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Physiological responses of Rhamdia quelen (Siluriformes: Heptapteridae) to anesthesia with essential oils from two different chemotypes of Lippia alba

ABSTRACT

The aim of this study was to evaluate if Lippia alba has different chemotypes according to the chemical composition of the essential oil (EO) considering collection site, and if the EO may have different effects on blood and plasma parameters in silver catfish, Rhamdia quelen, during and immediately after anesthesia. The citral (EO-C) and linalool (EO-L) chemotypes were identified, and both presented similar anesthetic effects for silver catfish. Fish were exposed to two concentrations of each EO, which induced slow and fast anesthesia (100 and 300 µL L-1, respectively). Blood ions did not change at any time of anesthesia induction and recovery and, therefore, the electrolyte balance was not altered. Blood gases oscillated through all exposure and recovery times, but there was an increase in pO2 after 10 min recovery in fish anesthetized with EO-C. Glucose increased in fish exposed to both EOs when compared with the control group. Overall, exposure to both EOs (except 100 µL L-1 EO-L at most times) reduced plasma cortisol levels compared to the control and/or ethanol groups. However, as plasma creatinine levels in fish anesthetized with EO-C were higher than control fish, the use of EO-L is preferable.

Keywords:
Blood gas; Cortisol; Glucose; Plasma ions; Silver Catfish

RESUMO

O objetivo deste estudo foi avaliar se Lippia alba apresenta diferentes quimiotipos de acordo com a composição química do óleo essencial (OE), considerando local de coleta e se o OE causa diferentes efeitos nos parâmetros sanguíneos e plasmáticos em jundiá, Rhamdia quelen, durante e imediatamente após a anestesia. Os quimiotipos citral (OE-C) e linalol (OE-L) foram identificados e ambos apresentaram efeito anestésico semelhante para jundiá. Os peixes foram expostos a duas concentrações de cada OE, que induziram anestesia lenta e rápida (100 e 300 mL L-1, respectivamente). Íons sanguíneos não se alteraram em nenhum tempo e consequentemente, o equilíbrio eletrolítico não foi alterado. Os gases sanguíneos oscilaram durante todo tempo de exposição e recuperação, mas houve aumento na pO2 após 10 min de recuperação em peixes anestesiados com OE-C. Níveis sanguíneos de glicose aumentaram nos peixes expostos a ambos OEs quando comparados com o grupo controle. De um modo geral, a exposição a ambos OEs (exceto 100 µL L-1 OE-L na maioria dos tempos) reduziu o cortisol plasmático comparado aos grupos controle e etanol. No entanto, como os níveis de creatinina plasmática em peixes anestesiados com OE-C foram maiores que nos peixes controle, é preferível o uso do OE-L.

Palavras-chave:
Cortisol; Gases sanguíneos; Glicose; Íons plasmáticos; Jundiá

Introduction

In aquaculture, anesthetics (synthetics or plant extratives) are widely employed: from light sedation, to reduce stress during handling and non-invasive procedures, to full anesthesia to avoid pain during surgery and larger interventions (Small, 2003Small BC. Anesthetic efficacy of metomidate and comparison of plasma cortisol responses to tricaine methanesulfonate, quinaldine and clove oil anesthetized channel catfish Ictalurus punctatus. Aquaculture . 2003; 218(1-4):177-185.; Roubach et al., 2005Roubach R, Gomes LC, Fonseca FAL, Val AL. Eugenol as an efficacious anaesthetic for tambaqui, Colossoma macropormum(Cuvier). Aquacult Res . 2005; 36(11):1056-1061.; Ross, Ross, 2008Ross LG, Ross B. Anaesthetic and sedative techniques for aquatic animals. 3rd ed. Oxford: Blackwell Publishing; 2008.; Kiessling et al., 2009Kiessling A, Johansson D, Zahl IH, Samuelsen OB. Pharmacokinetics, plasma cortisol and effectiveness of benzocaine, MS-222 and isoeugenol measured in individual dorsal aorta-cannulated Atlantic salmon (Salmo salar) following bath administration. Aquaculture . 2009; 286(3-4):301-308.; Neiffer, Stamper, 2009Neiffer DL, Stamper MA. Fish sedation, anesthesia, analgesia and euthanasia: consideration, methods and types of drugs. ILAR J. 2009; 50(4):343-360.; Silva et al., 2013aSilva LL, Garlet QI, Benovit SC, Dolci G, Mallmann CA, Bürger ME et al. Sedative and anesthetic activities of the essential oils of Hyptis mutabilis (Rich) Briq. and their isolated components in silver catfish (Rhamdia quelen). Braz J Med Biol Res . 2013a; 46(4):771-779.; Roohi, Imanpoor, 2015Roohi Z, Imanpoor MR. The efficacy of the oils of spearmint and methyl salicylate as new anesthetics and their effect on glucose levels in common carp (Cyprinus carpio L., 1758) juveniles. Aquaculture . 2015; 43:327-332.).

The anesthetic efficacy of several essential oils (EOs), such as Hyptis mutabilis (Silva et al., 2013aSilva LL, Garlet QI, Benovit SC, Dolci G, Mallmann CA, Bürger ME et al. Sedative and anesthetic activities of the essential oils of Hyptis mutabilis (Rich) Briq. and their isolated components in silver catfish (Rhamdia quelen). Braz J Med Biol Res . 2013a; 46(4):771-779.), Ocotea acutifolia (Silva et al., 2013bSilva LL, Garlet QI, Benovit SC, Dolci G, Mallmann CA, Bürger ME et al. Sedative and anesthetic activities of the essential oils of Hyptis mutabilis (Rich) Briq. and their isolated components in silver catfish (Rhamdia quelen). Braz J Med Biol Res . 2013a; 46(4):771-779.), Hesperozygis ringens (Silva et al., 2013bSilva LL, Silva DT, Garlet QI, Cunha MA, Mallmann CA, Baldisserotto B et al. Anesthetic activity of Brazilian native plants in silver catfish (Rhamdia quelen). Neotrop Ichthyol . 2013b; 11(2):443-451.; Toni et al., 2014Toni C, Becker AG, Simões LN, Pinheiro CG, Silva LL, Heinzmann BM et al. Fish anesthesia: Effects of the essential oils of Hesperozygis ringens and Lippia alba on the biochemistry and physiology of silver catfish (Rhamdia quelen). Fish Physiol Biochem . 2014; 40(3):701-714.), Aloysia triphylla (Parodi et al., 2014Parodi TV, Cunha MA, Becker AG, Zeppenfeld CC, Martins DI, Koakoski G et al. Anesthetic activity of Aloysia triphylla and effectiveness in reducing stress during transport of albine and grey strains of silver catfish, Rhamdia quelen. Fish Physiol Biochem . 2014; 40(2):323-334.) and Ocimum gratissimum (Silva et al., 2015Silva LL, Garlet QI, Koakoski G, Oliveira TA, Barcellos LJG, Baldisserotto B et al. Effects of anesthesia with the essential oil of Ocimum gratissimum L. in parameters of fish stress. Rev Bras Plant Med. 2015; 17(2):215-223.), has been demonstrated in fish. The EO of Lippia alba has been highlighted in the last decade through studies reporting its potential as antioxidant, anesthetic and sedative for fish (Cunha et al., 2010Cunha MA, Barros FMC, Garcia LO, Veeck APL, Heinzmann BM, Loro VL et al. Essential oil of Lippia alba: A new anesthetic for silver catfish, Rhamdia quelen. Aquaculture . 2010; 306(1-4):403-406.; Becker et al., 2012Becker AG, Parodi TV, Heldwein CG, Zeppenfeld CC, Heinzmann BM, Baldisserotto B.. Transportation of silver catfish, Rhamdia quelen, in water with eugenol and the essential oil of Lippia alba. Fish Physiol Biochem. 2012; 38(3):789-796.; Heldwein et al., 2014Heldwein CG, Silva LL, Gai EZ, Roman C, Parodi TV, Bürger ME et al. S-(+)-Linalool from Lippia alba: Sedative and anesthetic for silver catfish (Rhamdia quelen). Vet Anaesth Analg. 2014; 41(6):621-629.; Toni et al., 2014Toni C, Becker AG, Simões LN, Pinheiro CG, Silva LL, Heinzmann BM et al. Fish anesthesia: Effects of the essential oils of Hesperozygis ringens and Lippia alba on the biochemistry and physiology of silver catfish (Rhamdia quelen). Fish Physiol Biochem . 2014; 40(3):701-714.; Hohlenwerger et al., 2016Hohlenwerger JC, Copatti CE, Sena AC, Couto RD, Baldisserotto B, Heinzmann BM et al. Could the essential oil of Lippia alba provide a readily available and cost-effective anaesthetic for Nile tilapia (Oreochromis niloticus)? Mar Freshw Behav Physiol. 2016; 49(2):119-126.).

Lippia alba occurs in all regions of Brazil (Zétola et al., 2002Zétola M, Lima TC, Sonaglio D, Gonzalez-Ortega G, Limberger RP, Petrovick PR et al. CNS activities of liquid and spray-dried extracts from Lippia alba-Verbenaceae (Brazilian false melissa). J Ethnopharmacol . 2002; 82(2-3):207-215.; Oliveira et al., 2006Oliveira DR, Leitão GG, Santos SS, Bizzo HR, Lopes D, Alviano CS et al. Ethnopharmacological study of two Lippia species from Oriximiná, Brazil. J Ethnopharmacol . 2006; 108:103-108.; Neto et al., 2009Neto AC, Netto JC, Pereira PS, Pereira AMS, Taleb-Contini SH, França SC et al. The role of polar phytocomplexes on anticonvulsant effects of leaf extracts of Lippia alba (Mill.) N.E. Brown chemotypes. J Pharm Pharmacol. 2009: 61(7):933-939.; Cunha et al., 2010Cunha MA, Barros FMC, Garcia LO, Veeck APL, Heinzmann BM, Loro VL et al. Essential oil of Lippia alba: A new anesthetic for silver catfish, Rhamdia quelen. Aquaculture . 2010; 306(1-4):403-406.; Teles et al., 2012Teles S, Pereira JA, Santos CHB, Menezes RV, Malheiro R, Lucchese AM et al. Geographical origin and drying methodology may affect the essential oil of Lippia alba (Mill) N.E. Brown. Ind Crops Prod. 2012; 37(1):247-252.; Vale et al., 2012Vale TG, Furtado EC, Santos Júnior JG, Viana GSB. Central effects of citral, myrcene and limonene, constituents of essential oil chemotypes from Lippia alba (Mill.) N.E. Brown. Phytomedicine. 2012; 9(8):709-714.; Soares et al., 2016Soares BV, Neves LR, Oliveira MSB, Chaves FCM, Dias MKR, Chagas EC et al. Antiparasitic activity of the essential oil of Lippia alba on ectoparasites of Colossoma macropomum (tambaqui) and its physiological and histopathological effects. Aquaculture . 2016; 452:107-114.). Due to its genetic variation, wide geographical distribution and exposure to different soil and weather conditions, and distinct seasons of collection, L. alba can produce EOs with different chemical composition, which expresses the occurrence of distinct chemotypes (Pascual et al., 2001Pascual ME, Slowing K, Carretero E, Sánchez Mata D, Villar A. Lippia: traditional uses, chemistry and pharmacology: a review. J Ethnopharmacol . 2001; 76(3):201-214.; Hennebelle et al., 2008Hennebelle T, Sahpaz S, Joseph H, Bailleul F. Ethnopharmacology ofLippia alba. J Ethnopharmacol. 2008; 116(2):211-222.; Maffei et al., 2011Maffei ME, Gertsch J, Appendino G. Plant volatiles: production, function and pharmacology. Nat Prod Rep. 2011; 28(8):1359-1380.; Teles et al., 2012Teles S, Pereira JA, Santos CHB, Menezes RV, Malheiro R, Lucchese AM et al. Geographical origin and drying methodology may affect the essential oil of Lippia alba (Mill) N.E. Brown. Ind Crops Prod. 2012; 37(1):247-252.). There are numerous chemotypes of L. alba in Brazil, such as: citral, linalool, ß-caryophyllene, tagetenone, limonene, carvone, myrcene, γ-terpinene, camphor-1,8-cineole and estragole, which produce different pharmacological effects (Oliveira et al., 2006Oliveira DR, Leitão GG, Santos SS, Bizzo HR, Lopes D, Alviano CS et al. Ethnopharmacological study of two Lippia species from Oriximiná, Brazil. J Ethnopharmacol . 2006; 108:103-108.; Hennebelle et al., 2008Hennebelle T, Sahpaz S, Joseph H, Bailleul F. Ethnopharmacology ofLippia alba. J Ethnopharmacol. 2008; 116(2):211-222.; Vale et al. 2012Vale TG, Furtado EC, Santos Júnior JG, Viana GSB. Central effects of citral, myrcene and limonene, constituents of essential oil chemotypes from Lippia alba (Mill.) N.E. Brown. Phytomedicine. 2012; 9(8):709-714.; Viccini et al., 2014Viccini LF, Silveira RS, Vale AA, Campos JMS, Reis AC, Santos MO et al. Citral and linalool content has been correlated to DNA content in Lippia alba (Mill.) N.E. Brown (Verbenaceae). Ind Crops Prod . 2014; 59:14-19.). Thus, the distinct composition of same EO may result in different physiological and pharmacological effects during anesthesia.

Hematological and biochemical parameters of fish are valuable markers, since they can be used as indicators of physiological conditions, as well as in the control of diseases and stress manipulation (Aldrin et al., 1982Aldrin JF, Messager L, Laurencin FB. La biochimie clinique en aquaculture. Actes Colloq. 1982: 14, 219-326.; Tavares-Dias et al., 2008Tavares-Dias M, Moraes FR, Imoto ME. Hematological parameters in two neotropical freshwater teleost, Leporinus macrocephalus (Anostomidae) and Prochilodus lineatus (Prochilodontidae). Biosci J. 2008; 24(3):96-101.; Araújo et al., 2009Araújo CSO, Tavares-Dias M, Gomes ALS, Andrade SMS, Lemos JRG, Oliveira AT et al. Infecções parasitárias e parâmetros sanguíneos em Arapaima gigas Schinz, 1822 (Arapaimidae) cultivados no estado do Amazonas, Brasil. In: Tavares-Dias M, organizer. Manejo e sanidade de peixes em cultivo. Macapá: Embrapa Amapá; 2009. p.389-424.). Plasma cortisol is one of the most used indicators to evaluate stress in fish (Wendelaar Bonga, 1997Wendelaar Bonga SE. The stress response in fish. Physiol Rev. 1997; 77(3):591-625.) and the two major actions of this hormone are the control of the ionoregulatory balance and energy metabolism (Liew et al., 2015Liew HJ, Fazio A, Faggio C, Blust R, De Boeck G. Cortisol affects metabolic and ionoregulatory responses to a different extent depending on feeding ration in common carp, Cyprinus carpio. Comp Biochem Physiol A Mol Integr Physiol. 2015; 189:45-57.). The electrolytic imbalance can be observed by changes in plasma or blood ions (McDonald, Milligan, 1997McDonald G, Milligan L. Ionic, osmotic and acid base regulation in stress. In: Iwama GK, Pickering AD, Sumpter JP, Schreck CB, editors. Fish stress and health in aquaculture. Cambridge: Cambridge University Press; 1997. p.119-144.; Wendelaar Bonga, 1997Wendelaar Bonga SE. The stress response in fish. Physiol Rev. 1997; 77(3):591-625.; Takahashi et al., 2006Takahashi LS, Abreu JS, Biller JD, Urbinati EC. Efeito do ambiente pós-transporte na recuperação dos indicadores de estresse de pacus juvenis, Piaractus mesopotamicus. Acta Sci AnimSci. 2006; 28(4):469-475.). Glucose levels are also widely used as indicator of stress, hyperglycaemia being reported for several teleosts in this situation (Barton, Iwama, 1991Barton BA, Iwama GK. Physiological changes in fish from stress in aquaculture with emphasis on the responses and effects of corticosteroids. Annu Rev Fish Dis. 1991; 1: 3-26.). Stress also has an effect on other blood biochemical parameters such as levels of enzymes and substances with important metabolic functions, such as urea and creatinine, which indicate the overall health of the fish (Cnaani et al., 2004Cnaani A, Tinman S, Avidar Y, Ron M, Hulata G. Comparative study of biochemical parameters in response to stress in Oreochromis aureus, O. mossambicus and two strains of O. niloticus. Aquacult Res . 2004; 35(15):1434-1440.).

Physiological effects of the EO of L. alba cultivated in southern Brazil as anesthetic and sedative for silver catfish, Rhamdia quelen, was verified by many authors (Cunha et al., 2010Cunha MA, Barros FMC, Garcia LO, Veeck APL, Heinzmann BM, Loro VL et al. Essential oil of Lippia alba: A new anesthetic for silver catfish, Rhamdia quelen. Aquaculture . 2010; 306(1-4):403-406.; Heldwein et al., 2014Heldwein CG, Silva LL, Gai EZ, Roman C, Parodi TV, Bürger ME et al. S-(+)-Linalool from Lippia alba: Sedative and anesthetic for silver catfish (Rhamdia quelen). Vet Anaesth Analg. 2014; 41(6):621-629.; Toni et al., 2014Toni C, Becker AG, Simões LN, Pinheiro CG, Silva LL, Heinzmann BM et al. Fish anesthesia: Effects of the essential oils of Hesperozygis ringens and Lippia alba on the biochemistry and physiology of silver catfish (Rhamdia quelen). Fish Physiol Biochem . 2014; 40(3):701-714.; Salbego et al., 2014Salbego J, Becker AG, Gonçalves JF, Menezes CC, Heldwein CG, Spanevello RM et al. The essential oil from Lippia alba induces biochemical stress in the silver catfish (Rhamdia quelen) after transportation. Neotrop Ichthyol. 2014; 12(4):811-818.), but only the linalool chemotype. Therefore, it is of interest to investigate the anesthetic and physiological effects of EO obtained from other chemotypes of L. alba. Considering that a different chemotype of L. alba (myrcene-citral) cultivated in northern Brazil was identified by Oliveira et al. (2006Oliveira DR, Leitão GG, Santos SS, Bizzo HR, Lopes D, Alviano CS et al. Ethnopharmacological study of two Lippia species from Oriximiná, Brazil. J Ethnopharmacol . 2006; 108:103-108.), the aim of this study was to investigate a possible geographic effect in the EO composition (L. alba cultivated in northern and southern Brazil) and, if these EOs have different compositions, to evaluate their sedative and anesthetic effects in silver catfish, as well as their physiological effects on blood and plasma parameters.

Materials and Methods

Animals. One hundred sixty-eight juveniles silver catfish (Rhamdia quelen; 51.17 ± 1.69 g and 20.21 ± 1.40 cm) were obtained from a local fish farm and brought to the Fish Physiology Laboratory at the Universidade Federal de Santa Maria (UFSM). The species was identified at the Ichthyology Laboratory (Universidade Federal do Rio Grande do Sul) and a voucher specimen was deposited in this laboratory at number UFRGS 19612. The fish were maintained for one week in 250 L tanks (50 fish/tank) with continuous aeration; temperature 21 ± 2 °C; pH 6.5-7.5 and dissolved oxygen above 5.5 mg L-1. The animals were fed once a day with commercial feed and kept fasted for a period of 24 h prior to the experiments. The experimental protocol was approved by the Committee on Animal Experimentation - UFSM, under the registration number 074/2014.

Essential oils extraction and analysis. The specimens of Lippia alba linalool chemotype were cultivated at the Centro de Educação Superior do Norte (CESNORS-UFSM) - Frederico Westphalen, Rio Grande do Sul State, southern Brazil (27º23’48”S, 53º25’45”W), soil classified as Oxisol typical clayey. The climate is Cfa (humid subtropical) with average annual temperature of 19.1°C and rainfall of 1892 mm. Those from the citral chemotype were cultivated in Santarém, Pará state, northern Brazil (02º26’35”S, 054º54’54”W), soil classified as ultisol yellow Hapludox + yellow latosol Hapludox, but in the culture it was used black soil and cattle manure (3:1). The climate is Am (humid tropical) with average annual temperature of 25.9°C and rainfall of 2,150 mm.

Botanical identification of L. alba linalool chemotype was made by Gilberto Dolejal Zanetti (Department of Industrial Pharmacy, UFSM) and a voucher specimen (SMDB 10050) was deposited in the herbarium of the Department of Biology (UFSM). The L. alba citral chemotype was identified by Dr. Fatima Salimena (Universidade Federal de Juiz de Fora) and a voucher was registered in the herbarium of this institution under number CESJ 65276.

The essential oils were obtained by hydrodistillation of fresh leaves for 3h in a Clevenger apparatus (European Pharmacopoeia, 2007) and stored at -4oC until utilization. The analysis of the EOs was performed by gas chromatography-mass spectrometry-total ion chromatogram using an Agilent 6890 gas chromatograph coupled with an Agilent 5973 mass selective detector and employing a HP5-MS column (5% phenyl, 95% methylsiloxane, 30 m x 0.25 mm i.d. x 0.25 mm) as described by Silva et al. (2012Silva LL, Parodi TV, Rekcziegel P, Garcia VO, Bürger ME, Baldisserotto B et al. Essential oil of Ocimum gratissimum L.: anesthetic effects, mechanism of action and tolerance in silver catfish, Rhamdia quelen. Aquaculture . 2012; 350-353:91-97.). The constituents were identified by comparison of the Kovats retention index and their mass spectra with data from the mass spectral library (NIST, 2002NIST/EPA/NIH. Mass spectral library and search/analysis programs. John Wiley and Sons, Hoboken; 2002.) and the literature (Adams, 2001Adams RP. Identification of essential oil components by gas chromatography/quadrupole mass spectroscopy. 3rd ed. Illinois: Allured Publishing Corporation; 2001.).

Experiment 1. Anesthetic induction and recovery times. Anesthesia induction and recovery were tested at concentrations of 25, 50, 100, 200, 300 µL L-1 for both EOs. Eight fish were used for each concentration tested, and each juvenile was used only once. Sedation was characterized by the decreased reactivity to external stimuli, and anesthesia by total loss of equilibrium and cessation of locomotion, according to Small (2003Small BC. Anesthetic efficacy of metomidate and comparison of plasma cortisol responses to tricaine methanesulfonate, quinaldine and clove oil anesthetized channel catfish Ictalurus punctatus. Aquaculture . 2003; 218(1-4):177-185.). The EOs were previously diluted in 95% ethanol (1:10). Ethanol at the highest concentration used does not have any anesthetic effect in silver catfish (Cunha et al., 2010Cunha MA, Barros FMC, Garcia LO, Veeck APL, Heinzmann BM, Loro VL et al. Essential oil of Lippia alba: A new anesthetic for silver catfish, Rhamdia quelen. Aquaculture . 2010; 306(1-4):403-406.). After induction, fish were transferred to anesthetic-free aquaria to measure anesthesia recovery time. The fish were considered to be recovered when they returned to normal swimming and reacted to external stimuli.

Experiment 2. Exposure to anesthetics for physiological evaluation. Silver catfish were individually placed in an 8 L aquarium containing one of the EOs at 100 µL L-1 for up to 5 min or 300 µL L-1 for up to 2 min. These concentrations led to sedation and deep anesthesia, respectively (Cunha et al., 2010Cunha MA, Barros FMC, Garcia LO, Veeck APL, Heinzmann BM, Loro VL et al. Essential oil of Lippia alba: A new anesthetic for silver catfish, Rhamdia quelen. Aquaculture . 2010; 306(1-4):403-406.). Afterwards, fish from all groups were transferred individually to 8 L aquaria with anesthetic-free water for up to 10 min for recovery. There were also groups subjected to 26700 µL L-1 ethanol (the concentration used to dilute the highest EO concentration) and water only (control), which were handled as outlined above.

Blood analysis. Blood was collected from the caudal vein of silver catfish in less than 30 s with heparinized syringes at 1, 2 and 5 min of exposure (groups exposed to the concentration of 300 µL L-1 were not assessed at 5 min) and 5 and 10 min of recovery (total of 30 fish per treatment, n = 6 for each EO, concentration and collection time, each fish was sampled only once). Control and ethanol exposed fish were held tightly for blood collection. An aliquot of this blood was used to measure Na+, K+, Ca2 +, glucose, pH, partial pressures of O2 (pO2) and CO2 (pCO2) using the i-STAT® portable clinical analyzer with CG8+ cartridges (Abbott Laboratories, Chicago, IL, USA). The sample temperature was corrected to match the experimental water temperature (Roth, Rotabakk, 2012Roth B, Rotabakk BT. Stress associated with commercial longlining and recreational fishing of saithe (Pollachius virens) and the subsequent effect on blood gases and chemistry. Fish Res. 2012;115-116:110-114.). The use of i-STAT® and calculations for blood gases have been described for several fish species (Jacobs et al., 1993Jacobs E, Vadasdi E, Sarkozi L, Coman N. Analytical evaluation of i-STAT® portable clinical analyzer and use by non-laboratory health-care professionals. Clin Chem. 1993; 39(6):1069-1074.; Pidetcha et al., 2000Pidetcha P, Ornvichian S, Chalachiva S. Accuracy and precision of the i-STAT® portable clinical analyzer: an analytical point of view. J Med Assoc Thai. 2000; 83(4):445-450.; Harrenstien et al., 2005Harrenstien LA, Tornquist SJ, Miller-Morgan TJ, Fodness BG, Clifford KE. Evaluation of a point-of-care blood analyzer and determination of reference ranges for blood parameters in rockfish. J Am Vet Med Assoc . 2005; 226(2):255-265.; Kristensen et al., 2010Kristensen T, Rosseland BO, Kiessling A, Djordevic B, Massabau JC. Lack of arterial pO2 down regulation in Atlantic salmon (Salmo salar L.) during long-term normoxia and hyperoxia. Fish Physiol Biochem. 2010; 36(4):1087-1095.; Barbas et al., 2016Barbas LAL, Stringhetta GR, Garcia LO, Figueiredo MRC, Sampaio LA. Jambu, Spilanthes acmella as a novel anaesthetic for juvenile tambaqui, Colossoma macropomum: secondary stress responses during recovery. Aquaculture. 2016; 456:70-75.).

Plasma analysis. The remaining blood collected was centrifuged (800 x g for 10 min) and the plasma was used for analysis of creatinine and urea using an automated Vitros 250 (Ortho - Clinical Diagnostics) and Johnson & Johnson dry chemistry kits. All tests were carried out in duplicate.

Plasma cortisol was also determined in duplicate using an enzyme-linked immunosorbent assay (ELISA) kit (Diagnostics Biochem Canada Inc., Canada). This analysis was previously validated (Souza et al., 2015Souza CF, Salbego J, Gressler LT, Golombieski JI, Ferst JG, Cunha MA et al. Rhamdia quelen (Quoy & Gaimard, 1824) , submitted to a stressful condition: effect of dietary addition of the essential oil of Lippia alba on metabolism, osmoregulation and endocrinology. Neotrop Ichthyol . 2015; 13(4):707-714.). Absorbance was measured in a spectrophotometer at 450 nm, and intra- and inter-assay coefficients of variation were 6.3% and 5.2%, respectively.

Statistical analysis. Data are reported as mean ± SE. The homogeneity of variances among groups was determined with the Levene test. All treatment groups were compared by two-way analysis of variance (time x concentration) and Tukey’s test; or, when homogeneity of variances was not obtained, by the Scheirer-Ray-Hare extension of the Kruskal-Wallis test and the Nemenyi test. Analyses were performed using the STATISTICA software package, version 5.1 (StatSoft, Tulsa, OK, USA), and the minimum significance level was set at p < 0.05.

Results

Chemical composition of the essential oils. A total of 65 compounds were identified in the EO of L. alba collected in southern Brazil (EO-L) and 67 compounds in the EO of L. alba collected in northern Brazil (EO-C) (Tab. 1). The major component in the EO-L was β-linalool (50.56%), while the major compounds in the EO-C were E-citral (29.84%) and Z-citral (24.41%).

Tab. 1
Chemical composition of the essential oils of Lippia alba collected from southern (linalool chemotype - EO-L) and northern (citral chemotype - EO-C) Brazil. RI calc= calculated Kovats retention index; RI ref= reference Kovats retention index; (Adams, 2001Adams RP. Identification of essential oil components by gas chromatography/quadrupole mass spectroscopy. 3rd ed. Illinois: Allured Publishing Corporation; 2001.; NIST, 2002NIST/EPA/NIH. Mass spectral library and search/analysis programs. John Wiley and Sons, Hoboken; 2002.).

Anesthetic induction and recovery times. Both EO-C concentrations induced sedation faster than EO-L, but anesthesia was faster only at the lowest EO-C concentration. Fish anesthetized with EO-C took longer to recover than those anesthetized with EO-L (Fig. 1). Ethanol did not show any sedative or anesthetic effect.

Fig. 1
Time required for silver catfish (Rhamdia quelen) anesthesia induction and recovery (n=8 for each concentration tested) using different concentrations of essential oils from the linalool (EO-L) and citral (EO-C) chemotypes of Lippia alba. Stages are defined according to Small (2003Small BC. Anesthetic efficacy of metomidate and comparison of plasma cortisol responses to tricaine methanesulfonate, quinaldine and clove oil anesthetized channel catfish Ictalurus punctatus. Aquaculture . 2003; 218(1-4):177-185.). Values are mean ± SEM. Different letters indicate difference between concentrations and EOs in the same anesthetic stage. Based on two-way ANOVA followed by the Tukey post hoc test (p < 0.05).

Blood analysis. Blood pH, Na+, K+ and Ca2+ did not differ between any of the treatments and overall means were: pH (7.32 ± 0.04), Na+ (134.90 ± 0.89 mmol L-1), K+ (2.89 ± 0.12 mmol L-1), Ca2+(1.18 ± 0.06 mmol L-1).

Overall, blood pO2 levels of silver catfish anesthetized with both EOs and exposed to ethanol were lower than control fish, increasing after 10 min recovery (Figs. 2a-b). In contrast, an increase in pCO2 was observed for fish anesthetized with EO-C and no significant change was observed for silver catfish anesthetized with EO-L. At the end of 10 min of recovery fish anesthetized with EO-C still maintained blood PCO2 levels higher than control fish (Figs. 2c-d).

Fig. 2
Partial pressures of a-b oxygen (PO2) and c-d carbon dioxide (PCO2) in silver catfish (n = 6) (Rhamdia quelen) submitted to different concentrations of essential oils from the linalool (EO-L) and citral (EO-C) chemotypes of Lippia alba. Values are mean ± SEM. Different letters indicate significant difference between times within the same EO concentration. * indicate significant difference from control, # indicate significant difference from ethanol. Two-way ANOVA and Tukey’s test or Scheirer-Ray-Hare extension of the Kruskal-Wallis test and Nemenyi test.

Blood glucose levels increased in control and ethanol groups after 5 min, compared to initial values, returning to initial values at the end of the recovery period in the control group. Exposure to both EOs (except 300 µL L-1 EO-L) did not avoid this increase of blood glucose levels. At the end of the recovery period, the blood glucose levels of silver catfish exposed to ethanol and both EOs (except 300 µL L-1 EO-L) remained higher than the initial values and higher than those of the control group (Figs. 3a-b).

In the control group, plasma cortisol levels decreased after 2 min and remained low until the end of the recovery period. In fish exposed to ethanol, cortisol levels decreased up to 5 min after exposure and returned to the initial values after 5 min of recovery. Overall, exposure to both EOs (except 100 µL L-1 EO-L at most times) reduced plasma cortisol levels compared to the control and/or ethanol groups (Figs. 3c-d).

Fig. 3
a-b. Blood glucose (Glu) and c-d. plasma cortisol in silver catfish (Rhamdia quelen) (n = 6) subjected to different concentrations of essential oils from the linalool (EO-L) and citral (EO-C) chemotypes of Lippia alba. Values are mean ± SEM. Different letters indicate significant differences between times within the same treatment. * indicates significant difference from control, # indicates significant difference from ethanol. Two-way ANOVA and Tukey’s test or Scheirer-Ray-Hare extension of the Kruskal-Wallis test and Nemenyi test were used to determine statistical significance.

Plasma creatinine values of control fish increased significantly after 5 min and remained high at the end of the recovery period. Fish exposed to ethanol and both EOs showed significantly higher creatinine levels 1 min after exposure compared to control fish and these levels returned to control values at the end of recovery only in those exposed to EO-L (Figs. 4a-b). Plasma urea levels in the control group remained constant at all evaluation times. Fish exposed to ethanol showed a significant increase in plasma urea levels when compared to control fish after 5 min of exposure and returned to control values at the end of the recovery period. Plasma urea was significantly higher with most anesthesia treatments and recovery times in fish exposed to both EOs when compared to the control and ethanol groups (Figs. 4c-d).

Fig. 4
a-b. Plasma urea and c-d creatinine in silver catfish (Rhamdia quelen) (n = 6) subjected to different concentrations of essential oils from the linalool (EO-L) and citral (EO-C) chemotypes of Lippia alba. Values are mean ± SEM. Different letters indicate significant differences between times within the same treatment. * indicates significant difference from control, # indicates significant difference from ethanol. Two-way ANOVA and Tukey’s test or Scheirer-Ray-Hare extension of the Kruskal-Wallis test and Nemenyi test were used to determine statistical significance.

Discussion

Since EOs represent a chemical interface between plant and the surrounding environment, their syntheses are often affected by environmental conditions, thus expressing the occurrence of chemotypes or chemical races in the producing plant species (Gobbo-Neto, Lopes, 2007Gobbo-Neto L, Lopes NP. Plantas medicinais: fatores de influência no conteúdo de metabólitos secundários. Quím Nova. 2007; 30(2):374-381.). Although there are many examples of the occurrence of geographic variations of EOs chemical composition in several plants (Figueiredo et al., 2008Figueiredo AC, Barroso JG, Pedro LG, Scheffer JJC. Factors affecting secondary metabolite production in plants: volatile components and essential oils. Flavour Frag J. 2008; 23(4):213-226.), the distribution of chemotypes is often not locally limited. In some species, different chemotypes can grow side by side (Schmidt et al., 2004Schmidt A, Bischof-Deichnik C, Stahl-Biskup E. Essential oil polymorphism of Thymus praecox subsp. arcticus on the British Isles. Biochem Syst Ecol. 2004; 32(4):409-421.).

The present study demonstrates that the EO from L. alba cultivated by our group in southern Brazil has linalool as its main compound (50.56%), and so it belongs to chemotype linalool. On the other hand, L. alba collected in northern Brazil can be classified in the chemotype citral, once this is the major compound of its EO (54.26%). Some authors indicated that geographical distribution and exposure to different soil and weather conditions, season of collection can affect the chemical composition of L. alba EO (Pascual et al., 2001Pascual ME, Slowing K, Carretero E, Sánchez Mata D, Villar A. Lippia: traditional uses, chemistry and pharmacology: a review. J Ethnopharmacol . 2001; 76(3):201-214.; Hennebelle et al., 2008Hennebelle T, Sahpaz S, Joseph H, Bailleul F. Ethnopharmacology ofLippia alba. J Ethnopharmacol. 2008; 116(2):211-222.; Maffei et al., 2011Maffei ME, Gertsch J, Appendino G. Plant volatiles: production, function and pharmacology. Nat Prod Rep. 2011; 28(8):1359-1380.; Teles et al., 2012Teles S, Pereira JA, Santos CHB, Menezes RV, Malheiro R, Lucchese AM et al. Geographical origin and drying methodology may affect the essential oil of Lippia alba (Mill) N.E. Brown. Ind Crops Prod. 2012; 37(1):247-252.). However, specimens from the chemotypes citral, linalool and carvone, collected in different regions of Brazil, cultivated in similar conditions, maintained the same chemical composition, indicating that differences are due to genotypic variations (Tavares et al., 2005Tavares ES, Julião LS, Lopes D, Bizzo HR, Lage CLS, Leitão SG. Análise do óleo essencial de folhas de três quimiotipos de Lippia alba (Mill.) N. E. Br. (Verbenaceae) cultivados em condições semelhantes. Rev Bras. Farmacogn. 2005; 15(1):1-5.).

The EO-C anesthetized silver catfish within approximately 2 min at 300 µL L-1, inducing anesthesia faster than EO-L. Anesthesia recovery was slower with EO-C, but it can be considered and adequate anesthetic for silver catfish, because an ideal anesthetic must induce anesthesia up to 3 min and enable the recovery in about 10 min (Park et al., 2008Park MO, Hur WJ, Im SY, Seol DW, Lee J, Park IS. Anaesthetic efficacy and physiological responses to clove oil anaesthetized kelp grouper Epinephelus bruneus. Aquacult Res . 2008; 39(8):877-884.). The anesthetic effect of EO-L in silver catfish involves the modulation of the benzodiazepine (BDZ) site of the GABAergic system (Heldwein et al., 2012Heldwein CG, Silva LL, Reckziegel P, Barros FMC, Bürger ME, Baldisserotto B et al. Participation of the GABAergic system in the anesthetic effect of Lippia alba (Mill.) N. E. Brown essential oil. Braz J Med Biol Res. 2012; 45(5):436-443.). The EO-C blocks the excitability of rat sciatic nerves (Sousa et al., 2015Sousa DG, Sousa SDG, Silva RER, Silva-Alves KS, Ferreira-da-Silva FW, Kerntopf MR et al. Essential oil of Lippia alba and its main constituent citral block the excitability of rat sciatic nerves. Braz J Med Biol Res . 2015; 48(8):697-702.), but the anesthetic effect of the EO from Aloysia triphylla (which has citral as its major compound) in silver catfish is not related to a modulation of the BDZ site of the GABAA receptor (Santos et al., in pressSantos AC, Junior GB, Zago DC, Zeppenfeld CC, Silva DT, Heinzmann BM et al. Anesthesia and anesthetic action mechanism of essential oils of Aloysia triphylla and Cymbopogon flexuosus in silver catfish (Rhamdia quelen). Vet Anaesth Analg . Forthcoming 2017.).

All parameters examined in this study are within the range previously observed for silver catfish (Barcellos et al., 2001Barcellos LJG, Woehl VM, Wassermann GF, Quevedo RM, Ittzés I, Krieger MH. Plasma levels of cortisol and glucose in response to capture and tank transference in Rhamdia quelen (Quoy & Gaimard), a South American catfish. Aquacult Res. 2001; 32(2):121-123.; 2004Barcellos LJG, Conrad J, Kreutz LC, Souza C de, Rodrigues LB, Fioreze I, et al. Hematological changes in jundiá (Rhamdia quelen Quoy and Gaimard Pimelodidae) after acute and chronic stress caused by usual aquacultural management, with emphasis on immunosuppressive effects. Aquaculture . 2004; 237(1-4):229-236.). Blood Na+, K+ and Ca2+ of silver catfish were not affected by anesthesia with either EO tested. Similar results were found in the blood of Amazon catfish (Leiarius marmoratus) anesthetized with 10-200 µL L-1 eugenol (Honorato et al., 2014Honorato CA, Dambros A, Marcondes VM, Nascimento CA. Utilização do eugenol em jundiá da Amazônia (Leiarius marmoratus): implicações na sedação e avaliação hemogasométrica. Semin-Ciênc Agrár. 2014; 35(5):2759-2768.) and in the plasma of silver catfish anesthetized with 150 and 300 mg L-1 MS-222 (Gressler et al., 2014Gressler LT, Riffel APK, Parodi TV, Saccol EMH, Koakoski G, Costa ST et al. Silver catfish Rhamdia quelen immersion anaesthesia with essential oil of Aloysia triphylla (L’Hérit) Britton or tricaine methanesulfonate: effect on stress response and antioxidant status. Aquacult Res . 2014; 45(6):1061-1072.). However, silver catfish anesthetized with 150-450 µL L-1 of Hesperozygis ringens and Lippia alba (EO-L) showed altered plasma Na+ and K+ between 30-240 min of recovery (Toni et al., 2014Toni C, Becker AG, Simões LN, Pinheiro CG, Silva LL, Heinzmann BM et al. Fish anesthesia: Effects of the essential oils of Hesperozygis ringens and Lippia alba on the biochemistry and physiology of silver catfish (Rhamdia quelen). Fish Physiol Biochem . 2014; 40(3):701-714.) and anesthesia of tambaqui with 20 mg L-1 jambu extract induced blood ionoregulatory changes 2 h after recovery from anesthesia and Na+ levels did not return to control values, even after 72 h (Barbas et al., 2016Barbas LAL, Stringhetta GR, Garcia LO, Figueiredo MRC, Sampaio LA. Jambu, Spilanthes acmella as a novel anaesthetic for juvenile tambaqui, Colossoma macropomum: secondary stress responses during recovery. Aquaculture. 2016; 456:70-75.). Apparently ionoregulatory effects of anesthesia in blood or plasma are significant only after a few hours of recovery, when they can be detected.

During fish anesthesia, opercular movement (respiration) generally decreases compared to conscious fish, explaining the lower blood pO2 in silver catfish anesthetized with both EOs and the higher pCO2 in those anesthetized with EO-C. Through anesthetic recovery from both EOs there was an increase in blood pO2 levels and a reduction in pCO2 levels in those exposed to EO-C, which is expected for the recovery period with normal return of opercular movements. These same oscillation patters in pO2 and pCO2 from fish anesthesia, were found for red “pacu” (Piaractus brachypomus) anesthetized with MS-222 (150 mg L-1) (Hanley et al., 2010Hanley CS, Clyde VL, Wallace RS, Paul-Murphy J, Patterson TA, Keuler NS et al. Effects of anesthesia and surgery on serial blood gas values and lactate concentrations in yellow perch (Perca flavescens), walleye pike (Sander vitreus), and koi (Cyprinus carpio). J Am Vet Med Assoc. 2010; 236(10):1104-1108.) and “tambaqui” (Colossoma macropomum) anesthetized with waxy extract of “jambu” flowers (Spilanthes acmella) at 20 mg L-1 (Barbas et al., 2016Barbas LAL, Stringhetta GR, Garcia LO, Figueiredo MRC, Sampaio LA. Jambu, Spilanthes acmella as a novel anaesthetic for juvenile tambaqui, Colossoma macropomum: secondary stress responses during recovery. Aquaculture. 2016; 456:70-75.).

An increase in plasma levels of glucocorticoids such as cortisol is one of the main responses to stress (Barton, 2002Barton BA. Stress in fishes: a diversity of responses with particular reference to changes in circulating corticosteroids. Integr Comp Biol. 2002; 42(3):517-525.). Plasma cortisol increases significantly in juvenile R. quelen 5 - 30 min after handling (Koakoski et al., 2012Koakoski G, Oliveira TA, Rosa JGS, Fagundes M, Kreutz LC, Barcellos LJG. Divergent time course of cortisol response to stress in fish of different ages. Physiol Behav. 2012; 106(2):129-132.), but surprisingly, handling was not sufficient to raise the plasma cortisol in the control group of silver catfish in our study. Toni et al. (2015Toni C, Martos-Sitcha JA, Ruiz-Jarabo I, Mancera JM, Martínez-Rodríguez G, Pinheiro CG et al. Stress response in silver catfish (Rhamdia quelen) exposed to the essential oil of Hesperozygis ringens. Fish Physiol Biochem . 2015; 41(1):129-138.) observed no increase in plasma cortisol levels of silver catfish exposed for 6 h to 30 and 50 µL L-1 EO from Hesperozygis ringens and they proposed that the primary stress reaction only took place in the first minutes after contact with the EO, as observed in fish exposed to EO-L in the present study.

A study by Gesto et al. (2014Gesto M, Otero-Rodiño C, López-Patiño MA, Míguez JM, Soengas JL, Conde-Sieira M. Is plasma cortisol response to stress in rainbow trout regulated by catecholamine-induced hyperglycemia? Gen Comp Endocrinol. 2014; 205:207-217.) using stressed rainbow trout (Oncorhynchus mykiss) showed that when catecholamines were released in the blood no changes in cortisol levels were observed as glucose levels increased. As plasma cortisol levels did not increase significantly, we suppose that the increase of blood glucose in silver catfish observed in the present study might be due to catecholamine release. According to Morgan, Iwama (1997Morgan JD, Iwama GK. Measurements of stressed states in the field. In: Iwama GK, Pickering AD, Sumpter JP, Schreck CB, editors. Fish stress and health in aquaculture. Cambridge: Cambridge University Press; 1997. p.247-270.), an increase in blood glucose occurs in response to a stressor, in order to provide most of the energy demand to cope with this stress. Our results corroborate those obtained by Inoue et al. (2011Inoue LAKA, Boijink CL, Ribeiro PT, Silva AMD, Affonso EG. Avaliação de respostas metabólicas do tambaqui exposto ao eugenol em banhos anestésicos. Acta Amaz. 2011; 41(2):327-332.) and Honorato et al. (2014Honorato CA, Dambros A, Marcondes VM, Nascimento CA. Utilização do eugenol em jundiá da Amazônia (Leiarius marmoratus): implicações na sedação e avaliação hemogasométrica. Semin-Ciênc Agrár. 2014; 35(5):2759-2768.), who observed that anesthesia with eugenol increased plasma glucose compared to the sham control in “tambaqui” (20 and 60 mg L-1) and Amazon catfish (10-200 µL L-1). Anesthesia with 20 mg L-1 “jambu” extract also increased blood glucose levels in “tambaqui” (Barbas et al., 2016Barbas LAL, Stringhetta GR, Garcia LO, Figueiredo MRC, Sampaio LA. Jambu, Spilanthes acmella as a novel anaesthetic for juvenile tambaqui, Colossoma macropomum: secondary stress responses during recovery. Aquaculture. 2016; 456:70-75.). Several studies testing a variety of anesthetics on multiple fish species also demonstrated increased glycemia after anesthesia induction (Ortuno et al., 2002Ortunõ J, Esteban MA, Meseguer J. Effects of four anaesthetics on the innate immune response of gilthead seabream (Sparus aurata L.). Fish Shellfish Immunol. 2002; 12(1):49-59.; Deriggi et al., 2006Deriggi FG, Inoue LAKA, Moraes G. Stress responses to handling in Nile tilapia (Oreochromis niloticus Linnaeus): assessment of eugenol as an alternative anesthetic. Acta Sci Anim Sci. 2006; 28(3):269-274.; Barbosa et al., 2007Barbosa LG, Moraes G, Inoue LAKA. Respostas metabólicas do matrinxã submetido a banhos anestésicos de eugenol. Acta Sci Biol Sci. 2007; 29:255-260.; Park et al., 2008Park MO, Hur WJ, Im SY, Seol DW, Lee J, Park IS. Anaesthetic efficacy and physiological responses to clove oil anaesthetized kelp grouper Epinephelus bruneus. Aquacult Res . 2008; 39(8):877-884.).

An increase in plasma urea levels in silver catfish during anesthesia and recovery was observed for both EOs when compared to the control group. The same result was obtained for plasma creatinine levels, but these levels were much higher in silver catfish anesthetized with EO-C than in control fish, and these levels returned to control values after 10 min recovery in those exposed to EO-L. Anesthesia of goldfish (Carassius auratus) with 50 µL L-1 nanoencapsulated clove oil also increased serum urea levels (Gholipourkanani et al., 2015Gholipourkanani H, Gholinasab-Omran I, Ebrahimi P, Jafaryan H. Anesthetic effect of clove oil loaded on lecithin based nano emulsions in gold fish, Carassius auratus. J Fish Aquat Sci. 2015; 10(6):553-561.). Increases in urea and creatinine levels together are probably due to lesions caused in the kidney of fish (Das, Mukherjee, 2003Das BK, Mukherjee SC. Toxicity of cypermethrin in Labeo rohita fingerlings: biochemical, enzymatic and haematological consequences. Comp Biochem Physiol C Toxicol Pharmacol. 2003; 134(1):109-121.). Studies conducted by Borges et al. (2007Borges A, Scotti LV, Siqueira DR, Zanini R, Amaral FD, Jurinitz DF et al. Changes in hematological and serum biochemical values in Jundiá Rhamdia quelen due to sub-lethal toxicity of cypermethrin. Chemosphere. 2007; 69(6):920-926.) showed an increase in urea and creatinine levels in the serum of silver catfish exposed to cypermethrin, suggesting that these analyses can be useful for early detection of intoxication in fish. However, as nitrogen compounds are excreted as ammonia mainly through the gills (Nawata et al., 2007Nawata CM, Hung CCY, Tsui TKN, Wilson JM, Wright PA, Wood CM. Ammonia excretion in rainbow trout (Oncorhynchus mykiss): evidence for Rh glycoprotein and H+-ATPase involvement. Physiol Genomics. 2007; 31(3):463-474.), and time of exposure to the EOs was brief, the elevation of urea levels observed in both EO groups in our study may be due to changes in ammonia and creatinine gill excretion and not to renal lesions.

In summary, different chemotypes of L. alba were detected according to their place of cultivation. We suggest that the EO-L and EO-C can be safely used as anesthetics in silver catfish, because the alterations in most parameters returned to control values within 10 min. However, the EO obtained from different chemotypes of the L. alba presented different physiological responses in plasma creatinine and the use of EO-L is preferable because the high creatinine levels provoked by EO-C exposure. Additional studies with longer exposure and/or recovery times are necessary to improve our understanding of the effects of the EO of this chemotype on renal function.

Acknowledgments

The authors are grateful to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Comissão de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS-PRONEX), Ministério da Pesca e Aquicultura/Ministério da Ciência e Tecnologia/FINEP and INCT-ADAPTA (CNPq - FAPEAM).

References

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Publication Dates

  • Publication in this collection
    2017

History

  • Received
    09 June 2016
  • Accepted
    23 Dec 2016
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