmopolitan scattered pattern, inhabiting tropical, temperate and sub-Arctic soils, playing important roles in biogeochemistry (van den Hoogen et al., 2019). Importantly, many of these organisms are plant-parasitic entities that lead to drastic impacts on crops worldwide; thus, resulting in global annual losses around $125 billion (Barker et al., 1994; Crow et al., 2000; Chitwood, 2003). Some examples are the soybean-parasitic nematodes
In nature, some organisms exhibit a singular survival strategy to droughts known as anhydrobiosis (life without water) − a very stable state of suspended animation into which some species are able to enter when subjected to desiccation, which induces substantial water loss, resulting in a cellular water content of less than 0.1 g g−1 dry mass and metabolism ceases (Tunnacliffe and Lapinski, 2003). This phenomenon is reported in bacteria, fungi, plants, and animals (Tunnacliffe and Lapinski, 2003), including some nematodes species, such as
Notoriously, in the dry state, anhydrobiotic organisms exhibit polyextremotolerance, i.e. tolerance to harsh conditions for life, such as high and low temperatures (from −273°C to +151°C) (Rahm, 1923; Becquerel, 1950); ionizing radiation (Keilin, 1959); high hydrostatic pressures (Seki and Toyoshima, 1998); vacuum (Rebecchi et al., 2009); and hyperacceleration (Souza and Pereira, 2018). Biological activity is fully restored after rehydration. Anhydrobiosis is an important phenomenon in nematodes since it may allow the long-term survival of plant-parasitic and/or of free-living nematodes species during droughts, which may tend to be more intense and frequent in the light of climate change.
An unresolved question is whether, in fact, metabolism is completely interrupted during anhydrobiosis (Wharton, 2015). In order to answer this question, Barrett (1982) fed nematodes with radiolabeled glucose and searched for metabolic products during the period of desiccation. Similarly, Örstan (1998) stored desiccated bdelloid rotifers inside argon gas chambers and reported their survival even after several periods in the absence of oxygen – necessary for metabolism. Lastly, computer-based data revealed that the remaining water contents in desiccated colonies of
Gallium (Ga) is a soft metal categorized as a post-transition metal, sharing some physicochemical characteristics with Aluminium, Indium, and Thalium. In particular, Ga has low melting point (29.76°C) (Jefferson Lab, 2003) and is slowly oxidized (Moskalyk, 2003), when compared to other metals. Previously, we have shown that desiccated
Therefore, in the present study, our objective was to investigate the tolerance of desiccated (in anhydrobiosis) and hydrated (living)
Mixed populations of
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 (
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.
Gallium confinements. A: Gallium metal-containing test tube caps, B: metallic confinement (gray) scheme showing the membranes (white) with nematodes.
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 (
Two membranes per treatment (
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 (
Desiccated
Likewise, NC1, GT1, and GT2 population growth values (Fig. 2B) did not significantly (
Under hydrated settings (i.e. group HC1), we show that the
On the other hand, the NC1 group remained desiccated in a Ga-free dry condition (10% RH) and kept in suspended animation ensuring a high survival rate, which is expected since
Therefore, our results show for the first time, the high tolerance of desiccated
Since Ga-based semiconductors production has attracted attention of the microelectronics industry (Flora, 2000), an increase in Ga concentration in soil has been evidenced (Angelone and Bini, 1992; Kabata-Pendias and Pendias, 1984) and possibly correlated with its increasing global consumption (Jaskula, 2018), which may result in toxic levels of Ga in terrestrial and aquatic environments, threatening the local biodiversity. Therefore, we should encourage studies related to Ga toxicity and biological tolerance to heavy metals.
Although Ga toxicity has not been widely explored, its acute and chronic lethal concentrations (LC50) have already been investigated in a few prokaryotic and eukaryotic organisms (Bireg et al., 1980; Lin and Hwang, 1998; Onikura et al., 2005; García-Contreras et al., 2014). Here, we show that more than a half of the desiccated
Heavy metal tolerance can be observed in different nematodes species. Metals may stimulate sensorial apparatus (e.g. metal-ion receptors) (Sambongi et al., 1999) which, subsequently, trigger physiological responses (e.g. pharyngeal pumping ceasing) (Jones and Candido, 1999). In addition, as reported here, this tolerance can be strongly enhanced by anhydrobiosis. More importantly, some plant-parasitic nematodes, such as
Anhydrobiosis is a singular biological state promoted by desiccation, which renders some organisms tolerant to diverse physicochemical stresses. This phenomenon is especially important in the current scenario of a global warming threat, when droughts tend to occur more frequently. Therefore, understanding the process of anhydrobiosis in nematodes is crucial since several species play important roles in soil and aquatic substrate chemistry, and others impact economically important crops.