Aquaculture 494 (2018) 37–44

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Aquaculture

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Control of Hysterothylacium sp. (Nematoda: Anisakidae) in juvenile pirarucu T (Arapaima gigas) by the oral application of essential oil of Piper aduncum

Amanda Curiel Trentin Corrala, Marieta Nascimento de Queiroza, Sanny Maria de Andrade-Portob, German Augusto Murrieta Moreyc, Francisco Celio Maia Chavesd, Valdelira Lia Araújo Fernandese, Eduardo Akifumi Onoa, ⁎ Elizabeth Gusmão Affonsoe, a Nilton Lins University, Av. Professor Nilton Lins 3259, Parque das Laranjeiras, 69058-030 Manaus, AM, Brazil b Federal University of Amazonas – UFAM, Faculdade de Ciências Agrárias, Departamento de Ciências Pesqueiras, Laboratório de Sanidade de Animais Aquáticos, Avenida Rodrigo Otávio, 6200, Setor Sul, Coroado, 69080-000 Manaus, AM, Brazil c Federal University of Paraná – UFPR, Laboratório de Ecologia Molecular e Parasitologia Evolutiva, rua XV de novembro, 1299, 80060-000 Curitiba, PR, Brazil d Empresa Brasileira de Pesquisa Agropecuária – EMBRAPA, Rodovia AM 10, km 29, s/n, 69010-970 Manaus, AM, Brazil e National Amazonia Research Institute - INPA, Coordenação de Tecnologia e Inovação, Avenida André Araújo 2936, 69067-375 Manaus, AM, Brazil

ARTICLE INFO ABSTRACT

Keywords: The Hysterothylacium sp. (Nematoda, Anisakidae) may cause significant economic losses in fish pro- Farming duction. In order to control the nematode larvae in juvenile pirarucu (52 ± 3.2 g), the efficacy of essential oil Phytotherapic drug (EO) of Piper aduncum when added to feed was evaluated. Chemical analysis of the EO composition had been Helminths performed previously, showing that 92% of the substance was dillapiole. After the palatability test, the efficacy Amazon of the EO was evaluated (0, 32, 48, 56, and 64 mL/kg) at 7 and 15 days, based on the blood parameters and the parasite load of the fish. The results showed significant differences for hematocrit and corpuscular constants at 7 days and for total proteins at 15 days between the treatment groups and the control group (without EO). The parasitic indices showed a prevalence of 100% for stage L3 larva of the nematode Hysterothylacium sp. At 7 and 15 days, the values of average intensity, average abundance, and intensity of parasites decreased with increasing EO concentration, and at 15 days, these were significantly lower between the treatment group and the control group, with an efficacy of 76.21% for 64 mL/kg. Thus, the EO of P. aduncum demonstrates anthelmintic potential in the control of nematode larvae in pirarucu without affecting fish homeostasis.

1. Introduction spinulosa Diesing, 1939 (Araújo et al., 2009); Nilonema senticosum (Mathews et al., 2014); the monogenoids Dawestrema cycloancistrium The production of the pirarucu Arapaima gigas (Osteoglossiformes, Price & Nowlin, 1967; Dawestrema cycloancistrioides Kritsky, Boeger & Arapaimatidae), an endemic species in the Amazon region, has been Thatcher, 1985 (Araújo et al., 2009; Andrade-Porto et al., 2015; increasing each year despite the various obstacles encountered in its Marinho et al., 2013; Serrano-Martínez et al., 2015); Trichodina sp.; farming (IBGE, 2017). The initial phase of farming is considered the Trichodina heterodentata (Miranda et al., 2012); Ichthyobodo sp.; Ich- most critical for the survival due to nutritional and health problems, thyophthirius multifiliis (Marinho et al., 2013); Piscinoodinium pillulare and consequently, when post-larval loss is included, mortality can be as (Protozoa) (Araújo et al., 2009; Serrano-Martínez et al., 2015); Poly- high as 80% (Araújo et al., 2009; Marinho et al., 2013; Serrano- acanthorhynchus macrorhynchus Diesing, 1856 (Acanthocephala); Ca- Martínez et al., 2015; Andrade-Porto et al., 2015). ballerotrema brasiliense (Digenea); and Schizochoerus liguloides (Cestoda) Such losses have been attributed primarily to high infestation by (Marinho et al., 2013). Recently, the first record of larvae in the third and/or to infections caused by pathogens, particularly parasites. The stage (L3) of Hysterothylacium sp. (Nematoda, Anisakidae) was de- literature describes numerous species of parasites of pirarucu in farming scribed from South America, detected in the intestine, stomach, and systems, including the following intestinal : Camallanus tri- pyloric caeca of specimens of pirarucu juveniles grown in the Amazon dentatus Drasche, 1884; Terranova serrata Drasche, 1884; Goezia (Andrade-Porto et al., 2015). This nematode, when occurring at a high

⁎ Corresponding author at: Department of Technology and Innovation, National Amazonia Research Institute, Av. André Araújo 2936, P.O. Box 478, 69060-001 Manaus, AM, Brazil. E-mail address: [email protected] (E.G. Affonso). https://doi.org/10.1016/j.aquaculture.2018.04.062 Received 17 September 2017; Received in revised form 27 April 2018; Accepted 30 April 2018 Available online 11 May 2018 0044-8486/ © 2018 Elsevier B.V. All rights reserved. A.C.T. Corral et al. Aquaculture 494 (2018) 37–44 infection rate, can cause intestinal obstruction and lack of appetite in 45% powdered crude protein (CP) at 2-hour intervals. After this, the fish, causing significant economic losses in fish production; in addition, plankton was replaced by 2000 mL of water with increasing amounts of the presence of this nematode in food fish arises the concern to public feed at each feeding. After 15 days, the fish began to feed only on health because of indications that it may present a zoonotic potential powdered feed, and gradually, the size of the granules was increased (Balbuena et al., 2000; Felizardo et al., 2009; Andrade-Porto et al., until the fish were ingesting 4-mm granules of feed. 2015; Ribeiro et al., 2017). Recently, Ribeiro et al. (2017), assessed the During the fattening period, the fish were fed to satiation twice pathogenic potential of the third stage larvae of Hysterothylacium daily, with commercially extruded 4-mm feed for carnivorous fish (45% deardorffoverestreetorum in mammals and observed that this parasite CP) being used, until the fish reached the desired weight. Twenty-four can activate both cellular and humoral immune responses. Also, it can hours prior to the beginning of each experiment (palatability and effi- stimulate the immune system which may result in severe allergies and cacy tests), the fish went through food deprivation for gastrointestinal gastrointestinal manifestations. These results contribute to the better emptying. Before distributing the fi sh to the experimental units, a understanding of human anisakidosis, considering the lack of knowl- sample of 10 was removed to evaluate the parasitic load (Eiras edge regarding the immune response and allergic reaction of these et al., 2006). larvae in humans. This work was undertaken with the approval of the Committee for Several chemicals have been tested and used for the control of en- Ethics in Research in the Use of Animals (Comitê de Ética em Pesquisa doparasites and ectoparasites of fish. However, disadvantages exist, no Uso de Animais – CEUA) (Prot No. 023/2013). such as high treatment costs, toxicity for the or for humans, and the possibility of resistance by parasites to the active substances, in addition to the serious risks the chemicals pose to the environment, 2.2. Extracting the essential oil among others (Pahor-Filho et al., 2015). A practical, economical, and effective alternative that is gaining prominence in global aquaculture The leaves of P. aduncum were collected and dried at Embrapa are products from medicinal plants (Wang et al., 2011; Banerjee and Western Amazon (Embrapa Amazônia Ocidental) in Manaus, Brazil. For Saha, 2013; Reverter et al., 2014; Valladão et al., 2015; Andrade et al., complete removal of moisture, the leaves were kept at 45 °C, in ovens 2016; Hashimoto et al., 2016). Brazil, which holds the largest floristic with continuous air circulation, for 48 h and then were stored in bags at biodiversity of the planet and whose traditional population frequently 20 °C until the extraction of the essential oil. For this, the standard uses medicinal plants for treating various diseases, is taking advantage method of drag by water vapor was used in the Experimental Station of of this potential to produce phytotherapic drugs in disease control in Tropical Forestry of INPA. The hydrodistillation of the material was different sectors of animal production, including in fish farming (Souza- carried out in a semi-industrial-scale plant with a capacity of 20 kg, in Chagas et al., 2016; Ellse et al., 2016). which three machining operations were conducted, which resulted in Among the studied plants of medicinal interest, emphasis is being an oil extraction yield of 3.12%. given to the spiked pepper, Piper aduncum, which is widely distributed ff in the American tropics. The pharmacological e ects of its essential oil 2.3. Chemical composition of the essential oil of P. aduncum have shown efficacy in the control of pests and microorganisms, and it is important for agriculture because of its insecticidal, fungicidal, bac- The sample of essential oil of P. aduncum was subjected to analysis tericidal, acaricidal, molluscicidal, and parasiticidal characteristics to define its chemical composition using mass spectrometry (GC–MS (Orjala et al., 1993; Baldoqui et al., 1999; Almeida et al., 2009; Flores model GC2010/QP2010 Plus – Shimadzu Scientific Instruments, Kyoto, et al., 2009; Dal Picolo et al., 2014). The essential oil of P. aduncum Japan) at the Biotechnology and Natural Resources laboratory at the consists of monoterpene type hydrocarbons (piperitone, 1,8-cineole), Amazonas State University, Amazonas, Brazil. The injection of 1 μLof β sesquiterpene ( -caryophyllene and bicyclogermacrene), and phenyl- solution was performed in the splitless mode, with a column tempera- propanoids (dillapiole, myristicin, and apiole) that are commonly found ture from 50 to 290 °C and a gradual increase of 3 °C/min. The quan- in species of the genus Piperaceae (Burke and Nair, 1986; Vila et al., tification of the dillapiole present in oil was performed by means of a 2005; Rali et al., 2007; Potzernheim et al., 2012), and its concentration calibration curve, with a linear response observed over the working in leaves can vary from 0.25 to 4% (Cicció and Ballestero, 1997; Maia range (20–1000 μg/mg). et al., 1998). Although studies evaluating its efficacy and adverse ef- fects on the physiological homeostasis of fish are scarce, the pharma- cological potential of this plant in the form of extracts is well demon- 2.4. Preparation and administration of medicated feed strated in the treatment of different pathogens Silva et al., 2009, 2013; Volpe et al., 2015). The tested oil concentrations (32, 48, 56, 64, and 80 mL oil/kg feed) Therefore, in this study, the efficacy of the essential oil of P. were calculated from the total biomass of the fish. The essential oil was aduncum in the control of the nematode Hysterothylacium sp. in pirarucu diluted with grain alcohol 1:7.5 (oil:alcohol), evenly sprayed on the juveniles and its adverse effects on the metabolism of fish was assessed. pellets of commercially extruded feed for carnivorous fish (45% CP), Complementarily, the chemical composition of the essential oil was and kept under refrigeration, following the methodology suggested by identified. Dairiki et al. (2013). For the control feed, the same procedure was performed but without the addition of essential oil. 2. Materials and methods Medicated feed (with essential oil) and control feed (without es- sential oil) were administered for 7 and 15 days. Fish received the 2.1. Acclimatization and food conditioning of the fish medicated feed once a day, in the morning (9:00 am), and the feed without medication was offered in the afternoon (4,00 pm). The A. gigas juveniles (5.8 ± 0.7 g) acquired from a commercial fish amount of feed offered in each experimental unit was defined relative farm were acclimated in 2000-L tanks with water renewal and constant to the present biomass, which was determined from the values of the aeration at the Experimental Aquaculture Center of the INPA (National initial biometry. Amazonia Research Institute) in Manaus, Brazil. The pirarucu consumed feed at a rate of 5% of their live weight Fish were feeding on live wild zooplankton when acquired and were (LW) per day, and the medicated feed that was prepared accounted for weaned to dry feed following the protocol described by Cavero et al. 2% of their LW. This medicated preparation, offered in a smaller (2003), with the following modifications: in the first 7 days, the fish quantity of feed, guaranteed that all fish during the morning feeding were fed 2000 mL of plankton, mixed with commercial feed containing could consume this feed.

38 A.C.T. Corral et al. Aquaculture 494 (2018) 37–44

2.5. Palatability test The observation of the parasites through the stereomicroscope ex- amination revealed the occurrence of one uniform group of parasites For the palatability test, 60 fish were used (25.4 ± 3.5 g), dis- with the same morphology, color, tail shape and presence of larval tributed in 60-L cones, at a storage density of four fish/cone, with tooth. Considering the uniformity of the parasites observed and the continuous water flow for 7 and 15 days. The experimental design great number of specimens recorded in each treatment, it was not consisted of four essential oil concentrations: 32, 48, 64, and 80 mL/kg possible to prepare slides from all individuals. Therefore, a sample of 10 feed and the control group (without oil) in triplicates (Zheng et al., specimens were taken at random from the total of parasites collected 2009). from each treatment at 7 and 15 days. Thus, 80 slides were prepared for During feeding, palatability was observed through the acceptance/ taxonomic identification based on the morphology of the posterior ingestion of feed at all concentrations, and the amount of feed offered extremity of the worms. For general morphometric analysis, 20 speci- per experimental unit was recorded. The ingestion rate (feed ingestion mens were selected at random from the total amount of parasites found. percentage) was calculated according to Barros et al. (1998): ingestion For identification of the parasites, temporary slides of the nema- rate (%) = ingested feed × 100 / administered feed. todes were prepared, with the parasites first being diaphonized in 50%, 60%, and 70% phenic acid and next in lactic acid with glycerin and 2.6. Anthelmintic efficacy of P. aduncum lactophenol (Moravec, 1998). For preservation of the specimens in the temporary assemblies, a Petri dish served as a humidity chamber for Pirarucu juveniles (52 ± 3.2 g) were distributed in tanks of 310 L each evaluated sample. Permanent slides were prepared after the spe- each, at a density of nine fish/experimental unit in a continuous water cimens had been washed with 70% alcohol and mounted with Canadian flow system (renewal rate of 175 mL/min). The experimental design balm (Amato et al., 1991). was completely randomized, consisting of four treatment groups with The morphological identification of the nematodes and larval stage feed medicated with essential oil, based on the results of the palatability were carried out according to Deardorff and Overstreet (1980, 1981), tests: 32, 48, 56, and 64 mL/kg feed and the control group (without Moravec (1998) and Andrade-Porto et al. (2015) and the parasites were essential oil) in triplicates. The fish were fed on the medicated feed, compared with paratypes deposited by Andrade-Porto et al. (2015) in except for the control group, for a period of 7 and 15 days. the Collection of Non-Insect Invertebrates of INPA (No. 59 INPA - Wet At the end of each period, three fish from each experimental unit material). Photomicrographs of the specimens were taken under an were sampled for determination of blood parameters and later para- optical microscope (Carl Zeiss, Model Axio Lab A1) with an attached sitological analysis. During the whole experimental period, physical digital camera (Carl Zeiss, Model AxioCam ERc5s). All measurements and chemical analyses of the water were conducted. were performed and scaled with a micrometer eyepiece, and the mea- The anthelminthic efficacy was determined as described by Martins surements are presented in millimeters (mm), with averages presented et al. (2001) as the number of parasites of control group − number of in parentheses. parasites after the treatment/number of parasites of control group × 100%. 2.9. Water quality monitoring

2.7. Blood sample collection and determination of blood parameters Dissolved oxygen (DO), temperature (T), and electrical conductivity (EC) were determined with a digital multiparameter probe (YSI, Model Blood was collected by puncture of the tail vein using 3 mL syringes 85-25FT, Ohio, USA) and hydrogen ionic potential (pH) with a digital rinsed with ethylenediaminetetraacetic acid (EDTA) 10%, and the pH meter (YSI Model 60-10FT, Ohio, USA). Total ammonia con- samples were stored at 4 °C. From the total blood, the following para- centrations were determined according to Verdouw et al. (1978) and meters were analyzed: hematocrit (Ht%), by the microhematocrit nitrite concentrations, carbon dioxide concentrations, hardness, and method; erythrocyte count (RBC - erythrocytes/μL) in citrate for- alkalinity according to Boyd and Tucker (1992). The temperature maldehyde solution, using a hemocytometer; and hemoglobin con- (28.05 ± 0.30 °C), dissolved oxygen (5.3 ± 0.05 mg / L), pH centration ([Hb] - g/dL), using the cyanmethemoglobin method. The (4.4 ± 0.02), electrical conductivity (28.6 ± 0.27 μS/cm), total am- hematometric indices—mean corpuscular volume (MCV fL), mean monia (0.40 ± 0.07 mg/L), total nitrite (0.008 ± 0.004 mg/L), and corpuscular hemoglobin concentration (MCHC%), and mean corpus- CO2 (8.3 ± 2.9 mg/L) of the water from the experimental units in the cular hemoglobin (MCH pg)—were calculated from the values of Ht, palatability and efficacy tests did not present significant differences RBC, and [Hb], according to Wintrobe (1934). Plasma was used for the (p > 0.05) among the concentrations in the same period or between glucose analyses (mg/dL), with the enzymatic colorimetric method of periods (acclimatization, 7 days, and 15 days). All values obtained were glucose oxidase and total proteins (PPT – g/dL) by the biuret method within the limits considered appropriate for farming this species being used. These analyses were performed in a UV Visible Spectro- (Oliveira et al., 2012). photometer (BIOPLUS 2000, Bioplus, São Paulo, Brazil). 2.10. Statistics 2.8. Parasitological analyses—preparation and identification of parasites The palatability test, the blood parameters of pirarucu juveniles, Parasitological analyses were performed before and after the treat- and the parasitological indices, as well as the analyses of the physical ments. In each experimental unit, three randomly selected fish were and chemical variables of the water, were compared through analysis of euthanized by brain concussion, followed by cranial drilling, in ac- variance (ANOVA), followed by Tukey's test, where differences were cordance with the recommendations of the American Veterinary significant at 5% of probability, using StatSoft STATISTICA 6.0® soft- Medical Association – AVMA. The internal organs (intestines and sto- ware. mach) were removed and dissected in a stereomicroscope (Carl Zeiss, Model Stemi 2000-C). The parasites were fixed in 4% formaldehyde, 3. Results and discussion heated to 65 °C (Moravec, 1998), and for preservation, the specimens were stored in 70% alcohol with 5% glycerin (Amato et al., 1991). The The results of the exploratory analysis of the essential oil showed parasite prevalence, intensity, average intensity and average abundance the presence of two major substances: 1,2-methylenedioxy-5,6-di- were calculated in accordance with Bush et al. (1997) and the EO ef- methoxy-4-allylbenzene (dillapiole) and undec-4-ene,4,11,11-tri- ficacy evaluation was calculated in accordance with Martins et al. methyl-8-methylene-[1R-(1R*,4E,9S*)]-(CAS) 1-caryophyllene(−)-car- (2001). yophy (trans-caryophyllene). In addition, dillapiole accounted for

39 A.C.T. Corral et al. Aquaculture 494 (2018) 37–44

Fig. 1. Chromatograms of essential oil analysis of Piper aduncum,byGC–MS of HPLC grade dichloromethane. A = Caryophyllene and B = Dillapiole. X-axis presents time in minutes and y-axis the total ion current (TIC). approximately 92% of the product (Fig. 1). The quantification of dil- results demonstrating a decrease in their ingestion by fish, which may lapiole, using a calibration curve, was 791.2 μg/mg of the essential oil. compromise the use of the oral route for drug administration (Forwood Studies conducted with P. aduncum from the Amazon have described et al., 2013). The results obtained in the present study corroborate these the presence of up to 98% of dillapiole in the essential oil of this plant studies, proving the importance of testing for palatability of the sup- (Almeida et al., 2009). plemented feed in the target species. Thus, for the efficacy tests, feed The ingestion rate of the medicated feed during the palatability test medicated containing up to 64 mL/kg essential oil of P. aduncum can be is represented in Table 1. At 7 and 15 days of the experiment, sig- applied because ingestion at those levels did not differ significantly nificant differences (p < 0.05) were observed in the ingestion of the (p > 0.05) with the ingestion rate of the fish in the control group feed with the highest concentration (80 mL/kg); however, no sig- (without EO). Furthermore, 100% fish survival was observed in 7 and nificant differences (p > 0.05) were observed in the afternoon (feed 15 days of experimentation in all treatments. without medication). The feed with the highest concentration had the In the efficacy test of the essential oil of P. aduncum as an anthel- lowest ingestion rate of the medicated feeds (< 60%) in both periods (7 mintic for pirarucu, blood parameters indicating stress were evaluated and 15 days), with behavioral changes being observed, such as erratic in fish fed with and without the inclusion of EO in the feed, for 7 and swimming and rejection of the treated pellets. Furthermore, in the 15 days, and the results are shown in Table 2. Changes were observed in afternoon (use of feed without medication), although no decrease was the values of Ht, MCV, and MCH with 7-day treatment, and changes in observed in the intake rate, the animals presented lethargic behavior the values of the total protein of pirarucu were observed after 15 days of during food capture, which was not verified for the other concentra- treatment. Changes in the Ht values in the two highest concentrations tions. Therefore, the results indicated that feed with the highest con- of P. aduncum in relation to the control group may be the result of two centration of essential oil (80 mL/kg) became less palatable, preventing mechanisms: 1) an increase in MCV, causing a decrease in the plasma the ingestion of the medicated feed, which consequently compromised volume, and 2) a decrease in plasma volume, resulting in increased the action of the product. The alteration in the palatability of feed volume of erythrocytes, which may increase the hemoglobin con- medicated with different drugs is well described in the literature, with centration. The values of Ht and RBC were also verified in Oncorhynchus

Table 1 Ingestion rate (%) of Arapaima gigas fed on feed with different concentrations of essential oil of Piper aduncum (32, 48, 64, and 80 mL/kg) and the control group (no drug), in the morning (9:00 am) and afternoon (4:00 pm) for 7 and 15 days. Average ± standard deviation.

Period Ingestion rate (%) (7 days)

Control 32 (mL/kg) 48 (mL/kg) 64 (mL/kg) 80 (mL/kg)

⁎ Morning 95.95 ± 2.83a 94.38 ± 7.89a 88.33 ± 5.69a 86.42 ± 9.13a 64.28 ± 6.73b Afternoon 95.23 ± 1.38 94.28 ± 5.17 92.38 ± 2.21 92.38 ± 3.71 89.52 ± 9.71

⁎ Ingestion rate (%) (15 days)

⁎ Morning 92.22 ± 2.06a 87.67 ± 4.71a 82.11 ± 5.06a 78.3 ± 6.4a 60.11 ± 6.22b Afternoon 96.44 ± 2.18 89.55 ± 2.47 87.89 ± 3.59 87.3 ± 1.4 85.55 ± 5.94 ⁎ Different letters in the same row indicate significant difference (p < 0.05).

40 A.C.T. Corral et al. Aquaculture 494 (2018) 37–44

Table 2 Blood parameters of Arapaima gigas fed for 7 and 15 days on medicated feed containing different concentrations of essential oil of Piper aduncum. Hematocrit (Ht), Number of erythrocytes (RBC), hemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), corpuscular hemoglobin concentration (MCHC), glucose (Gl), and total protein (TP). Average ± standard deviation, N = 9.

⁎ Parameters Feed with essential oil of Piper aduncum (7 days)

Control 32 (mL/kg) 48 (mL/kg) 56 (mL/kg) 64 (mL/kg)

⁎ Ht (%) 24.33 ± 6.24b 26.83 ± 6.22ab 25.61 ± 5.94ab 30.89 ± 3.12a 28.88 ± 1.31a RBC (eryt./uL) 1.46 ± 0.31 1.33 ± 0.29 1.63 ± 0.13 1.52 ± 0.25 1.50 ± 0.15 Hb (g/dl) 9.98 ± 1.83 11.60 ± 2.16 9.96 ± 3.22 11.63 ± 2.85 12.58 ± 1.66 MCV (fL) 170.6 ± 47.8ab 208.42 ± 65.7a 159.18 ± 43.6b 206.7 ± 31.6a 193.9 ± 14.4ab MCH (pg) 69.9 ± 15.4ab 89.85 ± 22.62b 62.55 ± 22.87a 77.8 ± 22.3ab 85.49 ± 20.5ab MCHC (g/dL) 42.33 ± 9.07 43.91 ± 4.56 38.82 ± 8.29 37.35 ± 7.39 43.83 ± 7.88 Gl (mg/dl) 37.61 ± 3.03 36.28 ± 6.38 39.11 ± 2.86 38.78 ± 6.92 38.63 ± 2.26 TP (g/dL) 1.45 ± 0.53 1.60 ± 0.44 1.31 ± 0.50 1.37 ± 0.43 1.49 ± 0.24

Feed with oil of Piper aduncum (15 days)

Ht (%) 31.22 ± 5.23 29.63 ± 4.21 25.02 ± 4.75 27.35 ± 5.93 28.00 ± 9.08 RBC (eryt./uL) 1.91 ± 0.25 1.61 ± 0.15 1.45 ± 0.13 1.59 ± 0.20 1.80 ± 0.16 Hb (g/dl) 12.15 ± 1.52 11.44 ± 0.93 10.24 ± 1.93 11.26 ± 1.92 11.59 ± 2.45 MCV (fL) 164.4 ± 31.47 185.8 ± 40.05 180.35 ± 18.6 171.7 ± 32. 163.4 ± 33.4 MCH (pg) 63.83 ± 7.52 71.29 ± 9.68 70.82 ± 12.06 70.88 ± 10.47 64.57 ± 12.80 MCHC (g/dL) 39.30 ± 3.12 39.18 ± 5.83 44.55 ± 4.39 41.80 ± 3.54 43.22 ± 7.84 Gl (mg/dL) 37.22 ± 5.77 41.50 ± 3.03 37.78 ± 3.53 41.08 ± 6.16 37.35 ± 6.51 TP (g/dL) 1.60 ± 0.30 a 1.58 ± 0.09 ab 1.53 ± 0.28 ab 1.30 ± 0.30 a 1.18 ± 0.37 b ⁎ Different letters in the same row indicate significant difference (p < 0.05). mykiss after ingestion of feed with essential oils of Satureja khuzestanica et al., 2016). From the above, the way the oil is administered and the and Myrtus communis (Saei et al., 2016). With regard to the number of parts of the plant used may act on the behavior and/or physiology of erythrocytes of pirarucu infected by nematodes at the different sam- the test animal, which is important information for the pharmacological pling time-points (7 and 15 days) and with different concentrations of application of this plant in species with morphological adaptations to essential oil of P. aduncum, 1) the effect of the medicated feed is sug- air breathing, such as the pirarucu. gested to be of low toxicity, and 2) the treatment did not change the In farming systems, the presence of pathogens in different taxo- oxygen carrying capacity of the blood. These are corroborated with the nomic groups such as monogenoids, trichodinids, and nematodes results obtained in the hemoglobin and MCH concentrations. (See (Araújo et al., 2009; Marinho et al., 2013; Andrade-Porto et al., 2015)is Table 2). clearly demonstrated and requires commitment for managing health The glucose concentrations were not altered among the different conditions in the pirarucu. In the literature, the health problems diag- treatments with P. aduncum, with the feeding time with the drug (7 and nosed for this species have been associated with inadequate manage- 15 days), or with the infection by the parasite. Another parameter that ment of larvae and post-larvae, especially after the withdrawal of par- contributes to the evaluation of the health status of fish is total plasma ental care, during the feeding transition period (live food to feed), and proteins (Zhou et al., 2009). In general, the values of total protein in with environmental water quality (Araújo et al., 2009; Andrade-Porto this study are lower than those described for this same species in other et al., 2015). works (Tavares-Dias et al., 2007; Andrade et al., 2016). Despite the In this study, parasitological analysis revealed that, of the 135 ex- adverse effects of parasitosis on fish, the different concentrations of amined fish, 100% were infested with nematode larvae in all treatment essential oil of P. aduncum accentuated the decrease in the concentra- groups and at intensities that varied from 5 to 949 and 9 to 1221, in 7 tions of total plasma protein of the pirarucus in this study (Table 2). In and 15 days of the experiment, respectively, with no fish mortalities general, the maintenance of proteins depends on intracellular me- being observed (Table 4). This high intensity of infestation shows the chanisms, either in the formation process or in the degradation process, real situation faced by most producers of pirarucu, with big losses oc- that depend on specific proteins that can be affected by environmental curring in the larvae and post-larvae of this species. Moreover, the wide stressors or management conditions (Iwama et al., 2004). In the lit- variation in the number of parasites (5–1221) found per fish evaluated erature, hypoproteinemia has been observed in studies under chemo- prior to the experiments can be related to the intraspecific character- and phytotherapic drug effects in fish (Sudová et al., 2009). istic of the species, as all were from the same offspring and maintained In addition to the action against pathogens, P. aduncum has been under the same environmental conditions. The nematode infestation cited in the literature for its anesthetic effect from its alkaloids that act can stimulate cellular/humoral responses in the host, which is able to on the central nervous system of animals (Lorenzi and Matos, 2002). eliminate certain infectious stages (Larsen et al., 2002). However, in this study, hyperactivity of the animals was observed im- The average intensity indices and average parasite abundance mediately after the ingestion of the feed medicated with EO, which may showed no significant differences (p > 0.05) between the treatments be related to the effect of substances derived from the plant's secondary (32, 48, 56, and 64 mL/kg) at 7 days, but all presented significant dif- metabolism, and these may act separately or in synergism (Dewick, ferences compared to the control group (p < 0.05). However, at 2009). It is possible that dillapiole, which at 92% is the main compo- 15 days, these indices showed significant differences (p < 0.05) be- nent of the EO of P. aduncum, followed by caryophyllene (Fig. 1A), may tween the tested concentrations of P. aduncum and the control group have been responsible for this reaction in the animals. Other studies on and between the groups with lower concentration treatments and the the use of the essential oil and compounds isolated from plants have treatment with 64 mL/kg (Table 3). This difference may have been demonstrated anesthetic action in fish, such as menthol, a major com- caused by the cumulative effect of the drug, enhanced by exposure ponent of Mentha spp., in Oreochromis niloticus (Hashimoto et al., 2016), time, providing a maximal response (Hollinger, 2003). and the essential oil of Mentha piperita (40 mg/L) in A. gigas (Malheiros A total of 37,574 nematode larvae were recorded in the body cavity

41 A.C.T. Corral et al. Aquaculture 494 (2018) 37–44

Table 3 Prevalence (P%), Intensity (I), average Intensity (AI) ± standard deviation and Average abundance (AA) ± standard deviation of nematodes in Arapaima gigas after 7 and 15 days of treatment with different concentrations of essential oil of P. aduncum and control (without essential oil). N = 135.

Concentrations (mL/kg) 7 Days

⁎ ⁎ P(%) I AI AA

Control 100 20–702 536.22 ± 19.66a 536.22 ± 19.66a 32 100 5–949 519.67 ± 26.65b 519.67 ± 26.65b 48 100 112–856 498.44 ± 23.46b 498.44 ± 23.46b 56 100 100–760 456.44 ± 22.79b 456.44 ± 22.79b 64 100 11–798 450.67 ± 23.55b 450.67 ± 23.55b

15 Days

Control 100 9–1221 567.00 ± 36.05a 567.00 ± 36.05a 32 100 20–600 401.67 ± 20.63b 401.67 ± 20.63b 48 100 124–708 338.67 ± 18.10b 338.67 ± 18.10b 56 100 9–603 260.11 ± 18.00b 260.11 ± 18.00b 64 100 9–288 134.89 ± 10.43c 134.89 ± 10.43c ⁎ Different letters in the same column indicate significant difference (p < 0.05) within the time-point.

Table 4 Morphometric measurements of larvae of Hysterothylacium sp. in pirarucu ju- veniles, Arapaima gigas N = 20.

Characters Measurements (mm)

Length 2.25–2.75 (2.65 ± 0.35) Width 0.1–0.16 (0.12 ± 0.02) Larval tooth 0.002 Esophagus 0.18–0.30 (0.24 ± 0.1) Intestinal cecum 0.062–0.20 (0.12 ± 0.06) Ventricular appendix 0.78–1.2 (1.0 ± 0.3) Distance from the anus to the end of the 0.055–0.085 (0.075 ± 0.025) posterior region (tail) of the 135 pirarucus in this study and no parasites were found in the internal organs. The morphometric analysis revealed the presence of larva in stage L3, identified as Hysterothylacium sp., family Anisakidae (Table 4; Fig. 2). These nematodes are important agents of parasitic diseases in humans (zoonoses), known as anisakiasis (Ubeira et al., 2000), and their genus, Hysterothylacium, has already been described for several Neotropical freshwater fish species (Luque et al., 2011). With wide geographical distribution, this genus has been recorded in the Mediterranean (Rello et al., 2008), Indic and South Pacific(Shamsi et al., 2013); Northwestern Pacific(Yoshinaga et al., 1989; Shih and Jeng, 2002); Norwegian (Mo et al., 2014) and Danish waters (Køie, 1993; Skov et al., 2014); Southern Pacific(Carvajal and Gonzalez, 1990), Atlantic Ocean (Eiras and Rego, 1987; Luque et al., 2011), Fig. 2. Anterior (A) and posterior (B) region of the L3 larva of Hysterothylacium sp. of a pirarucu juvenile, Arapaima gigas. Bar Scale 0.5 mm (A). Bar Scale Persian Gulf (Petter and Sey, 1997) and Northern Pacific(Moser and 0.1 mm (B). Hsieh, 1992). Andrade-Porto et al. (2015) reported, for the first time, the presence of larvae of Hysterothylacium sp., with high prevalence (98%), in the intestine, stomach, and pyloric caeca of pirarucu juveniles obtained from fish farms in the Amazon. Table 5 The genus Hysterothylacium has a variety of intermediate, definitive, Efficacy percentage (%) of essential oil of Piper aduncum administered in the and paratenic hosts. Under natural conditions, these parasites reach feed and the control group (without essential oil) against nematodes of sexual maturity in the digestive tract of bony fish (Køie, 1993)or Arapaima gigas in experiments of 7 and 15 days. marine mammals (Deardorff and Overstreet, 1982), whereas copepods, Treatment (mL/kg) Efficacy (%) polychaetes, and other invertebrates are frequently used as inter- mediate hosts. The high infestation with larvae of Hysterothylacium sp. 7 days 15 days recorded in this work suggests that the source of infection is through Control 0 0 feeding in the nursery phase of the pirarucu, where small crustaceans 32 3.08 29.15 infected with this parasite are part of the diet, as reported by Andrade- 48 7.04 40.27 Porto et al. (2015). Table 5 presents values of the calculation of efficacy 56 14.87 54.12 (%) of the essential oil of P. aduncum in the control of nematode larvae 64 15.95 76.21 after 7 and 15 days of treatment. After 7 days of the experiment, no

42 A.C.T. Corral et al. Aquaculture 494 (2018) 37–44 significant difference (p > 0.05) was observed between the treatment (Crustacea:Branchiura) with plant derivatives. Aquaculture 414–415, 202–209. groups, except at the highest concentration (64 mL/kg), or between the Barros, M.M., Pezzato, L.E., Salaro, A.L., 1998. Feeding behavior of red tilapia ff ffi (Oreochromis niloticus × Oreochromis aureus) fed with di erent additives. Rev. Bras. treatment groups and the control group, with low e cacy of P. Zoot. 27 (4), 635–641. aduncum for the control of the nematodes, including at the higher Boyd, C.E., Tucker, C.S., 1992. Water Quality and Pond Soil Analyses for Aquaculture. concentration. At 15 days, the treatment groups were significantly dif- Auburn University, United States of America. ff Burke, B., Nair, M., 1986. Phenylpropene, benzoic acid and flavonoid derivatives from ferent (p < 0.05) relative to the control group, and a di erence existed fruits of jamaican Piper species. Phytochemistry 25, 1427–1430. in the highest concentration (64 mL/kg), which promoted 76.21% ef- Bush, A.O., Lafferty, K.D., Lotz, J.M., Shostaak, A.W., 1997. Parasitology meets ecology ficacy. In both experimental periods, no fish mortality was recorded. on this terms: Margolis et al. revisited. J. Parasitol. 83, 575–583. The oral administration of drugs, through the addition of pharma- Carvajal, J., Gonzalez, L., 1990. Presence of Hysterothylacium sp. (Nematoda: Anisakidae) in a cage-cultured coho salmon in Chile. Rev. Chil. Hist. Nat. 63, 165–168. cological bases to the feed, is an important route for controlling en- Cavero, B.A.S., Ituassú, D.R., Pereira-Filho, M., Roubach, R., Bordinhon, A.M., Fonseca, doparasites of fish. However, this approach presents a disadvantage in F.A.L., Ono, E.A., 2003. Use of live food as starter diet in feed training juvenile pir- – the treatment of diseases that cause a loss of appetite or anorexia in fish, arucu. Act. Amaz. 38 (8), 1011 1015. Cicció, J.F., Ballestero, C.M., 1997. Constituyentes volátiles de las hojas y espigas de Piper which may lead to a lower ingestion or even to no ingestion of the aduncum (Piperaceae) de Costa Rica. Rev. Biol. Trop. 45 (2), 783–790. medicated feed (Shalaby et al., 2006; Yamamoto et al., 2011; Ishimaru Dairiki, J.K., Majolo, C., Chagas, E.C., Chaves, F.C.M., Oliveira, M.R., Morais, I.S., 2013. et al., 2013). In addition, food ingestion may be impaired if the drug Procedimentos para inclusão de óleos essenciais em rações para peixes. Bol. Pesq. Embrapa Amaz. Ocid. 42, 1–8. changes the taste or odor of the feed, which is then avoided by the Dal Picolo, C.R., Bezerra, M.P., Gomes, K.S., Passero, L.F.D., Laurenti, M.D., Martins, animal. In this study, the infestation by nematodes did not impair food E.G.A., Sartorelli, P., Lago, J.H., 2014. Antileishmanial activity evaluation of ingestion during the palatability test; however, as discussed above, the adunchalcone, a new prenylated dihydrochalcone from Piper aduncum L. Fitoterapia – ffi 97, 28 33. taste of medicated feed changed the ingestion rate. Despite the e cacy Deardorff, T.L., Overstreet, R.M., 1980. Hysterothylacium and Iheringascaris (both of the EO of P. aduncum, particularly with 15 days of treatment, which previously = Thynnascaris) (Nematoda: Anisakidae) from the Northern Gulf of indicated its successful application for the control larvae in the stage L3 Mexico. Proc. Biol.Soc. Wash. 93, 1035–1079. Deardorff, T.L., Overstreet, R.M., 1981. Larval Hysterothylacium (=Thynnascaris) of Hysterothylacium sp. in pirarucu, increasing ingestion of the medi- (Nematoda: Anisakidae) from Fishes and Invertebrates in the Gulf of Mexico. vol. 48. cated feed could improve the therapeutic response of the product in Proceedings of Helminthological Society of Washington, pp. 113–126. future trials. Therefore, techniques for improving the palatability of Deardorff, T.L., Overstreet, R.M., 1982. Hysterothylacium pelagicum sp. n. and medicated feeds are strongly recommended. Hysterothylacium cornutum (Stossich, 1904) (Nematoda: Anisakidae) from marine fishes. Proc. Helmint. Soc. Washington 49, 246–251. Eiras, J.C., Rego, A.A., 1987. The histopathology of Scomber japonicus infection by 4. Conclusion Nematobothrium scombri (Trematoda: Didymozoidae) and of larval anisakid nematode infections in the liver of Pagrus pagrus. Mem. Inst. Osw. Cruz. 82, 155–159. fi ff Eiras, J.C., Takemoto, R.M., Pavanelli, G.C., 2006. 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