Universidade Federal de Santa Maria

Ci. e Nat., Santa Maria v.42, e55, 2020


ISSN 2179-460X

Received 21/09/19  Accepted: 30/03/20  Published:13/08/20






Influence of seasonality on protein and carbohydrate accumulation in the liver of Leptodactylus macrosternum (ANURA: Leptodactylidae)


José Lucas de AraújoI

 Márcio Frazão ChavesII

José Jailson Lima BezerraIII

Jakeline Moreira da SilvaIV

Geraldo Jorge Barbosa de MouraV



IUniversidade Federal Rural de Pernambuco. Programa de Pós-graduação em Melhoramento Genético de Plantas. Recife-PE, Brasil.

IIUniversidade Federal de Campina Grande. Cuité-PB, Brasil.

IIIUniversidade Federal de Pernambuco. Programa de Pós-graduação em Biologia Vegetal. Recife-PE, Brasil.

IVUniversidade Federal Rural de Pernambuco. Programa de Pós-graduação em Melhoramento Genético de Plantas. Recife-PE, Brasil.

VUniversidade Federal Rural de Pernambuco. Programa de Pós-graduação em Ciência Animal Tropical-UFRPE. Programa de Pós-graduação em Ciência Ecologia-UFRPE. Programa de Pós-graduação em Ciências Ambientais-UFRPE. Programa de Pós-graduação em Planejamento Ambiental-UCSAL. Recife-PE, Brasil.




Leptodactylus macrosternum is considered an environmental bioindicator, providing information on possible disturbances and conservation status of ecosystems. Therefore, the present study quantifies proteins and carbohydrates present in the liver cells of this species according to seasonal variations. Thirty-two (32) adult males were collected in a Caatinga area, at Horto Florestal Olho D’água da Bica, Cuité city, Paraíba State, northeastern Brazil, between January and December 2013. Small liver fragments were removed from the animals, weighed, measured, and sent to histological analysis. Carbohydrates were stained with the PAS reaction and proteins were stained with xylidine. The proteins and carbohydrates deposited in the livers were statistically quantified by the Kruskal-Wallis test, followed by the Dunn test when necessary. Protein values were highlighted for animals collected during the months of August (140985.9 ± 35395.4 pixels) and December (142731.8 ± 31865.7248). In turn, the amount of carbohydrates in liver fragments did not differ significantly during the sampled months, remaining just above 150000 pixels. Notwithstanding, this variable differed significantly for rainfall and temperature, r = 0.01 / p = 0.03 and r = 0.02 / p = 0.04, respectively, suggesting that L. macrosternum acts as an important bioindicator of seasonal variations.

Keywords: Caatinga. Histochemistry. Anurans.



Leptodactylus macrosternum é considerado bioindicador ambiental, fornecendo informações sobre possíveis perturbações e status de conservação dos ecossistemas. Mediante o exposto, objetivou-se quantificar as proteínas e carboidratos presentes nas células do fígado desta espécie de acordo com as variações estacionais. Foram coletados 32 machos adultos em área de Caatinga, no Horto Florestal Olho D’água da Bica, cidade de Cuité, estado da Paraíba, nordeste do Brasil entre os meses de janeiro a dezembro de 2013. Pequenos fragmentos de fígado dos animais foram removidos, pesados, medidos e encaminhados à rotina histológica. Os carboidratos foram corados com reação de PAS e as proteínas foram coradas com xilidina. A quantificação das proteínas e dos carboidratos depositadas nos fígados dos animais foram estatisticamente analisadas pelo teste Kruskal-Wallis e quando necessário completado pelo Teste de Dunn. Os valores referentes a as proteínas tiveram destaque para os animais coletados durante os meses de agosto (140985, 9 ± 35395, 4 pixels) e dezembro (142731.8 ± 31865.7248). Enquanto que a quantidade de carboidratos nos fragmentos de fígado não apresentaram diferenças significativas durante os meses amostrados, permanecendo pouco acima de 150000 pixels. Em relação a pluviosidade e temperatura foram observadas diferenças significativas, r = 0,01/p = 0,03 e r = 0,02/p = 0,04 respectivamente, sobre a quantidades de carboidratos depositados, sugerindo que L. macrosternum atua como um importante bioindicador de variações estacionais.

Palavras-chave: Caatinga. Histoquímica. Anuros.




There are 6,771 known species of amphibians in the world, of which 5,858 belong to the order Anura (FROST, 2010). Anurans have intrinsic adaptive characteristics such as elongated hind limbs and fused vertebrae adapted for jumping. They play an important ecological role in the control of insects and other invertebrates, which are the main members of their diet. Moreover, they are one of the groups most sensitive to environmental variation, being thus widely used as environmental biomarkers (ETEROVICK; SAZIMA, 2004) .

Anurans of the family Leptodactylidae are the most threatened. Among the causes of their decline is the reduction of habitats and climate change (STUART et al., 2004). According to Li et al. (2013), humidity and temperature are some of the factors responsible for climate change, being extremely important for anuran survival. Therefore, assessing anuran tolerance to climatic extremes seems to be a fundamental issue for their conservation (NAVAS et al., 2007).

In this sense, knowledge of energy patterns, different factors, and its relationship with the biology of these animals provide information that can complement the understanding of the structuring principles of communities in their ecosystems (BIONDA et al., 2011). Based on the dynamics of populations and communities, these principles concern the distribution of groups of species, their internal and environmental relationships. Thus, they are a primary and essential tool for more applied studies, aimed at the survey, conservation, and management of biodiversity (HOCKEY; CURTIS, 2009).

In Caatinga areas, the rainfall distribution pattern determines the activity of anurans. During drought, the anuran species that occur in this region adapt physiologically and behaviorally to thermal variations and food and water shortages in temporary puddles (PRADO et al., 2000; BERTOLUCI; RODRIGUES, 2002). In this perspective, studies on the history, ecology, and evolution of amphibians in these areas are extremely necessary to help understand the species distribution patterns as well as their conservation in this ecosystem (RODRIGUES, 2003).

Considering their vulnerability to anthropogenic factors and their high microenvironmental specificity, several species of the family Leptodactylidae have an important role in many habitats (whether terrestrial or aquatic, in tropical, subtropical, and temperate regions), participating in maintenance and control of other species (VITT; CALDWELL, 2001). An example is Leptodactylus macrosternum (MIRANDA-RIBEIRO, 1926), considered an environmental bioindicator, which provides information on possible disturbances and conservation of ecosystems (HEYER et al., 2000).

It is noteworthy that during drought conditions, the anuran species L. macrosternum is one of those which adapt to variations and shortages in temporary puddles, as mentioned above (SUN; NARINS, 2005; AMÉZQUITA et al., 2011). Quantification of key elements such as carbohydrate and protein in the liver can provide important information about the reproductive and/or nutritional status of individuals in a population (GUIMARÃES, 2010). Different environmental factors regulate the daily and seasonal activities of anurans, such as rainfall, relative humidity, temperature, and photoperiod (HATANO et al., 2002; BOQUIMPANI-FREITAS et al., 2002; XIMENEZ; TOZETTI, 2015; BOTH et al., 2008; VAN SLUYS et al., 2012; TOZETTI et al., 2010). For example, low air temperature directly affects ectothermic animals due to reduced metabolic activity, which may decrease vocalization or foraging activities (OLIVEIRA et al., 2013).

It is known that histochemical studies of anurans are scarce, as well as the determination of proteins and carbohydrates present in the liver of some species, which may indicate how they adapt to the varied ecosystems and seasonal fluctuations (VIEIRA et al., 2007). Given the above, the present study quantifies the proteins and carbohydrates present in the liver of L. macrosternum, allowing a better understanding of the metabolic functions of the individuals analyzed. In addition, we sought to associate seasonal variation with protein and carbohydrate accumulation in the liver cells of animals collected in the mesoregion of Agreste Paraibano, Brazil.



2.1 Study Area

Specimens of Leptodactylus macrosternum were collected in the Horto Florestal Olho D’água da Bica (HFODB), in Cuité city, Paraíba State (Figure 1). The site is located in the mesoregion of Agreste Paraibano, in the microregion of Western Curimataú (6°29'06" S / 36°9'24" W), at an altitude of 667 meters above sea level, comprising a total area of ​​758.6 km². The climate is hot and dry, with temperatures ranging from 17 to 28 °C. The average monthly rainfall is 76.35 mm, and relative humidity around 70% (TEIXEIRA, 2003).


Figure 1 Location of the collection area, Horto Florestal Olho D’agua da Bica, Cuité city, Paraíba State

Source: CHAVES et al., 2017.


The hydrography of the studied region is quite peculiar, with ephemeral rivers, weirs, in addition to natural water sources. Among these sources is the Olho D’água da Bica, a perennial spring located near the Education and Health Center (CES) of the Federal University of Campina Grande (UFCG). The region around the source is an arboreal and shrubby Caatinga area, with the presence of streams, dams, wetlands, and hillside areas (COSTA, 2009).


2.2 Specimens Evaluated

This study analyzed 32 adult males of the species L. macrosternum, which were collected every two weeks between January and December 2013, in the Horto Florestal Olho D’água da Bica, with prior authorization from IBAMA/SISBIO (44134-1) (Table 1).


Table 1 Number of specimens collected.

Collection months

Number of individuals collected


6 individuals


11 individuals


7 individuals


3 individuals


3 individuals


2 individuals



2.3 Preparation of the Slides

After collecting the samples, the male L. macrosternum individuals were taken to the Amphibian Biosystematics Laboratory (LABAN) of the Federal University of Campina Grande, Cuité Campus (PB). To prepare the slides, small liver fragments were removed from the animals, weighed, measured, and sent to histological analysis (RIBEIRO; LIMA, 2000).

Liver fragments were fixed in Bouin solution (mixture of 71% picric acid, 37% formaldehyde, and 5% glacial acetic acid) for 24 hours and then dehydrated in increasing alcohol series (70%-30min, 80%-30min, 90%-30min, 95%-30min, and 100%-30min) (HOPWOOD, 1990). Subsequently, they were embedded in paraffin and sectioned at 5.0 μm in a microtome. The sections were colored using specific dyes, where the PAS (Periodic acid-Schiff) reaction was used to analyze carbohydrates, and xylidine (pH 7.5) to analyze proteins. Each individual was cut into ten histological sections. The histological slides were observed under a light microscope with an image capture system. To quantify the proteins and carbohydrates present in liver fragments, a total of 320 photomicrographs (160 for carbohydrates and 160 for proteins) were analyzed. These images were submitted to the GIMP 2.0 editor (GNU Image Manipulation Program, UNIX platforms) to convert digital images into a gray scale (black and white). This color segmentation allows measuring the number of pixels in the selected fabric as described by Temitope (2013).

Climate Data

Monthly meteorological data of temperature (ºC) and rainfall (mm) were obtained from the database of the Executive Agency of Water Management of Paraíba State (AESA), from the meteorological station located next to the sampling site.

Statistical Analysis

After verifying the existence of outliers, the Shapiro-Wilk test was used to assess data normality. When necessary, the data were submitted to normalization (x + 0.5)1/2 (Tukey and Dunn, respectively). The amount of proteins and carbohydrates in the liver was quantified by the Kruskal-Wallis test, followed by the Dunn test when necessary.

The relationship between climate variables and protein and carbohydrate quantification was assessed by the simple linear regression test. Spearman correlation tests (nonparametric data) were used for the evaluated parameters. The statistical significance considered was P ≤ 0.05, and all analyses were made according to Krebs (1999).




3.1 Protein and carbohydrate quantification

Histological analysis of the liver cells of L. macrosternum showed the presence of Kupffer cells, also known as pigmented macrophages (Figure 2, A and B). According to Agius and Roberts (2003), this points to different types of granules in the cytoplasm, which may indicate diversified chemical substances, such as melanin, resulting from the degradation of phagocytized cellular material. Thus, these cells act as a defense system and their lack or death leads to diseases.

Specifically in amphibians, liver melanomacrophages, called Kupffer cells, are endowed with several cytoplasmic granules, which may indicate the presence of different chemical substances, in addition to dark granules with histochemical and ultrastructural properties similar to melanin (GUIDA et al., 2004). Hepatocytes were also found to contain pigment granules, whose function is related to protein synthesis for maintenance or export (CORSARO et al., 2000). Gartner and Hiat (2003) state that hepatocytes are distributed in plaques as a “wall”, characterized by a starry morphology with an oval nucleus, and also consider that these hepatic structures are the main cells of the liver. Therefore, the images observed in this study corroborate the descriptions of the aforementioned authors, demonstrating that this pattern is conserved within vertebrates.


Figure 2Visualization of cellular structures present in the liver of Leptodactylus macrosternum (MIRANDA-RIBEIRO, 1926)

(a)                (b) 

 (a) Visualization of structures present in the liver (using xylidine dye for histochemical analysis of proteins); (b) Visualization of structures present in the liver (using the PAS reaction for histochemical analysis of carbohydrates) of male Leptodactylus macrosternum individuals (MIRANDA-RIBEIRO, 1926) collected from the HFOB area, Cuité city, Paraíba State, from January to December 2013.


The monthly values ​​of protein quantities in the analyzed liver fragments (Figure 3) were significantly lower ​​during the month of July (p = 0.04, Kruskal-Wallis). In addition, the amount of proteins (given in pixels) was considerably high in the months of May, June, August, November, and December, ranging between 120000 and 140000 pixels. The highest values ​​observed for this parameter occurred in the months of August (140985.9 ± 35395.4 pixels) and December (142731.8 ± 31865.7248).


Figure 3 – Monthly mean values and standard deviation of the amount of proteins deposited in the liver of male Leptodactylus macrosternum individuals (MIRANDA-RIBEIRO, 1926) collected from the HFODB area, Cuité city, Paraíba State, from January to December 2013

* indicates significant statistical differences by the Kruskal-Wallis test (p<0.05).


The amount of carbohydrates deposited in the livers of the animals did not differ significantly during the sampled months, remaining just above 150000 pixels. However, this parameter was relevant in the months of June (176251.13 ± 6904.78) and May (172161.13 ± 53484.26) (Figure 4).


Figure 4 – Monthly mean values ​​and standard deviation of the amount of carbohydrates deposited in the liver of male Leptodactylus macrosternum individuals (MIRANDA-RIBEIRO, 1926) collected from the HFODB area, Cuité city, Paraíba State, from January to December 2013


According to Dayton and Fitzgerald (2001), rainfall irregularity and the formation and duration of water bodies limit various aspects of the life of anurans, such as acquisition of resources, accumulation of reserves and, mainly, reproductive activity. In this sense, low rainfall intensity as well as its unpredictability in semiarid environments can determine some physiological adjustments in anurans, limiting the occurrence of these animals in some months of the year. This justifies the absence of L. macrosternum individuals between January-April and September-October, as observed during field trips (SANTOS et al., 2007; GIARETTA et al., 2008).

Quantification ​​of proteins and carbohydrates accumulated in the liver of L. macrosternum specimens can also be used as an indicator of various activities performed by these animals such as feeding, reproductive foraging, among others. Therefore, it can be said that these values ​​may be related to the mobilization of the energy reserves necessary for vitellogenesis, reproduction, or even preparation for a period of low food availability (MADALOZZO et al., 2013).


3.2 Relationship between protein/carbohydrate and seasonality

There were significant relationships between rainfall and carbohydrate amounts (r = 0.01; p = 0.03), and between temperature and carbohydrate amounts (r = 0.02; p = 0.04). These factors may be associated with a greater intensity in the feeding of L. macrosternum, with subsequent accumulation of fats and carbohydrates in the liver (CHAVES et al., 2017). Variation in rainfall and temperature was not significantly related to the amount of proteins deposited in the liver of L. macrosternum (Table 2).


Table 2 Amount of proteins and carbohydrates deposited in the liver of males of the species Leptodactylus macrosternum (MIRANDA-RIBEIRO, 1926) as a function of rainfall and temperature in the Horto Florestal Olho D’Água da Bica, Cuité city, Paraíba State, Brazil, between May-August and November-December 2013

















* = Statistically significant values; r = Value of the statistical analysis; p = Significance test.


In addition to interfering with carbohydrate accumulation in the liver of L. macrosternum, other studies have indicated that, in semiarid areas, rainfall is the environmental condition that most interferes with the reproductive activity of anurans, followed by temperature. These factors thus determine the most favorable time for reproduction and, consequently, the spatial distribution of these animals (SASSO-CERRI et al., 2004; MOORE et al., 2005; CHAVES et al., 2017).




The techniques used in this study proved to be relevant to understand the physiological behavior of anurans at different times of the year. The species L. macrosternum proved to be sensitive to seasonal variations, as environmental factors were significantly related to the amounts of carbohydrates and proteins deposited in the liver of the animals analyzed.




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