Idiosyncratic liver pigment alterations of five frog species in response to contrasting land use patterns in the Brazilian Cerrado

Introduction

Human-driven land use changes are causing biodiversity loss around the world (Newbold et al., 2015; Powers & Jetz, 2019). Brazil is one of the countries with the highest proportion of net loss of tree cover in South America, with a loss of 8% from 1982 to 2016 (Song et al., 2018). At the same time, there was a 12% increase in short vegetation cover (Song et al., 2018), which includes shrubs and herbaceous vegetation. This trend was especially significant in the Brazilian Cerrado, of which less of 2% are protected (Beuchle et al., 2015; Françoso et al., 2015; Damasco et al., 2018). Accordingly, more than half of the original 2 million km² of the Cerrado were transformed into planted pastures and annual cultures by 2005 (Klink & Machado, 2005). Central Brazil is a thriving region for industrial agricultural activities (Dias et al., 2016) as one of the largest exporters of soybean and cattle meat in the world (Contini & Martha, 2010; Reynolds et al., 2016). One of the consequences of land use change for export-oriented agricultural activities is the intensive use of agrochemicals (Schiesari et al., 2013; Aranha & Rocha, 2019). Therefore, land use changes, along with agrochemicals, are currently the main threats for biodiversity conservation and the sustainable use of natural resources in this biome (Reynolds et al., 2016).

Water quality in the Cerrado has been drastically affected by the intense use of fertilizers, with a significant increase in nitrogen and pesticides (Hunke et al., 2015). As a result, freshwater habitats receive a great load of contaminants, which impact several aspects of aquatic biodiversity (De Marco et al., 2013; Bichsel et al., 2016). For example, there is evidence that the replacement of natural habitats by urban and agricultural areas decrease not only amphibian populations, but also their genetic diversity (Eterovick et al., 2016). Additionally, land use changes can promote rapid transformation in biological communities beyond species composition. Specifically, it can alter phenotypic aspects of several animal groups (Borges et al., 2019a), which impact how species interact and adapt to a changing environment. These phenotypic changes include DNA damages (Borges et al., 2019b) and developmental abnormalities (Borges et al., 2019a). However, little is known about the effects of contrasting land uses on internal phenotypic aspects of organisms inhabiting Neotropical agroecosystems.

Amphibians are good bioindicators of environmental quality because they show rapid responses to environmental stress (Halliday, 2000; Brodeur & Vera Candioti, 2017). In addition, they have permeable skin and eggs, making them vulnerable to aquatic contamination and infections. As such, these animals are useful for monitoring changes in both the aquatic and terrestrial environments because they depend on the two environments to complete their life cycle (Brodeur & Vera Candioti, 2017). Amphibians are rapidly declining worldwide due to human-induced changes in the environment (Catenazzi, 2015; Alton & Franklin, 2017). Several factors are involved in this decline, including climate change, increased incidence of ultraviolet (UV) radiation due to ozone depletion, invasive species, environmental contamination, diseases, and habitat change or loss (Collins & Crump, 2009; Alton & Franklin, 2017).

Previous studies have analyzed the effects of agrochemicals on tadpole developmental abnormalities (Borges et al., 2019a) or genotoxic effects in adult anurans. However, environmental alterations that promote morphological and physiological changes at the tissue level are poorly understood. The liver plays a key role in the detoxification of xenobiotics (Pérez-Iglesias et al., 2018; Fanali et al., 2018), as well as other functions related to the metabolism (Hinton, Segner & Braunbeck, 2001; Fenoglio et al., 2005). The detoxification is performed by hepatocytes and melanomacrophages (MMs) and may be either enzymatic or not. Melanomacrophage centers and their pigments (melanin, lipofuscin, and hemosiderin) are involved in the hepatic response to various toxic compounds. Thus, the liver is an important organ to evaluate the response of organisms to environmental pollutants (Fenoglio et al., 2005).

Melanomacrophages are cells that occur in the hepatic tissue and produce and store melanin in their cytoplasm. The area and occurrence of MMs in the liver increase in response to environmental stressors, such as temperature (Santos et al., 2014), UV radiation (Franco-Belussi, Sköld & De Oliveira, 2016), and xenobiotics (Çakıcı, 2015; Pérez-Iglesias et al., 2016). In addition to melanin, MMs contain catabolic substances originated from the degradation of red blood cells: hemosiderin and lipofuscin, originated from the degradation of polyunsaturated membrane lipids (De Oliveira & Franco-Belussi, 2012). Therefore, the density of MMs can be a useful morphological biomarker for the effect of environmental stressors (De Oliveira et al., 2017). Morphofunctional changes in the hepatic parenchyma happen as a result of contaminants, suggesting the action of detoxification by MMs due to the processing action of some enzymes, besides the protective action of melanin (Fenoglio et al., 2005). Additionally, the functions of MMs are also related to the maintenance of homeostasis, regulating fibrosis, controlling basophiles, and participating in the recycling of red blood cells (Gutierre et al., 2018).

Changes in the amount of catabolic substances within MMs may be associated with changes in phagocytic activity (Bucke, Vethaak & Lang, 1992; Fenoglio et al., 2005). For example, lipofuscin is produced as a result of the degradation of cellular components. Thus, its increase may hinder cell renewal and promote an accumulation of damaged cellular components in the tissue. The accumulation of lipofuscin can increase cellular sensitiveness to oxidative stress, since this molecule binds to metals, such as iron and copper (Terman & Brunk, 2004). Hemosiderin is an intermediate metabolite generated during the recycling of components in blood production. Thus, the accumulation of hemosiderin indicates a disorder in blood cell recycling (Pérez-Iglesias et al., 2016). In addition, environmental factors that alter the concentration of these substances in the liver may strongly contribute to the decrease in animal health. Consequently, this can affect individual fitness and promote population decline in the long term. Therefore, analyzing liver cell physiology may be useful for detecting the effects of environmental changes by human actions. However, while previous studies (Franco-Belussi, De Lauro Castrucci & De Oliveira, 2013; Santos et al., 2014; Gregorio et al., 2016; Pérez-Iglesias et al., 2016) have analyzed the variation of melanin, lipofuscin, and hemosiderin in response to climatic factors and xenobiotics in laboratory experiments using model species, no study has investigated how these three substances vary at the same time in response to contrasting land use patterns in wild amphibian populations.

Here, we tested the influence of contrasting land use regimes associated with aquatic contaminants on liver cell morphology and physiology of five frog species in the Brazilian Cerrado. These can be hidden effects of changes associated with agricultural intensification that are often neglected in biodiversity assessments that only consider species abundance and occurrence. We expect that frogs collected in ponds embedded in areas with intense agricultural activity will have increased hepatic metabolism because of potential aquatic contaminants, increased solar incidence and temperature. As a result, individuals will have higher amounts of melanin, due to its protective effects against free radicals in tissues, along with higher amounts of hemosiderin and lipofuscin, because these two substances reflect changes in hepatic metabolism.

Materials and Methods

Study area and specimen sampling

Field work was carried out in two regions: three ponds in the surroundings of Rio Verde (17°48′11.28″ S; 50°56′24.95″ W), and two ponds within the Emas National Park (18°15′32.11″ S; 52°53′14.13″ W; Fig. 1), Goiás, central Brazil. The set of ponds were selected in both regions because they had the same species composition, allowing us to compare the effects of contrasting land use in a paired design (our dataset is available at Franco-Belussi et al. (2020)). Ponds in the National Park were selected based on a previous survey (Kopp, Signorelli & Bastos, 2010) that sampled the same ponds and recorded the presence of our target species. Sampling sites in the Rio Verde region were selected because they were inserted into an agricultural matrix and also harbored the same species. Ponds were separated by at least 1.25 km and a maximum of 3.20 km in Rio Verde and by 15.30 km in the National Park. Samplings were conducted during the breeding season between November 2013 and March 2014. Field work consisted of 1 week of sampling in each region each year.

The region around the city of Rio Verde is a thriving agricultural area, mainly covered with planted pastures, and monocultures of soybean, corn, and sorghum. The Emas National Park is an enclave of Cerrado Protected Area with several typical vegetation types of this biome, varying from open formation, such as campo limpo and campo rupestre, to closed-canopy formations, such as Cerradão and Seasonal Deciduous Forest (Valente, 2006). The Instituto Chico Mendes de Conservação da Biodiversidade provided colleting permit (Sisbio #34485-1) and Emas National Park provided housing and authorization for field work.

We used a paired design in which we collected five adult males of the following species in each region: Boana albopunctata, Dendropsophus minutus, Scinax fuscomarginatus, Leptodactylus fuscus, and Physalaemus cuvieri. These species were previously selected because they occurred in both regions (Kopp, Signorelli & Bastos, 2010). Additionally, all of them are widely distributed throughout South America (AmphibiaWeb, 2020) and seem to be generalists, occurring in a wide range of habitats, from open, natural formations, to bush Savannas, to peri-urban areas (Haddad et al., 2013). All species are classified as Least Concern in both the IUCN Red list (IUCN, 2010) and Brazilian National Red list (ICMBio, 2018). B. albopunctata, S. fuscomarginatus, and D. minutus are hylid tree-frogs that call perched on the vegetation, while L. fuscus and P. cuvieri are medium-sized leptodactylids that are found calling and usually foraging on the ground near temporary ponds (Haddad et al., 2013). Both B. albopunctata and D. minutus deposit egg masses directly on water, while L. fuscus and P. cuvieri build foam nests in which they deposit eggs on the margin (in the case of P. cuvieri) or on subterranean chambers (in the case of L. fuscus; Haddad et al., 2013) of water bodies. Thus, adults, eggs, and larvae of these species have different degrees of contact with contaminated water. Consequently, these species can potentially be useful as environmental indicators, since they are highly abundant within their geographic range and seem to respond quickly to environmental disturbance.

Results

The amount of each pigment varies among species (Fig. S1A), but P. cuvieri and B. albopunctata had the highest areas of melanin, while D. minutus and L. fuscus had relatively more lipofuscin. We found both great intra- and interspecific variation in the amount of each substance (Fig. 2). Most species had higher amounts of lipofuscin and melanin than hemosiderin, except B. albopunctata that had lower amounts of lipofuscin than the other species. Interestingly, L. fuscus and D. minutus showed little intraspecific variation in the amount of all three substances, while P. cuvieri and B. albopunctata had high intraspecific variation. The means for the three substances was similar for L. fuscus and D. minutus, whereas the other three species had very distinct means. Interestingly, the relative proportion of the three pigments also varied between sampling regions, because animals from the agricultural area had more melanin and lipofuscin, but less hemosiderin (Fig. S1B). Species whose liver pigments more strongly varied between sampling regions were P. cuvieri, B. albopunctata, and S. fuscomarginatus (Fig. 3).

The total inertia of the dc-CA model was 0.955. The first axis of dc-CA accounted for 72% of the variation in the trait-environment relationship, while the second accounted for 26%. The maximized fourth-corner correlation along the first and second axes are 0.83 and 0.50, respectively. Melanin was positively correlated with natural and planted pastures, but negatively correlated with man-made buildings and agriculture (Table 1). Hemosiderin was negatively correlated natural and planted pastures, and man-made buildings, but positively correlated with agriculture. The correlation pattern of lipofuscin was almost identical to hemosiderin with small changes in the strength of correlation with some land use types (Table 1).

The abundance of P. cuvieri was positively correlated with the percentage of planted pasture in the landscape (Fig. 4). This was also the species with the highest amount of melanin in the liver (Fig. 4), which was positively correlated (fourth-corner correlation = 0.473, Table 1) with the percentage of planted pasture (Fig. 4). In contrast, L. fuscus had the lowest amount of melanin (Fig. 4) whose abundance was negatively correlated with the percentage of planted pasture in the landscape (Fig. 4).

Conversely, the abundance of D. minutus and S. fuscomarginatus were positively correlated with area of agriculture and negatively with anthropogenic area, while the abundance of B. albopunctata was positively correlated with anthropogenic area and negatively with area of agriculture (Fig. 4). D. minutus and S. fuscomarginatus had higher amounts of hemosiderin and lipofuscin, suggesting higher hepatic metabolism, which was positively correlated with agriculture and negatively with percentage of anthropogenic area (Fig. 4). Conversely, B. albopunctata had fewer hepatic catabolite substances (Fig. 4).

Discussion

We found that P. cuvieri occurred in sites with planted pastures and man-made buildings, had higher amounts of melanin, while the abundance of L. fuscus was positively correlated with man-made buildings and negatively correlated with planted pastures. Additionally, D. minutus and S. fuscomarginatus had high amount, while B. albopunctata had low amount of lipofuscin and hemosiderin and its abundance was positively correlated with man-made buildings. Taken together, these results demonstrate the effects of environmental changes on MMs and hepatic metabolism of frog species.

The amount of melanin was highest in P. cuvieri and B. albopunctata, but low in L. fuscus, D. minutus, and S. fuscomarginatus. Despite this interspecific variation, there was a clear pattern of change in melanin within species between sampling regions. There was also a high positive correlation between melanin and planted pastures (0.473), and a less strong correlation with man-made buildings (−0.033), and natural pastures (0.139). Melanin is a complex polymer that absorbs and neutralizes free radicals, besides participating in the innate immune response and protection of tissues in ectotherms (Cesarini, 1996). Changes in the amount of melanin was found in frogs experimentally exposed to several xenobiotics. In these experiments, the variation in melanin was due to several compounds (reviewed in De Oliveira et al. (2017)), such as polycyclic aromatic hydrocarbons (PAHs; Fanali et al., 2018), herbicides (e.g., atrazine and glyphosate; Pérez-Iglesias et al., 2019; Bach et al., 2018), drugs, and endocrine disrupters (Gregorio et al., 2016). Here, we found large amounts of atrazine in the area under agriculture influence (more than 5,000 times the limit accepted by Brazilian legislation, i.e., 5,349.940 µg/L). Atrazine is an endocrine disruptor, which has immunotoxic and immunosuppressive effects even at concentrations usually found in the environment (i.e., <500 µg/L; Rohr & McCoy, 2010). This substance can change swimming ability, cause malformations, and promote nuclear alterations at higher concentrations in tadpoles (Pérez-Iglesias et al., 2019). Atrazine causes oxidative stress in several tissues, which can evolve to function loss. The replacement of natural vegetation by agriculture can increase UV incidence and temperature (Lipinski, Santos & Schuch, 2016). This is an indirect effect of land use change that can affect the amount of internal melanin in frogs.

Although our statistical model explained much variation in the three substances, other climatic factors that have been changing because of human activities, such as temperature and UV radiation can also change the amount of melanin in internal tissues of frogs (Franco-Belussi, Provete & De Oliveira, 2017). Changes in these environmental variables may promote changes in hepatic metabolism, such as increasing glycogen and lipid levels (Mizell, 1965; Barni & Bernocchi, 1991; Fenoglio, Bernocchi & Barni, 1992; Barni et al., 2002). Adaptation to natural conditions (i.e., hibernation) may promote an increase in the amount of melanin in the liver by increasing the number of cells (i.e., hyperplasia) or the increase in cell size (i.e., hypertrophy), besides an increase in the production of melanin in cell interior (Barni et al., 2002). These changes occur as a mechanism of metabolic defense against free radicals. For example, with increased storage of lipids and glycogen in hepatic tissue due to environmental changes that occur naturally in hibernating animals (Mizell, 1965; Barni & Bernocchi, 1991; Fenoglio, Bernocchi & Barni, 1992; Barni et al., 2002). Thus, the liver of anurans is a plastic organ, besides being sensitive to the alterations of the natural biological rhythms (i.e., reproduction and hibernation), coordinating these mechanisms to maintain the homeostasis of the organism during adaptation to environmental changes (Barni et al., 2002). Therefore, cells that produce and store melanin seem to be involved in the adaptation to environmental stressors at the tissue level.

Dendropsophus minutus and S. fuscomarginatus had high amounts, whereas B. albopunctata had low amounts of hemosiderin and lipofuscin. Lipofuscin and hemosiderin are products of cellular catabolism and can be used to measure hepatic metabolism, since both substances may be altered as a result of environmental stress following habitat alteration (Santos et al., 2014; Pérez-Iglesias et al., 2016). Low amounts of these substances indicate decreased phagocytic activity of cells (Bucke, Vethaak & Lang, 1992; Fenoglio et al., 2005), while accumulation of hemosiderin is related to increased turnover of blood cells (Fenoglio et al., 2005). Therefore, the increase in recycling of blood cells in D. minutus and S. fuscomarginatus indicates that biotic or abiotic factors resulting from anthropic changes may be causing changes in the liver of these two species, but less so in B. albopunctata.

The idiosyncratic responses of species may be related to differences in life history traits. Internal melanin varies among species and organs in anurans, possibly due to its adaptive function conferred by the protective functions of the pigment (Provete et al., 2012; Franco-Belussi, Provete & De Oliveira, 2017). For example, Physalaemus and Leptodactylus are terrestrial and can have more contact with xenobiotics; while Boana, Scinax, and Dendropsophus are arboreal and putatively less exposed to aquatic xenobiotics (Silva et al., 2013). However, it is noteworthy that any drastic change in land use appears to promote metabolic alterations related to liver physiological processes in anurans. Therefore, MMCs seem to be efficient biomarkers indicating alterations in the liver in response to contrasting land use types. Actions for mitigating the negative effects of industrial agricultural activities must be taken if we want to reach the goal of making environmentally responsible agricultural products (ONU 2050 goals), especially in grassy biomes (Parr et al., 2014; Overbeck et al., 2015).

Conclusion

Our a priori hypothesis was that frogs from the area with intense agricultural activity would have higher amounts of melanin, hemosiderin, and lipofuscin, because of the increase in hepatic metabolism necessary to deal with potential contaminants and higher solar incidence promoted by loss of vegetation cover. We found that the amount of melanin, hemosiderin, and lipofuscin indeed varied between regions, but each species seemed to respond differently to these contrasting lands use types. The species whose liver metabolism most changed across different land use type were P. cuvieri, B. albopunctata, and S. fuscomarginatus. Therefore, these species should be used for evaluating environmental alterations. Aquatic contaminants may alter organismal health that cannot be assessed by only recording species presence in each environment, since the internal morphology of individuals can be damaged. Alterations in hepatic metabolism can compromise population viability and species persistence in the environment in the long term. Our results reinforce the need to include multiple biological aspects of species (e.g., morphology, physiology) in environmental monitoring programs.

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