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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

ABSTRACT

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.

Keywords:
Amapá; Delta; Fish transaminases; Tralhoto; Viviparity

RESUMO

Anableps anableps é um teleósteo vivíparo típico de estuários do Delta do Rio Amazonas, sendo representativo deste bioma na ilha de Maracá, com potencial para o biomonitoramento. O objetivo deste estudo foi aplicar diferentes biomarcadores em machos e fêmeas desta espécie e verificar a possível influência sazonal em aspectos de sua fisiologia. Para a coleta dos peixes foram realizadas três expedições, de abril/2018 (estação chuvosa), setembro/2018 (estação seca) até fevereiro/2019 (estação chuvosa). Foram obtidos parâmetros biométricos, índices gonadossomático (IGS), hepatossomático (IHS), viscerossomático (IVS) e amostras de sangue para dosagem de triglicerídeos, proteínas totais, glicose e a atividade das enzimas aspartato aminotransferase (AST), alanina aminotransferase (ALT) e fosfatase alcalina (ALP). O IGS dos machos foi maior na estação chuvosa e das fêmeas na estação seca, isso provavelmente ocorreu devido ao processo de embriogênese. Os machos aumentaram a biomassa na estação seca e apresentaram homogeneidade metabólica, já as fêmeas apresentaram hiperglicemia, hipertrigliceridemia e maior atividade da ALT. Os biomarcadores testados mostraram-se promissores para o biomonitoramento, sugerindo de forma preliminar que há um assincronismo sazonal entre machos e fêmeas de A. anableps na alocação de recursos energéticos em diferentes momentos do seu ciclo de vida.

Palavras-chave:
Amapá; Delta; Peixes transaminases; Tralhoto; Viviparidade

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., 2020Häder DP, Banaszak AT, Villafãne VE, Narvartre MA, González RA, Helbling EW. Anthropogenic pollution of aquatic ecosystems: Emerging problems with global implications. Sci Total Environ. 2020; 713:136586. https://doi.org/10.1016/j.scitotenv.2020.136586
https://doi.org/10.1016/j.scitotenv.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., 2020Foesch MDS, Francelino MR, Rocha PA, Gomes ARL. River water contamination resulting from the Mariana disaster, Brazil. Floresta e Ambiente. 2020; 27(4):e20180132. https://doi.org/10.1590/2179-8087.013218
https://doi.org/10.1590/2179-8087.013218...
), fires in all biomes (da Silva Junior et al., 2020da Silva CA Junior, Teodoro PE, Delgado RC, Teodoro LPR, Lima M, Pantaleão LPR et al. Persistent fire foci in all biomes undermine the Paris agreement in Brazil. Sci Rep . 2020; 10:16246. https://doi.org/10.1038/s41598-020-72571-w
https://doi.org/10.1038/s41598-020-72571...
), and deforestation (for example in the Amazon River Basin region; Exbrayat et al., 2017Exbrayat JF, Liu YY, Willians M. Impact of deforestation and climate on the Amazon Basin’s aboveground biomass during 1993-2012. Sci Rep. 2017; 7:15615. https://doi.org/10.1038/s41598-017-15788-6
https://doi.org/10.1038/s41598-017-15788...
), and river silting (Santos et al., 2020Santos A, Lopes PMO, Silva MV, Jardim AMRF, Moura GBA, Fernandes GST et al. Causes and consequences of seasonal changes in the water flow of the São Francisco river in the semiarid of Brazil. Environ Sustain Indic. 2020; 8:100084. https://doi.org/10.1016/j.indic.2020.100084
https://doi.org/10.1016/j.indic.2020.100...
).

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., 2016Mansur AV, Brondízio ES, Roy S, Hetrick S, Vogt ND, Newton A. An assessment of urban vulnerability in the Amazon Delta and Estuary: a multi-criterion index of flood exposure, socio-economic conditions and infrastructure. Sustain Sci. 2016; 11:625-43. https://doi.org/10.1007/s11625-016-0355-7
https://doi.org/10.1007/s11625-016-0355-...
).

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, 2014Costa C, Teixeira JP. Biomonitoring. In: Wexler P, editor. Encyclopedia of Toxicology. Academic Press; 2014. p.483-84. http://dx.doi.org/10.1016/B978-0-12-386454-3.01000-9
http://dx.doi.org/10.1016/B978-0-12-3864...
). 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., 2016Ruaro R, Gubiani EA, Cunico AM, Moretto Y, Piana PA. Comparison of fish and macroinvertebrates as bioindicators of neotropical streams. Environ Monit Assess . 2016; 188:45. https://doi.org/10.1007/s10661-015-5046-9
https://doi.org/10.1007/s10661-015-5046-...
).

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 and biodiversity indicator (Costa, Teixeira, 2014Costa C, Teixeira JP. Biomonitoring. In: Wexler P, editor. Encyclopedia of Toxicology. Academic Press; 2014. p.483-84. http://dx.doi.org/10.1016/B978-0-12-386454-3.01000-9
http://dx.doi.org/10.1016/B978-0-12-3864...
); 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., 2008Zhou Q, Zhang J, Fu J, Shi J, Jiang G. Biomonitoring: an appealing tool for assessment of metal pollution in the aquatic ecosystem. Anal Chim Acta. 2008; 606:135-50. https://doi.org/10.1016/j.aca.2007.11.018
https://doi.org/10.1016/j.aca.2007.11.01...
). 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, 1938Turner CL. Adaptations for viviparity in embryos and ovary of Anableps anableps. J Morphol . 1938; 62(2):323-49. https://onlinelibrary.wiley.com/doi/abs/10.1002/jmor.1050620208
https://onlinelibrary.wiley.com/doi/abs/...
; Knight et al., 1985Knight FM, Lombardi J, Wourms JP, Burns JR. Folicullar placenta and embryonic growth of the viviparous four-eyed fish (Anableps). J Morphol. 1985; 185:131-42. https://doi.org/10.1002/jmor.1051850110
https://doi.org/10.1002/jmor.1051850110...
). 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., 2005Ikeda RGP, Silva JMB, Miranda SCS. Morfometria do tralhoto, Anableps anableps (Linnaeus, 1758), do estuário de Caratateua - Curuçá - Pará. Bol Téc Cient CEPNOR, Belém. 2005; 5(1):93-103.), which includes the Amazon River Delta, Brazil.

This species resides in estuaries and performs intertidal migration as a surface swimming species (Krumme et al., 2014Krumme U, Calderón MA, Echterhoff A. Intertidal migration of the four-eyed fish Anableps anableps in north Brazilian mangrove creeks. Mar Ecol Prog Ser. 2014; 509:271-87. https://doi.org/10.3354/meps10863
https://doi.org/10.3354/meps10863...
); however, it does not migrate across long distances, being restricted to environments of low salinity (Watanabe et al., 2014Watanabe LA, Vallinoto M, Neto NA, Muriel-Cunha J, Saint-Paul U, Schneider H et al. The past and present of an estuarine-resident fish, the ‘‘four-eyed fish’’Anableps anableps (Cyprinodontiformes, Anablepidae), revealed by mtDNA sequences. PLoS ONE. 2014; 9(7):e101727. https://doi.org/10.1371/journal.pone.0101727
https://doi.org/10.1371/journal.pone.010...
). 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., 2015Michel KB, Aerts P, Gibb AC, Wassenbergh SV. Functional morphology and kinematics of terrestrial feeding in the largescale foureyes (Anableps anableps). J Exp Biol. 2015; 218:2951-60.). 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, 2007Brenner M, Krumme U. Tidal migration and patterns in feeding of the four-eyed fish Anableps anableps L. in a north Brazilian mangrove. J Fish Biol. 2007; 70:406-27. https://doi.org/10.1111/j.1095-8649.2007.01313.x). Such a food plasticity reveals a generalist strategy (Figueiredo et al., 2019Figueiredo MB, Nunes JLS, Almeida ZS, Paz AC, Piorski NM, Reis MR. Feeding ecology of Anableps anableps (Actinopterygii: Cyprinodontiformes: Anablepidae) off the north-eastern coast of Brazil. Acta Ichthyol Piscatoria. 2019; 49(3):213-19. https://doi.org/10.3750/AIEP/02477
https://doi.org/10.3750/AIEP/02477...
).

Cavalcante and coauthors (2012Cavalcante AN, Santos NB, Almeida ZS. Biologia reprodutiva de tralhoto, Anableps anableps, na baía de São marcos, Maranhão, Brasil. Bol Inst Pesca. 2012; 38(4):285-96. Available from: https://www.pesca.agricultura.sp.gov.br/38_4_285-296.pdf
https://www.pesca.agricultura.sp.gov.br/...
) 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., 2014Hook SE, Gallagher EP, Batley GE. The role of biomarkers in the assessment of aquatic ecosystem health. Integr Environ Assess Manag. 2014; 10(3):327-41. https://doi.org/10.1002/ieam.1530
https://doi.org/10.1002/ieam.1530...
), 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., 2019Milinkovitch T, Geffard O, Geffard A, Mouneyrac C, Chaumot A, Xuereb B et al. Biomarkers as tools for monitoring within the Water Framework Directive context: concept, opinions and advancement of expertise. Environ Sci Pollut Res . 2019; 26:32759-63. https://doi.org/10.1007/s11356-019-06434-x
https://doi.org/10.1007/s11356-019-06434...
).

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., 2016Madliger CL, Cooke SJ, Crespi EJ, Funk JL, Hultine KR, Hunt KE et al. Success stories and emerging themes in conservation physiology. Conserv Physiol. 2016; 4(1):cov057. https://doi.org/10.1093/conphys/cov057
https://doi.org/10.1093/conphys/cov057...
). 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., 2002Levesque HM, Moon TW, Campbell PGC, Hontela A. Seasonal variation in carbohydrate and lipid metabolism of yellow perch (Perca flavescens) chronically exposed to metals in the field. Aquat Toxicol. 2002; 60(3-4):257-67. https://doi.org/10.1016/S0166-445X(02)00012-7
https://doi.org/10.1016/S0166-445X(02)00...
).

Another field studies compared females of Hoplias malabaricus (Bloch, 1794) (Gomes et al., 2015Gomes ADO, Tolussi CE, Ribeiro CS, Honji RM, Moreira RG. The role of ovarian steroids in reproductive plasticity in Hoplias malabaricus (Teleostei: Characiformes: Erythrinidae) in tropical reservoirs with different degrees of pollution. Gen Comp Endocr. 2015; 222(1):1-10. https://doi.org/10.1016/j.ygcen.2014.10.008
https://doi.org/10.1016/j.ygcen.2014.10....
) and Astyanax fasciatus (currently Psalidon fasciatus (Cuvier, 1819)) (Tolussi et al., 2018Tolussi CE, Gomes ADO, Kumar A, Ribeiro CS, Lo Nostro FL, Bain PA et al. Environmental pollution affects molecular and biochemical responses during gonadal maturation of Astyanax fasciatus (Teleostei: Characiformes: Characidae). Ecotoxicol Environ Saf. 2018; 147:926-34. https://doi.org/10.1016/j.ecoenv.2017.09.056
https://doi.org/10.1016/j.ecoenv.2017.09...
) 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 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., 2010Correia TG, Narcizo AM, Bianchini A, Moreira RG. Aluminum as an endocrine disruptor in female Nile tilapia (Oreochromis niloticus). Comp Biochem Physiol C Toxicol Pharmacol. 2010; 151(4):461-66. https://doi.org/10.1016/j.cbpc.2010.02.002
https://doi.org/10.1016/j.cbpc.2010.02.0...
; Vieira et al., 2013Vieira VARO, Correia TG, Moreira RG. Effects of aluminum on the energetic substrates in neotropical freshwater Astyanax bimaculatus (Teleostei: Characidae) females. Comp Biochem Physiol C Toxicol Pharmacol . 2013; 157(1):1-08. https://doi.org/10.1016/j.cbpc.2012.08.003
https://doi.org/10.1016/j.cbpc.2012.08.0...
). 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., 1990Gill TS, Tewari H, Pande J. Use of the fish enzyme system in monitoring water quality: Effects of mercury on tissue enzymes. Comp Biochem Physiol C Toxicol Pharmacol . 1990; 97 (2):287-92. https://doi.org/10.1016/0742-8413(90)90143-W
https://doi.org/10.1016/0742-8413(90)901...
). 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., 2021Ebrahimzadeh M, Heidari B, Nazarhaghighi F, Valipour A. Physiological responses of the goldfish (Carassius auratus) during subacute exposure to organic pollutants. Bull Environ Contam Toxicol. 2021; 106:773-78. https://doi.org/10.1007/s00128-021-03164-0
https://doi.org/10.1007/s00128-021-03164...
).

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, 2017Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio). Plano de manejo Estação Ecológica de Maracá-Jipióca. Macapá, AP; 2017. Available from: https://www.icmbio.gov.br/portal/images/stories/plano-de-manejo/Portaria-plano-de-manejo/plano_de_manejo_esec_maraca_jipioca_icmbio.pdf
https://www.icmbio.gov.br/portal/images/...
).

The Maracá Island (study area; Fig. 1) is a federal conservation and integral protection unit called Ecological Station of Maracá-Jipióca (01°49’51” and 02°12’54”N 50°11’02” and 50°34’22”W) under the management of the Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio). It is a coastal marine biome that covers the islands of Maracá and Jipióca (submerged). There is a small stream (Igarapé do Inferno) that divides the Maracá Island into north and south (ICMBio, 2017Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio). Plano de manejo Estação Ecológica de Maracá-Jipióca. Macapá, AP; 2017. Available from: https://www.icmbio.gov.br/portal/images/stories/plano-de-manejo/Portaria-plano-de-manejo/plano_de_manejo_esec_maraca_jipioca_icmbio.pdf
https://www.icmbio.gov.br/portal/images/...
).

FIGURE 1
| Ecological Station of Maracá-Jipióca. Black dot shows the location of the Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio) research base.

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, 2017Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio). Plano de manejo Estação Ecológica de Maracá-Jipióca. Macapá, AP; 2017. Available from: https://www.icmbio.gov.br/portal/images/stories/plano-de-manejo/Portaria-plano-de-manejo/plano_de_manejo_esec_maraca_jipioca_icmbio.pdf
https://www.icmbio.gov.br/portal/images/...
). 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, 2015Mesquita EMC, Isaac-Nahum VJ. Traditional knowledge and artisanal fishing technology on the Xingu River in Pará, Brazil. Braz J Biol. 2015; 75(3):S138-57. http://dx.doi.org/10.1590/1519-6984.01314BM
http://dx.doi.org/10.1590/1519-6984.0131...
), with dimensions of 30 m/40 mm and 4 m/30 mm, respectively (ICMBio, 2019Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio). Ministério do Meio Ambiente/Instituto Chico Mendes de Conservação da Biodiversidade. Portaria Nº 3, de 2 de janeiro de 2019. Dispõe sobre regras comunitárias comuns e específicas para gestão integrada de uso e manejo dos recursos naturais e pesqueiros para a gestão da Resex Marinha de Gurupi-Piriá no Estado do Pará e dá outras providências. Diário Oficial da União, Brasília DF, January 4; 2019. p.88. Available from: https://www.in.gov.br/materia/-/asset_publisher/Kujrw0TZC2Mb/content/id/57876342/do1-2019-01-04-portaria-n-3-de-2-de-janeiro-de-2019-57876156
https://www.in.gov.br/materia/-/asset_pu...
). 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., 2015Ikeda RGP, Silva JMB, Miranda SCS. Morfometria do tralhoto, Anableps anableps (Linnaeus, 1758), do estuário de Caratateua - Curuçá - Pará. Bol Téc Cient CEPNOR, Belém. 2005; 5(1):93-103.).

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 (C10H14N2Na2O8.2H2O, 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 (1996Vazzoler AEAM. Biologia da reprodução de peixes teleósteos: Teoria e prática. Maringá: EDUEM; 1996.), and celomic fat for viscerosomatic (VSI) following Silva et al. (2015Silva EM, Monserrat JM, Sampaio LA, Tesser MB. Crescimento e metabolismo do nitrogênio em juvenis de Trachinotus marginatus alimentados com diferentes níveis proteicos. Arq Bras Med Vet Zootec. 2015; 67(1):131-39. https://doi.org/10.1590/1678-7069
https://doi.org/10.1590/1678-7069...
). Condition factor (CF) was calculated as Fulton (1904): K=100 W/L3 (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 protein content followed the Biuret reaction (Gornall et al., 1949Gornall AG, Bardawill CJ, David MM. Determination of serum proteins by means of the biuret reaction. J Biol Chem. 1949; 177:751-66. https://doi.org/10.1016/S0021-9258(18)57021-6
https://doi.org/10.1016/S0021-9258(18)57...
); glucose (Trinder, 1969Trinder P. Determination of glucose in blood using glucoseoxidase with an alternative oxygen acceptor. Ann Clin Biochem. 1969; 6:24-27. https://doi.org/10.1177/000456326900600108
https://doi.org/10.1177/0004563269006001...
) and triglycerides (Bucolo, David, 1973Bucolo G, David H. Quantitative determination of serum triglycerides by the use of enzymes. Clin Chem. 1973; 19:476-82. https://doi.org/10.1093/clinchem/19.5.476
https://doi.org/10.1093/clinchem/19.5.47...
) followed a colorimetric enzyme test. The protocols of activity of AST and ALT kits followed the method described by Bergmeyer (1974Bergmeyer HU. Methods of enzymatic analysis. Verlag Chemie, Weinheim; 1974.), and ALP followed the method of McComb et al. (1979McComb RB, Bowers GN, Posen S. Measurement of alkaline phosphatase activity. In: Alkaline Phosphatase. Springer, Boston, MA; 1979. p.289-372. https://doi.org/10.1007/978-1-4613-2970-1_7
https://doi.org/10.1007/978-1-4613-2970-...
). 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).

The lowest detection level and intra- and inter-assay coefficients of variation (% CV) were 45.4 ± 14.36 mg dL-1, 8.05% (n = 3) and 11.77 % (n = 5) for glucose; 37.01 ± 0.05 mg dL-1, 3.46 % (n = 3) and 16.03 (n = 4) for triglycerides; 1.30 ± 0.17 g dL-1, 8.63 % (n = 4) and 17.50 % (n = 6) for total proteins; 45.97 ± 13.64 U L-1, 14.00 % (n = 4), and 16.74 % (n = 4) for AST; 8.72 ± 0.82 U L-1, 6.96% (n = 2), 17.80% (n = 4) for ALT; and 26.58 ± 1.29 U L-1, 15.51 % (n = 4), 20.23 ± (n = 4) for ALP.

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).

TABLE 1
| Biometric analysis and body index of males and females of Anableps anableps during seasonal cycle. Total length and body weight were compared inter-sex and gonadosomatic index (GSI), hepatosomatic index (HSI), viscerosomatic index (VSI), and Fulton´s condition factor (K) was compared between animals of the same sex. Results are expressed as mean and standard deviation. Different letters indicate statistical differences (Kruskal-Wallis with a Dunn’s post hoc test, P < 0.05; Mann-Whitney test P < 0.05 for GSI, HSI, VSI, and K males).

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).

TABLE 2
| Concentration of plasma energetic substrates and plasma activity of liver enzymes of males and females of Anableps anableps during expeditions in the dry (September 2018) and rainy seasons (February 2019). Results are expressed as mean and standard deviation. Different letters indicate statistical differences for intra and inter-sex comparisons in different seasons. Hepatic enzymes alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST).

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).

TABLE 3
| Pearson correlation test for biometric parameters, body indexes, and plasma of males and females of Anableps anableps in a seasonal cycle. P < 0.05 indicates a significative correlation. Alanine aminotransferase (ALT), Gonadosomatic (GSI); viscerossomático (IVS); Viscerosomatic (VSI).

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 (2008Nascimento FL, Assunção MSI. Ecologia reprodutiva dos tralhotos Anableps anableps e Anableps microlepis (Pisces: Osteichthyes: Cyprinodontiformes: Anablepidae) no rio Paracauari, ilha de Marajó, Pará, Brasil. Bol Mus Para Emílio Goeldi, sér Ciências Naturais. 2008; 3(3):229-40.), 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 (2012Cavalcante AN, Santos NB, Almeida ZS. Biologia reprodutiva de tralhoto, Anableps anableps, na baía de São marcos, Maranhão, Brasil. Bol Inst Pesca. 2012; 38(4):285-96. Available from: https://www.pesca.agricultura.sp.gov.br/38_4_285-296.pdf
https://www.pesca.agricultura.sp.gov.br/...
), 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, 2020Famoofo OO, Abdul Wo. Biometry, condition factors and length-weight relationships of sixteen fishs pecies in Iwopin fresh-water ecotype of Lekki Lagoon, Ogun State, Southwest Nigeria. Heliyon. 2020; 6:e02957. https://doi.org/10.1016/j.heliyon.2019.e02957
https://doi.org/10.1016/j.heliyon.2019.e...
).

The categorized interpretation of the condition factor, according to Le Cren (1951Le Cren ED. The length-weight relationship and seasonal cycle in gonadal weight and condition in the perch (Perca fluviatilis). J Anim Ecol. 1951; 20:201-19. https://doi.org/10.2307/1540
https://doi.org/10.2307/1540...
), 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., 2018Hagmayer A, Furness AI, Reznick DN, Pollux BJA. Maternal size and body condition predict the amount of post-fertilization maternal provisioning in matrotrophic fish. Ecol Evol. 2018; 8:12386-96. https://doi.org/10.1002/ece3.4542
https://doi.org/10.1002/ece3.4542...
). 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., 2010Anderson ET, Stoskopf MK, Morris JA Jr, Clarke EO, Harms CA. Hematology, plasma biochemistry, and tissue enzyme activities of invasive red lionfish captured off North Carolina, USA. J Aquat Anim Health. 2010; 22(4):266-73. http://dx.doi.org/10.1577/H10-0291
http://dx.doi.org/10.1577/H10-0291...
). 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., 2016Sopinka NM, Donaldson MR, O´Connor CM, Suski CD, Cooke SJ. Stress indicators in fish. In: Schreck CB, Tort L, Farrell AP, Brauner CJ, editors. Biology of stress in fish - Fish Physiology. London: Academic Press; 2016. p.405-62. http://dx.doi.org/10.1016/B978-0-12-802728-8.00011-4
http://dx.doi.org/10.1016/B978-0-12-8027...
). 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., 2008Fernandes C, Fontaínhas-Fernandes A, Rocha E, Salgado MA. Monitoring pollution in Esmoriz-Paramos lagoon, Portugal: Liver histological and biochemical effects in Liza saliens. Environ Monit Assess. 2008; 145:315-22. https://doi.org/10.1007/s10661-007-0041-4
https://doi.org/10.1007/s10661-007-0041-...
).

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., 2018Akbary P, Yarahmadi SS, Jahanbakhshi A. Hematological, hepatic enzymes activity and oxidative stress responses of gray mullet (Mugil cephalus) after sub-acute exposure to copper oxide. Environ Sci Pollut Res. 2018; 25:1800-08. https://doi.org/10.1007/s11356-017-0582-1). 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., 2017Zhang YL, Zhang HL, Wang LY, Gu BY, Fan QX. Changes of ammonia, urea contents and transaminase activity in the body during aerial exposure and ammonia loading in Chinese loach Paramisgurnus dabryanus. Fish Physiol Biochem. 2017; 43:631-40. https://doi.org/10.1007/s10695-016-0317-0
https://doi.org/10.1007/s10695-016-0317-...
). 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., 2018Akbary P, Yarahmadi SS, Jahanbakhshi A. Hematological, hepatic enzymes activity and oxidative stress responses of gray mullet (Mugil cephalus) after sub-acute exposure to copper oxide. Environ Sci Pollut Res. 2018; 25:1800-08. https://doi.org/10.1007/s11356-017-0582-1).

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., 2020Esteves-Silva PH, Oliveira MSB, Gentil-Vasconcelos HC, Costa-Campos CE, Tavares-Dias M. New records of hosts for Excorallana longicornis and Nerocila acuminata (Crustacea: Isopoda) in brackish fish from the coast of the State of Amapá (Brazil), with an update on the geographic distribution of Nerocila acuminata. J Parasit Dis. 2020; 44:420-28. https://doi.org/10.1007/s12639-020-01192-x
https://doi.org/10.1007/s12639-020-01192...
) and that parasitism situations as this have been reported as capable of causing a plasma increase in AST and ALT activity (Rastiannasab et al., 2016Rastiannasab A, Afsharmanesh S, Rahimi R, Sharifian I. Alternations in the liver enzymatic activity of Common carp, Cyprinus carpio in response to parasites, Dactylogyrus spp. and Gyrodactylus spp. J Parasit Dis . 2016; 40(4):1146-49. https://doi.org/10.1007/s12639-014-0638-9
https://doi.org/10.1007/s12639-014-0638-...
). 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.

ACKNOWLEDGMENTS

This study was supported by the Fundação de Amparo à Pesquisa do Estado do Amapá (FAPEAP), and by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (FAPEAP: Process 250.203.038/2017; CNPq: Process 61000/2013-5). The authors would like to thank the ES Maracá-Jipióca staff and the ICMBio manager Mr. Iranildo Coutinho for their help in the field activities and the EcoDelta (UNIFAP) research team.

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ADDITIONAL NOTES

  • HOW TO CITE THIS ARTICLE

    Guedes MEG, Correia TG. 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. Neotrop Ichthyol. 2021; 19(4):e210078. https://doi.org/10.1590/1982-0224-2021-0078

Edited by

Juan Miguel Mancera

Publication Dates

  • Publication in this collection
    13 Dec 2021
  • Date of issue
    2021

History

  • Received
    12 Apr 2021
  • Accepted
    25 Aug 2021
Sociedade Brasileira de Ictiologia Neotropical Ichthyology, Núcleo de Pesquisas em Limnologia, Ictiologia e Aquicultura, Universidade Estadual de Maringá., Av. Colombo, 5790, 87020-900, Phone number: +55 44-3011-4632 - Maringá - PR - Brazil
E-mail: neoichth@nupelia.uem.br