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WHERE I WORK
13 October 2020

Biologist Cristina Cuiabália Neves and her team are dedicated to maintaining a nature reserve that is home to many endangered and threatened species.

Patricia Maia Noronha

Patricia Maia Noronha is a freelance writer in Lisbon, Portugal.

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Cristina Cuiabália Neves is a biologist and manager at Sesc Pantanal in Mato Grosso, Brazil. Credit: Maria Magdalena Arréllaga for Nature

Fires are the biggest challenge between June and October in Sesc Pantanal, a privately owned natural heritage reserve of 108,000 hectares in the state of Mato Grosso, Brazil. This year, in the first three months of the dry season, more than 50% of the land was damaged by flames, and the drier areas were burnt to the ground. It was the worst scenario in 20 years.
This picture was taken in August. I’m on top of a tanker truck leading the team of firefighters that work with me and the rest of the park-management staff during this season. We monitor more than 1,000 square kilometres of land, lakes, bays and rivers in this reserve, so we use motorcycles, tractors, boats and an aeroplane.
Around 700 species of animal live here. Among them are 12 endangered species, including the jaguar, the marsh deer and the giant anteater. Many are injured or killed by the fires. This year, even a jaguar, which is usually fast enough to escape, was burnt. It is now recovering at our facilities.
I was born in the city of Cuiabá, the state capital. My mother taught geography there at the Federal University of Mato Grosso, and I used to go with her on field trips to Pantanal, a vast area of wetlands and grasslands that contains the nature reserve. I see it as a member of my family. When I did my PhD at the University of São Paulo in 2014, I knew I wanted to study how to control the main threats to Pantanal biodiversity: fishing, hunting, drug trafficking and fires.
Since its creation as a reserve in 1998, Sesc Pantanal has supported 65 research projects that help us to understand how we can nurture the fauna and flora that live there. Conservation has always been our main goal, but now we are also educating the community and our visitors. We know that most of our fires are started outside the reserve, to clear space for pastures. In Brazil, where there is much agriculture and cattle farming, we need to find sustainable ways to reduce the pressure on the land — and to make it less vulnerable to fires.

Nature 586, 466 (2020)
doi: 10.1038/d41586-020-02859-4

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Soil status, plant development and root architecture
Measurements of the gravimetric soil water content of the subsoils in both variants of the soil rhizotrons showed clear differences depending on the irrigation scenario: For the variants with top-irrigation, the water availability in the topsoil corresponded to a pF value of 2.0 at the beginning of the experiment, while for the variants with sub-irrigation the pF value was 2.2 (Fig. 2).
Figure 2

Changes of (a) pF Values and (b) gravimetric soil moisture contents in dependency of the specific bulk density (topsoil 1.1 g cm3; subsoil 1.4 g cm) plotted over time for the two soil rhizotron trials (grey = sub-irrigation; black = top-irrigation).

Full size image

The initial pF value of the subsoils was approximately 2.1 in both variants. Irrigation affected the time course of pF values: It remained within the range of the field capacity (pF 2.1–2.2 at day 44) in the irrigated top- and subsoils, respectively (Fig. 2a). The other, non-irrigated complementary soil layers dried out and the pF values increased to 2.8–2.9 from approximately day 20 onwards. Changes in gravimetric water content reflected these scenarios: gravimetric soil moisture remained at 5% in the irrigated topsoil but dropped to 2% (the matric potential declined by − 53 kPa) in the variants with subsoil irrigation. Also, the irrigation of the subsoil almost maintained a constant water content (the matric potential declined by − 5 kPa, only), while the subsoil dried out upon top-irrigation (the matric potential declined by − 61 kPa). Consequently, our setup allowed a comparison of plant growth and related P acquisition from soil with either sufficient water supply in top- or subsoil, respectively.
The 10 cm thick layer of topsoil, which was implemented in all rhizotron types, supported similar developments of wheat plants in all rhizotrons. Progressing plant developmental changes in both the aboveground plant parts and the root architecture were observed once the sand was accessed by roots: Since then, plant growth was significantly reduced in the sandy rhizotrons compared with those filled with soil as illustrated by the measured plant parameters after 44 days (Table 1, quantitatively evaluated only for the end of the experiment).
Table 1 Characteristics of plants, 33P uptake and water inputs due to the different forms of irrigation from different rhizotron trials (n = 3) after 44 days; different letters indicate significant differences among different rhizotron trials (p  More

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The evaluation of T. qataranse growth parameters suggests that Pb has no adverse effect on the plant at concentrations of less than 100 mg/L Pb. However, at 100 mg/L, the metal disturbed healthy growth and, in particular, interfered with root development. Consistent with our findings, a similar study using Z. fabago reported that Pb negatively affects root development14. The root plays a vital role in plant health and development, influencing other tissues’ response to stress conditions. Despite it being one of the most critical parameters in the assessment of plant health, a significant reduction in total chlorophyll content was observed (Fig. 1c). However, Pb toxicity symptoms (e.g., leaf chlorosis and root darkening) were not apparent across any of the treatments. Typically, Pb accumulation in plants raises the level of chlorophyllase, an enzyme that negatively affects chlorophyll. An increased level of chlorophyllase slows down photosynthesis and, therefore, affects overall growth and development. Consequently, due to slow metabolic activities, cell division is adversely affected and healthy growth is inhibited15.
Pb accumulates differently in plant tissue parts, especially in the root22. Concerning T. qataranse Pb accumulation, overall data across all treatments indicates that T. qataranse preferentially concentrates Pb in the root (up to 2,784 mg/kg). Our result is consistent with the reports of many similar studies. For instance, known plant species, including Nerium oleander L. and Brassica juncea, accumulate higher Pb concentrations in their roots than other tissue parts16. Also, in a study involving different plants, Finster, et al.17 determined that the roots always accumulate more Pb than other plant parts, including the fruits, where only traces of the metal translocate the shoot. Our result is also in agreement with the work of Langley-Turnbaugh and Belanger18. Kumar, et al.19 and Pourrut, et al.20 conducted several critical reviews of Pb toxicity in plants and determined that several factors contribute to restricted metal translocation in plants. Of such, Casparian strip endodermis restriction is by far the most limiting for Pb. Notheless, the ability of T. qataranse to accumulate more than 1000 mg/kg Pb suggests that it is a Pb hyperaccumulator21.
Additionally, the root BCF across all treatments was higher than that of the shoot (Fig. 2b). The BCF indicates that, to some degree, T. qataranse sequestrate Pb from growth medium that contains up to 1600 mg/kg Pb. However, it was optimal in the 50 mg/kg treatment. At this concentration, the growth medium had up to 800 mg/kg Pb. The TF under all treatments was less than 1 (Fig. 2c), meaning that T. qataranse can not sufficiently transfer Pb to its aerial parts. In the current study, the restriction of Pb translocation finding is consistent with our previous report on T. qataranse where field samples were analyzed for various metals accumulation, including Pb22. Some of the factors that affect metal bioavailability and uptake include plant and metal types; metal form, concentration, and age in the soil; pH; and organic matter content. However, pH and total organic matter content are the most critical in terms of metal bioavailability and uptake of Pb. The pH significantly affects the behavior of Pb by dictating its chemical form. Metals, including Pb, are more soluble at low or near-neutral pH values. At pH  > 8, metals tend to precipitate in the soil. Similarly, a high TOC limits the bioavailability of Pb11. In this work, the pH and TOC in the growth medium were 7.35 and 1.87%, respectively. Therefore, given the neutral pH and low TOC, their effects on Pb bioavailability and uptake by T. qataranse was insignificant and can be eliminated.
Various response mechanisms enable plants to withstand metal toxicity, of such, metal avoidance and uptake are the most common. Before compartmentalization, Pb is translocated to a degree that can be described by the TF23. Pb mainly precipitates on the root cell wall and only the free ions are transported to other parts via the xylem and phloem cells24. Previous works confirmed that Pb disrupts cellular homeostasis by replacing essential cations and altering metal-containing enzyme activity. In plants, the primary sources of ROS are chloroplasts, mitochondria, and peroxisomes. Pb toxicity interferes with electron transport chains in turn increasing ROS accumulation. Nearly every stage of the central dogma of plants (DNA, RNA, protein) is affected by Pb toxicity25.
The antioxidant system is one mechanism used by plants for protection against metal toxicity. In this study, the result of the SOD, CAT, APX, GPX, and GR assay show increased activity of all five enzymes. SOD activity was the highest, up to ten times higher than the control (0 mg/kg Pb), particularly in the root (Fig. 3a), suggesting the critical role of SOD in T. qataranse antioxidative defense. Having the highest enzymatic activity be in the root changes root organic constituents due to Pb complexation. This indicates that, as suggested in our previous work, as an uptake mechanism, Pb ions bind to T. qataranse root by complexation through cationic exchange with hydroxyl and carboxyl functional groups7. This is a well-established complexation mechanism of transition metals, including Pb1. In comparison, GR demonstrated the least activity (Fig. 3e). Such differences can be attributed to the specific roles each enzyme has in ameliorating Pb stress. Many other studies reported a similar increase in the activities of one or all the enzymes analyzed in this work following plants’ exposure to Pb; examples are increases in the activity of CAT, SOD, APX, and GPX in Ceratophyllum demersum L.26 and the cotton plant27; SOD, APX, GPX, and GR in Oryza sativa L.28; and APX, CAT, and GR in Triticum aestivum9. Changes in enzymatic activity account for the elimination of ROS and the improvement of stress conditions in plants. Therefore, the enhanced activities of all enzymes suggest that their role is crucial in ameliorating Pb toxicity in T. qataranse. Other studies support this conclusion, including Ferrer, et al.16 who attribute enhanced CAT and APX activities to the efficient ROS scavenging capability of Z. fabago exposed to Pb. In addition, Nikalje and Suprasanna30 reviewed several other similar studies involving halophytes . However, to the best of our knowledge, our work is the first on T. qataranse. In addition, primarily due to GR activity suggesting the utilization of glutathione, we can conclude that Pb detoxification in T. qataranse may partly involve glutathione metabolism30. Glutathione, which exists in either the reduced (GSH) or oxidized form (GSSG), acts as an antioxidant and chelating bioligand majorly accountable for metals detoxification. Enzymes involved in glutathione metabolism mediates detoxification Glutathione-S-transferases (GSTs) are a major phase II GSH-dependent ROS scavenging enzymes. They play significant roles in GSH conjugation with exogenous and endogenous species found during oxidative stress, including H2O2 and lipid peroxides9,10. Consistent with our findings, a more recent review by Kumar and Prasad14 discussed several other studies, some of which include the use of model species, Arabidopsis thaliana, and Oryza sativa, all of which support our findings.
It is worth mentioning that part of the discussion presented in this work is limited to the perspective of deciphering Pb tolerance and uptake mechanisms from metal translocation and plant antioxidative systems. However, both molecular and biochemical mechanisms play significant roles in toxic metals detoxification, including Pb. For instance, glutathione metabolism is known to regulates the biosynthesis of phytochelatins (PC), which bind Pb and transports it to vacuoles where detoxification can occur. Additionally, genes, such as glutamate cysteine ligae 1 (GSH1), glutamate cysteine ligae 1 (GSH2), phytochelatins synthase 1 (PCS1), and phytochelatins synthase 2 (PCS2), are actively involved in GSH-dependent PCs synthesis. Other primary and secondary metabolites that act as antioxidants, such as Tocols, flavonoids, anthocyanidins, and ascorbic acid, are essential to protecting plants against oxidative damage as well. The functions of these metabolites are well documented8,9,20. Further, we recognize that proteins regulate ROS signaling and the expression of such proteins changes due to Pb exposure11. Due to metal stress in plants, increased protein synthesis is essential in cellular metabolic processes. Mitogen-activated protein (MAP) kinase pathways regulate such processes, serving as a signaling system against oxidative stress. Signaling occurs through multiple stages of the reaction, modifying gene expression and, ultimately, protein synthesis4. Therefore, our future work will focus on the differential expression of proteins, particularly those that are related to stress responses, such as the heat shock protein family, due to Pb exposure in T. qataranse. More

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In the present work, we performed quantitative metabolomic analysis for eleven biological tissues of S. lucioperca. The advantage of the quantitative approach over commonly used semi-quantitative measurements is that the obtained data on the metabolite concentrations expressed in nmoles per gram of a tissue can be directly used by any researcher as a reference to the baseline level of metabolites in that tissue. Quantitative data also allow for the comparing the tissues with very dissimilar metabolomic compositions.
The metabolomic analysis performed in the present work demonstrates that although the majority of metabolites are common for all tissues, their concentrations in tissues may vary at a large scale. Moreover, there are some tissue-specific compounds with very high abundance in only 1–2 types of tissue. The examples of such metabolites are glycine, histidine, creatine, and betaine in muscle, ovothiol A in lens, NAA in lens and brain, glucose in liver. Apparently, these compounds are important for biological functions specific for these particular tissues.
Two groups of metabolites, osmolytes and antioxidants, play the key role in the cell protection against osmotic and oxidative stresses. In this work, the following compounds were conventionally assigned to osmolytes: taurine, myo-inositol, NAH, NAA, betaine, threonine-phosphoethanolamine (Thr-PETA), and Ser-PETA. Obviously, this assignment is rather arbitrary: some of these compounds, besides osmotic protection, perform other cellular functions, including cell signaling, providing substrate for biosynthesis, and so on17,18,19. At the same time, the tissues under study contain metabolites with concentrations of the same level or even higher than the concentrations of compounds assigned to osmolytes: lactate, glucose, acetate, creatine. These metabolites are mostly related to the reactions of cellular energy generation, and their concentrations should strongly depend on the fish activity. For that reason, in this work we did not include them into the list of osmolytes. Figure 6 shows the concentrations of osmolytes in different fish tissues (excluding acellular tissues AH and VH), and demonstrates that the composition of osmolytes in tissues strongly depends on the cell type.
Figure 6

Concentrations of major osmolytes (in µmol/g) in S. lucioperca tissues.

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We found in the fish tissues the following compounds with antioxidative properties: glutathione (GSH), ascorbate, OSH, and NADH. The detection of minor amounts of one more well-known thiol antioxidant, ergothioneine, in the gills of another freshwater fish, R. rutilus lacustris, has recently been reported15; however, in the present work ergothioneine was not found neither by NMR nor by LC–MS in any of the studied tissues of S. lucioperca. NADH was found only in NMR spectra of liver, muscle, heart, and lens, and its concentration in these tissues does not exceed 15 nmol/g. GSH, OSH, and ascorbate are present in much higher concentrations in the majority of the fish tissues, so these three compounds play the main role in the cellular defense against the oxidative stress. The presence and the relative abundance of GSH and OSH in the fish tissues were confirmed by LC–MS data.
The metabolomic features of particular fish tissues are discussed below.
AH and VH
AH and VH are acellular fluids with minimal metabolic activity. AH is produced in the ciliary epithelium through both the active secretion and the passive diffusion/ultrafiltration of blood plasma20,21,22,23. Consequently, the metabolomic composition of AH is similar to that of plasma10. VH is also connected with blood via the hematoophthalmic barrier24 and with AH, and one can see (Table 1, Fig. 2) that the metabolomic compositions of AH and VH are close to each other. Thus, it is safe to assume that the levels of metabolites in AH and VH reflect their levels in blood plasma, which circulates through the majority of fish tissues. Significant deviations of metabolite levels in tissue as compared to plasma should be attributed to the intracellular metabolic activity specific for this particular tissue.
Lens
The eye lens is one of the most anatomically isolated tissues. The lens mostly consists of metabolically inert fiber cells without nuclei and organelles with the exception of metabolically active epithelial monolayer. The data present in Table 1 indicate that the lens contains very high levels of proteinogenic amino acids: for some amino acids (for example, branched-chain amino acids, glutamine, aspartate) their levels in the lens are more than ten-fold higher than that in AH. Moreover, the concentrations of the majority of amino acids in the lens are higher than in any other fish tissue. The elevated levels of amino acids in the lens has been noticed many years ago25, and it was attributed to the active amino acid transport from AH to lens26,27,28,29. These amino acids are presumably needed to synthesize high protein content (up to 40% of the total lens weight), which is in turn needed to provide high refraction coefficient.
The fish lens contains a unique set of osmolytes and antioxidants. The lens osmolytes are myo-inositol, NAH, NAA, Thr-PETA, and Ser-PETA. We have previously shown15 that the concentrations of osmolytes in the fish lens undergo significant seasonal variations. At the late winter time, when the fish was caught for this study, the most abundant lens osmolyte is myo-inositol. High concentrations of this compound are also found in other fish tissues, including brain, gill, and spleen. Thr-PETA and Ser-PETA are also among the most abundant metabolites in the majority of the fish tissues. In opposite, NAH and NAA are present in high concentrations only in the fish lens and brain. At the same time, the concentration of taurine, which is the most abundant osmolyte in all other fish tissues, in the lens is rather low.
The major antioxidant of the fish lens is OSH12. It has been shown that the level of OSH in S. lucioperca lens vary from 3 µmol/g at autumn to 1.5 µmol/g at winter15, which is in a good agreement with our present data (Table 1). The concentration of the second most abundant lens antioxidant, GSH, is 3–4 times lower than that of OSH. Taking into account the properties of OSH30,31,32,33, it has been proposed12,34 that OSH is a primary protector against the oxidative stress, while the main function of GSH in the lens is the maintenance of OSH in the reduced state. It should be noticed that although OSH was also found in other fish tissues (Table 1, Fig. 2), its concentration in these tissues is significantly lower than in the lens. Therefore, in respect to fish, OSH can truly be called “lenticular antioxidant”.
High concentrations of amino acids, osmolytes, antioxidants and some other compounds in the lens indicate that these metabolites are either synthesized in metabolically active epithelial cells, or pumped into the lens from AH against the concentration gradient with the use of specific transporters also located in the epithelial layer. The fiber cells of the lens are metabolically passive, and fresh metabolites can appear in these cells only due to the diffusion from the epithelial layer toward the lens center. Therefore, one can expect that the concentrations of the most important metabolites decrease from the lens cortex toward the lens nucleus. To check this assumption, we measured the metabolomic profiles for cortex and nucleus separately. The measurements were performed for three lenses; then the ratios of the metabolite concentrations in the cortex to that in the nucleus were calculated and averaged. The results of the calculations are shown in Fig. 7 (only for metabolites with the highest and the lowest cortex/nucleus ratios) and Supplementary Table S2 (for all metabolites). Indeed, the levels of the majority of metabolites in the lens nucleus are significantly lower than in the cortex. For five metabolites, namely ATP, NAA, inosinate, ADP, and GSH the difference exceeds the factor of thirty; that means that these compounds are almost completely depleted during their diffusion toward the lens nucleus.
Figure 7

Barplot for statistically significant differences in the metabolomic content of lens cortex and nucleus. Bars show the averaged ratio of metabolite concentrations in the cortex to that in the nucleus of the S. lucioperca lens.

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Brain
The brain tissue similarly to the lens is isolated from the vascular system by means of the hematoencephalic barrier. However, in opposite to the lens, brain is very metabolically active tissue, as in particular indicated by the high level of lactate (14 µmol/g). Similar lactate concentrations were found only in muscle and heart (Table 1). Besides lactate, the most abundant metabolites of the fish brain are osmolytes myo-inositol, Ser-PETA, taurine, Thr-PETA, NAH, and NAA; the concentrations of these compounds in brain are in the range from 2.5 to 13 µmol/g (Table 1). The brain tissue also contains high levels of glutamate and creatine, which are used by brain cells for the cellular energy generation. The level of antioxidant ascorbate in brain (400 nmol/g) is significantly higher than in other fish tissues, which indicates the importance of ascorbate for the brain correct operation. Besides ascorbate, the brain tissue also contains OSH and GSH, but at significantly lower concentrations (100–200 nmol/g).
Blood-rich organs: liver, spleen, milt, muscle, heart, gill, kidney
Figure 4 demonstrates that from the metabolomic viewpoint, gill, kidney, milt, and spleen are the most similar tissues. However, the quantitative analysis indicates significant differences. In particular, spleen does not contain measurable by NMR amounts of antioxidants OSH and GSH. The levels of osmolytes are also different: the concentration of taurine in spleen is threefold higher than in gill and milt, while the level of myo-inositol in milt is much lower than in spleen and gill (Fig. 6). Significant differences are also found for some amino acids (alanine, creatine), organic acids (lactate, GABA), and nucleosides (ATP, ADP, AMP, inosine).
One of the important liver functions is the maintaining the glucose level in blood regulated by producing glucose from stored glycogen. Correspondingly, the level of glucose in liver is extremely high (40 µmol/g), which is higher than in any other tissue by at least an order of magnitude. Liver also contains elevated (as compared to other tissues) concentrations of threonine, glutamate, succinate, fumarate, AMP, and nicotinamide.
The biological functions of muscle and heart are relatively similar; however, the metabolomic compositions of these tissues differ significantly. The main osmolyte in muscle cells is taurine (30 µmol/g), while in heart the osmotic protection is shared between taurine (15 µmol/g) and Ser-PETA (12 µmol/g). Muscle contains very high levels of glycine and histidine. Glycine is known to protect muscles from wasting under various wasting conditions35,36, while histidine and histidine-related compounds were reported to play the role of intracellular proton buffering constituents in vertebrate muscle37. Very likely that both glycine and histidine also participate in the osmotic protection of the muscle cells. The level of creatine—the energy source—in muscle (23 µmol/g) is five-fold higher than in heart. More