Plasma energetic substrates and hepatic enzymes in the four-eyed fish Anableps anableps (Teleostei: Cyprinodontiformes) during the dry and rainy seasons in the Amazonian Island of Maracá, extreme north of Brazil

Anableps anableps is a viviparous teleost typical from Amazon Delta estuaries. It is representative of this biome in Maracá, which offers a potential for biomonitoring. The aim of this study is to apply different biomarkers to males and females of this species and verify possible seasonal influences on their physiology. To collect fish, three expeditions were carried out from the rainy season of April 2018 to the rainy season of February 2019. Biometric parameters and gonadosomatic (GSI), hepatosomatic (HSI), and viscerosomatic (VSI) indexes were calculated, and blood samples were taken to measure triglycerides, total proteins, glucose, and activity of the enzymes aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP). The GSI of males is higher in the rainy season and of females in the dry season. This is probably related to the embryogenesis process. Males show an increase in biomass during the dry season, a metabolic homogeneity, and females show an increase in plasma glucose, triglycerides, and ALT activity. The tested biomarkers are potential for biomonitoring, preliminarily suggesting that there is a seasonal asynchronism between males and females of A. anableps as for the allocation of energy resources at different times of their life cycle.


INTRODUCTION
Currently, several types of anthropogenic threats are affecting aquatic ecosystems globally, including chemical pollutants, climate change, fishing, introduction of exotic species, human population growth, and pathogens (Häder et al., 2020). In this scenario, recent ecological disasters have occurred in the neotropical region and are especially marked in Brazil, among which the mining accident in Mariana (Foesch et al., 2020), fires in all biomes (da Silva Junior et al., 2020), and deforestation (for example in the Amazon River Basin region; Exbrayat et al., 2017), and river silting (Santos et al., 2020).
Some of these impacts occur in conservation and integral protection units, as an example, we mention the marine coastal island of Maracá. This island is susceptible to all the environmental threats mentioned above, since the Amazon Delta Estuary is vulnerable to multiple environmental and anthropogenic pressures (Mansur et al., 2016).
Therefore, the biomonitoring of aquatic ecosystems is an important tool to allow an ecophysiological assessment of biodiversity and of landscape in face of unpredictable or unspecified environmental threats. It requires the verification and interpretation of biological and physiological responses of target organisms (Costa, Teixeira, 2014). One of the main challenges in implementing biomonitoring programs is the selection of a species or a taxonomic group to be considered as a bioindicator. This is a decisive factor in its applicability (Ruaro et al., 2016).
A sentinel species, that is, the one chosen for aquatic biomonitoring, must meet some premises: it must indicate environmental disturbances and be a good ecological 3/14 ni.bio.br | scielo.br/ni and biodiversity indicator (Costa, Teixeira, 2014); at the same time, it should meet some additional postulates, such as relative abundance, ease of capture, sampling and maintenance in laboratory, representativeness of the studied site, long life cycles, and relevance in the food chain (Zhou et al., 2008). In addition, this species must also meet some ecotoxicological requirements and have a relative physiological sensitivity to environmental stressors, allowing the basal and primary detection of effects and impacts on the food chain.
In this context, Anableps anableps (Linnaeus, 1758) Cyprinodontiformes: Anablepidae, commonly known by its indigenous name ("tralhoto"), or four-eyed fish, is a viviparous teleost species. Therefore, it is a livebearer species, in which superfetation is relatively absent or unusual and sexual dimorphism is evident, as the male has a tubular gonopod (Turner, 1938;Knight et al., 1985). The genus Anableps has the largest representatives in this order. They are pelagic animals distributed in the equatorial portion of the neotropical region (Ikeda et al., 2005), which includes the Amazon River Delta, Brazil.
This species resides in estuaries and performs intertidal migration as a surface swimming species (Krumme et al., 2014); however, it does not migrate across long distances, being restricted to environments of low salinity (Watanabe et al., 2014). Since it is a brackish water fish and inhabits mangrove areas, this species has morphological and jaw adaptations that allow it to jump out of the water and capture prey on land, especially in mudbanks (Michel et al., 2015). For this reason, they are notoriously common on the island of Maracá. In water columns or in substrates, it feeds on detritus, red algae, insects, fish, and Grapsidae crabs (Brenner, Krume, 2007). Such a food plasticity reveals a generalist strategy (Figueiredo et al., 2019).
Cavalcante and coauthors (2012) report studies that show the ecological importance, the reproductive strategy, and the abundance of A. anableps as its main characteristics. Therefore, it can be considered as a potential bioindicator species, especially because of its low occurrence in anthropically impacted environments. In addition to an appropriate choice of the bioindicator species for biomonitoring, the selection of biomarkers that express the physiological condition and the health of an organism is essential for an ecophysiological prognosis of an ecosystem (Hook et al., 2014), as they are defined as biological, biochemical, physiological responses, among others, presented by organisms in face of an environmental stressor. Hence, it can be used as a tool to support ecological assessment programs (Milinkovitch et al., 2019).
In light of the above, from the perspective of conservation physiology, it is possible to predict changes and impacts on wild animal populations through physiological metrics and approaches in order to contribute to conservation outcomes and management programs (Madliger et al., 2016). Among such metrics, fish metabolism and energetic substrates stand out. A study comparing individuals of yellow perch Perca flavescens (Mitchill, 1814) captured in different seasons (spring and autumn) in six lakes in Québec-Canada found seasonal changes in condition factor, plasma fatty acids, and several liver and metabolic biomarkers (Levesque et al., 2002).
Another field studies compared females of Hoplias malabaricus (Bloch, 1794) (Gomes et al., 2015) and Astyanax fasciatus (currently Psalidon fasciatus (Cuvier, 1819)) (Tolussi et al., 2018) in a reservoir (reference area) with another reservoir impacted by domestic sewage, industrial effluents, deforestation, and soil occupation in four different seasons in state of São Paulo (Brazil), showed the applicability of plasma biomarkers, as well as 4/14 ni.bio.br | scielo.br/ni body index markers, such as gonadosomatic (GSI), hepatosomatic (HSI), evidencing negative impacts on the physiology of these species due to the interaction between chemical contaminants and seasonal variations.
Energetic substrates, such as proteins, lipids and carbohydrates from plasma and tissues, and the body indexes mentioned above, have also been used as biomarkers in bioassays with fish exposed to metals, such as aluminum, and have been shown to be adequate when evidencing physiological impacts on reproduction, metabolism, and the endocrine system (Correia et al., 2010;Vieira et al., 2013). Also, for several decades many fish hepatic enzymes, such as transaminases and alkaline phosphatase, have been used to monitor ecosystems and water quality (Gill et al., 1990). Currently, studies on the same subject continue to be carried out and they have pointed out that these enzymes, considered as biomarkers, are powerful tools in environmental biomonitoring of aquatic ecosystems (Ebrahimzadeh et al., 2021).
The present study aims to investigate in different seasons the metabolism of energy substrates and activity of liver enzymes in the plasma of males and females of A. anableps in the estuarine ecosystem of island of Maracá. The reasons for this study are that the island of Maracá encompasses an aquatic ecosystem susceptible to anthropic influences and abiotic seasonal variations, the potential of the A. anableps for the context presented here, and the fact that there is no information available on characteristics of the basic physiology of this species, except for some data on zootechnical parameters, feeding and eye physiology.

MATERIAL AND METHODS
Study area and field expeditions. The state of Amapá is in the extreme north of the Brazilian territory, inserted inside the Amazon region. According to the National Waters Agency, the city of Amapá has a portion of its territory in the Araguari River basin and in coastal hydrographic basin. The latter includes the Maracá Island (ICMBio, 2017). The west side of the island is separated from the continent by the Carapori or Varadouro canal, and the east side gives direct access to the Atlantic Ocean (ICMBio, 2017).
Three field expeditions were conducted to the ES of Maracá-Jipióca in an annual seasonal cycle: April 2018 (rainy season), September 2018 (dry season), and February 2019 (rainy season). It is important to note that there are only two distinct seasons in the ES: dry (August to November) and rainy (December to July) (ICMBio, 2017). The fish were collected in the island using artisanal fishing technology, that is, fixed gillnets and cast nets (cone-shaped nets, locally known as "tarrafa") (Mesquita, Isaac-Nahum, 2015), with dimensions of 30 m/40 mm and 4 m/30 mm, respectively (ICMBio, 2019). The collection was performed during the passing from the high tide to the low tide around the mangrove stream areas for about an hour so that the passage of fish through the tidal canal was obstructed by the fishing nets (Ikeda et al., 2015).

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ni.bio.br | scielo.br/ni Sampling procedures. After the fish have been caught (voucher number: LABOT-UNIFAP #003), they were anesthetized with benzocaine 100 mg L -1 still at the collection site. A blood sample was obtained by venipuncture of the dorsal caudal vessel considering the lateral line of the fish as an anatomical reference using 3ml disposable syringes and needles (0.80 x 25 mm) -Becton Dickinson (NJ, EUA). The syringes contained 5% EDTA-Na (C 10 H 14 N 2 Na 2 O 8 .2H 2 O, Sigma) as anticoagulant. Then, the samples were centrifuged for 5 minutes at 655.1 g to separate the plasma, and then immediately stored in a freezer and kept frozen at -20 ºC until further analysis in the laboratory facilities of the Universidade Federal do Amapá (UNIFAP).
Biometric parameters, such as total length and body weight, were recorded. The liver, gonads (embryos and testicles) and all digestive tract organs in the fish abdominal cavity were removed and weighed to determine the body indexes (%): hepatosomatic -HSI; gonadosomatic -GSI (embryos for females) according to Vazzoler (1996), and celomic fat for viscerosomatic (VSI) following Silva et al. (2015). Condition factor (CF) was calculated as Fulton (1904): K = 100 W/L 3 (W (g) = body weight, L (cm) = total length).
Plasma biochemistry analysis. The plasma energetic substrates glucose, total proteins, and triglycerides and the activity of the hepatic enzymes alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured using commercial kits for humans (Life Biotechnologies, MG, Brazil). Total 6/14 ni.bio.br | scielo.br/ni protein content followed the Biuret reaction (Gornall et al., 1949); glucose (Trinder, 1969) and triglycerides (Bucolo, David, 1973) followed a colorimetric enzyme test. The protocols of activity of AST and ALT kits followed the method described by Bergmeyer (1974), and ALP followed the method of McComb et al. (1979). The analyses were validated according to the manufacturer's instructions in 1.5 mL glass cuvettes on a digital spectrophotometer (UV-Visible light, 5100 Tecnal SP, Brazil).

Statistical analysis.
The results are expressed as mean ± standard deviation in a 95% confidence level (p <0.05). Shapiro-Wilk normality test and a Brown-Forsythe homoscedasticity test were applied to treat the data. One-way analysis of variance (ANOVA) was performed to compare three or more groups (different seasons and same sex), two-way analysis of variance (ANOVA) compared three or more groups (seasons and sex), and Student t test to compare between two groups. The nonparametric data were submitted respectively a test of Kruskal-Wallis followed by Dunn's test and Mann-Whitney test. The relationship between biometric and plasma parameters was analyzed by Pearson´s correlation test (p <0.05).

RESULTS
We collected thirty-three specimens of A. anableps during the three expeditions: the rainy season in April 2018 (only females n = 9), the dry season in September 2018 (males n = 5 and females n = 7), and the rainy season in February 2019 (males n = 5 and females n = 7). All results presented are considered preliminary because this is a study with a small sample size.
The biometric analysis (Tab. 1) showed that the females in the rainy season (April 2018) have a longer total length compared to males in both seasons (P = 0.029) and females in the dry season (P = 0.029). Regarding rainy season females (2018), a higher body weight was recorded compared to males (P = 0.002) in the rainy season (2019) and females of the dry (2018) and rainy season (2019) (P = 0.008). Females in the dry season have a lower CF compared to females in the rainy seasons (P = 0.001). However, regarding body indexes, females in the dry season compared to females in the other seasons showed a high GSI (P = 0.012) and a higher VSI (P = 0.011) compared to females in the rainy season (2018). There was no statistical difference for HSI (P = 0.32) between females. The number of embryos between seasons was not significantly different (P = 0.28).
Comparing males, the body weight of dry season animals was greater than that in the rainy season (P = 0.002). There were no statistical differences for total length (P = 0.899), HSI (P = 0.392), VSI (P > 0.999), and CF (P = 0.732). However, GSI was higher in males in rainy season (P = 0.011).
The analysis of plasma energetic substrates (Tab. 2) showed no statistical differences for total proteins between all groups (P = 0.156). However, females in the dry season have a higher concentration of glucose (P < 0.005) and triglycerides (P < 0.001) compared to animals of both sexes in the dry and rainy seasons. Females in the rainy season have a higher triglycerides concentration compared to males in the same season (P < 0.050).
The activity of plasma liver enzymes (Tab. 2) showed that only females in the dry season have a higher ALT than females in the rainy season (P < 0.05). However, for this enzyme, there were no differences in the other comparisons between males and females (P = 0.274). There were also no differences for AST (P = 0.256) and ALP (P = 0.268). Defrosting and logistical difficulties in preserving the samples in the island of Maracá (rainy season of 2018, females) and hemolysis in AST samples for males of the rainy season of 2019 impeded these analyses.
The Pearson's correlation analysis showed a significative positive correlation only between total length and body weight of males and females in the rainy season in 2019 (Tab. 3).

DISCUSSION
We designed this field study to test whether the teleost Anableps anableps meets the requirements of a species with a potential for ecological biomonitoring in the scope of conservation physiology by assessing basic aspects of the energy metabolism of the plasma in seasonal cycles (rainy and dry seasons) in the island of Maracá. The field approaches present preliminary results, suggesting that males have a stable plasma metabolic physiology between seasons more than females do, which, in turn, showed high body indexes and an increased metabolic profile during the dry season. However, in view of the perspective adopted in this study, a more rigorous and robust biomonitoring approach is needed to confirm the results obtained for this species in the island of Maracá. The capture and in situ handling of A. anableps specimens to obtain data on their physiology required specific logistics, in addition to challenges that made the conduction of this study difficult, such as the rusticity of the research support base and the navigation on small boats to collection sites in face of extremely agitated tidal dynamics, including the phenomenon of "pororoca" and the presence of wild animals in the island. In addition, the difficulty of keeping the samples refrigerated in liquid nitrogen cylinders and stored in a freezer at -20 ºC using portable generators uninterruptedly was an aggravating factor and resulted in substantial sample losses. Biometric analyses and body indexes of males of A. anableps show that there were no allometric variations, as well in HSI and VSI between the dry and rainy seasons. Together with the very homogeneous plasma results, this preliminarily denotes an apparent physiological condition that is little variable or imperceptible to the biomarker considered. However, GSI, K, and body weight allowed us to infer an interrelation of these parameters with the energy investment and the reproductive cycle of these animals in a seasonal perspective.
It is interesting to note that the males of A. anableps in the dry season invested in the increase in body biomass, which suggests that this is not their reproductive peak. This is corroborated by the much lower GSI than that observed in the rainy season. At the same time, the condition factor was inverse to that of GSI in both seasons. Although the correlation between these parameters was not significant according to the Pearson's test, the allocation of energy resources for gonadal development was clearly not prioritized in that season.
According to Nascimento, Assunção (2008), A. anableps reproduces throughout the year without a defined period and there is no reproductive synchronism between males and females, that is, the peak of greater gonadal development of both sexes is inverse, which is in agreement with our preliminary findings. While males showed a greater gonadal development in the rainy season, in females this occurred in the dry season. This can be explained since this is a viviparous species and therefore related to the embryogenic process, that is, the gonadal development of females is greater after copulation. Females in this season (September 2018) also showed a higher number of embryos, which is in agreement with Cavalcante and coauthors (2012), who pointed out that September is the reproductive peak of females due to a greater gonadal development and a higher number of embryos. These results, together with the higher concentration of lipids and carbohydrates in the plasma in that season, suggest this period as the possible reproductive peak and the highest energy investment in females.
As for males, the K showed an inverse relation to GSI. This parameter allows monitoring a series of factors related to the health and ecophysiological status of fish. It is known that it is environmentally influenced by the reproductive cycle, specific characteristics intrinsic to each species, food, and even seasonal variation of abiotic factors and environmental or nonspecific stressors (Famoofo, Abdul, 2020).
The categorized interpretation of the condition factor, according to Le Cren (1951), with the standard value (k = 1.0), the females in the rainy season (2018-2019), and males in the dry season would be in adequate body conditions, while the females in the dry season and males in the rainy season (2019), would be in precarious body conditions.
Matrotrophy is a marked characteristic in A. anableps. Specializations in the follicular placenta, which has a portal circulatory system, ensure an adequate supply of nutrients to the embryos, a condition that denotes a high reproductive energy investment (Hagmayer et al., 2018). In this scenario, still little explored in the literature on Anablepidae, our results suggest that there is a simultaneous accumulation of visceral fat, a greater gonadal-embryonic development, and a high concentration of energetic substrates in the blood of females in the dry season.
We consider that our physiological results are original, preliminary, and contribute to the development of reference values for A. anableps, since basic plasma biochemistry parameters can be used to assess fish health status and are extremely important to compose the biochemical profile of a species (Anderson et al., 2010). Therefore, it can be used as baseline data in a future ecophysiological biomonitoring in Amazon Delta.
Usually, the high concentration of glucose in the plasma of teleosts, together with cortisol, is used to identify conditions of physiological stress, which may be due to environmental stressors or capture (Sopinka et al., 2016). However, we consider that there may have been a metabolic adjustment in A. anableps females, whose increased ALT activity implied gluconeogenesis and plasma hyperglycemia, since transamination is an important biochemical reaction in several metabolic processes, such as the formation of metabolites, substrate amino acids, and gluconeogenesis (Fernandes et al., 2008).
Liver enzymes, such as ALT, AST and ALP, have been used in biomonitoring to detect organ damage and impairment to fish health (Akbary et al., 2018). Studies have shown that increased ALT activity in teleost plasma is implicated in the catabolism of amino acids and the formation of alanine (Zhang et al., 2017). When there is a change in the metabolism of proteins and carbohydrates due to stressful situations, changes in AST and ALT activity occur (Akbary et al., 2018).
Although it is not possible to determine the causes for the plasma changes observed here in females in the dry season, whether due to a seasonal physiological adjustment or a non-specific stressor, it is worth mentioning the fact that in a parasitological study, specimens of A. anableps were found on the island of Maracá infected by ectoparasites (Esteves-Silva et al., 2020) and that parasitism situations as this have been reported as capable of causing a plasma increase in AST and ALT activity (Rastiannasab et al., 2016). This cause-effect relationship must be analyzed with caution in view of the considerations presented here.
In conclusion, the GSI of males of Anableps anableps is higher in the rainy season and of females in the dry season, and these differences are probably related to embryogenesis. This is in line with the literature, suggesting that the reproductive peak for each sex is in opposite periods. Plasma biomarkers (energetic substrates and liver enzymes) contribute to the composition of reference values for the physiology of this species and indicate that seasonal variation does not affect males. However, females show a greater mobilization of energetic substrates in the dry season, which coincides with the greatest gonadal development and the largest number of embryos. These results, although preliminary, show the applicability of the tested biomarkers and the relevance of A. anableps for ecophysiological biomonitoring studies on the island of Maracá and the urgent need for more physiological information on this species.