Treatments for the control of Neoechinorhynchus buttnerae (Acanthocephala) in tambaqui Colossoma macropomum

Intensive fish farming with inadequate management can imbalance the fish-pathogen-environment relationship, culminating in diseases outbreaks. In the case of tambaqui Colossoma macropomum farming, it may be affected by acanthocephalosis, a parasitic infection without a specific and safe treatment yet. The objective of this study was to evaluate the effect of the active ingredients in trichlorfon, albendazole, piperazine, and ivermectin, as well as the non-traditional chemists calcium hydroxide and diatomaceous earth incorporated to the feed, to control Neoechinorhynchus buttnerae (Acanthocephala) in tambaqui. A 63-day trial was conducted in a completely randomized experimental design, with 7 treatments, 3 replicates each, and 54 fish per treatment. Fish were sampled for weight gain and parasite count on days 0, 15, 35, and 63. In general, the efficacy of anthelmintics was low in the treatment of N. buttnerae in tambaqui. Nevertheless, among the tested products, calcium hydroxide and diatomaceous earth showed promising results (48.2% and 36.4% efficacy, respectively), followed by ivermectin and piperazine (15.9% and 12.6%, respectively). Future studies are warranted to test longer drug administration periods, novel products, and diverse experimental conditions to treat N. buttnerae in tambaqui, to provide information to develop strategies for disease management.


Introduction
Brazil is one of the major aquaculture producer in South America, with 581,000 tons per year, and estimated to increase production by 89% until 2030, reaching 1097 million tons (FAO 2018). The Amazonian tambaqui Colossoma macropomum (Serrasalmidae) is the second most farmed species in the country, the first being Nile tilapia Oreochromis niloticus (Valenti et al. 2021). However, in the last 2 years, tambaqui share in the aquaculture scenario decreased significantly: 3.2% and 5.5%, in 2020 and 2021, respectively, representing 31.2% of total national production in 2021 (Peixe 2021;Peixe 2022). Yet, the Brazilian North and Midwest Regions remain the main producers of the species, notably the states of Rondônia, Maranhão, and Mato Grosso (Peixe 2021).
As any other animal production, disease issues in tambaqui farming are a concern. Direct and indirect economic losses due to diseases in the Brazilian fish farms are estimated at USD 84 million per year (Tavares- Dias and Martins 2017). Intensive fish farming systems without good management practices may imbalance the fish-pathogen-environment relationship, culminating in increased susceptibility to diseases Costa et al. 2019;Souza et al. 2021;Tavares-Dias et al. 2021). The main parasitic organisms reported in farmed C. macropomum are Acanthocephala, Copepoda, Monogenoidea, Myxozoa, and Crustacea (Rocha et al. 2018;Chagas et al. 2019;Fujimoto et al. 2019). If not properly addressed, the parasitic infections will cause significant economic losses and eventually threaten the long-term sustainability of the industry (Fridman et al. 2014). For example, acanthocephalosis outbreaks have already been reported in the past few years (Chagas et al. 2015Gomes et al. 2017;Valladão et al. 2020).
Acanthocephalans are the smallest group of fish endoparasites, with nearly 1100 species (Bush et al. 2001). The life cycle of acanthocephalans is indirect, requiring an arthropod as intermediate host and a vertebrate as definitive host (Santos et al. 2013). So far, Neoechinorhynchus buttnerae (Eoacanthocephala: Neoechinorhynchidae) is the only acanthocephalan species reported to be infecting tambaqui Dias et al. 2015;Jerônimo et al. 2017;Lourenço et al. 2017;Pereira and Morey 2018;Valladão et al. 2020). The clinical signs observed in N. buttnerae infection are stiffening and thickening of the fish intestinal wall, due to the mechanical damage caused by their proboscis, triggering an intense inflammatory process (Jerônimo et al. 2017). Also, mucus production is increased in response to the infection (de Matos et al. 2017).
Very few studies have tested anthelmintic drugs to treat acanthocephalan infections (Chagas et al. 2015;Valladão et al. 2020;Farias et al. 2021). In vitro tests against N. buttnerae have been conducted with avermectins, praziquantel, and levamisole (Oliveira et al. 2019), oleoresins from Copaifera (Seixas et al. 2020), and essential oils of Aloysia triphylla, Adenocalymna alliaceum, Allium sativum, Croton cajucara, Curcuma longa, Lippia gracilis, Lippia origanoides, Ocimum gratissimum, Mentha arvensis, and Piper aduncum (Oliveira et al. 2021). While in vivo tests were conducted with albendazole and praziquantel , fenbendazole and levamisole (Sebastião et al. 2022), essential oils of Mentha piperita, Lippia alba, and Zingiber officinale (Costa et al. 2020) and a food additive, Acantex® (Sebastião et al. 2020). In this way, additional studies with anthelmintic drugs are warranted to test the efficacy in controlling the N. buttnerae parasitic infection in tambaqui. The objective of this study was to evaluate the effect of the active ingredients in trichlorfon, albendazole, piperazine, and ivermectin, as well as calcium hydroxide and diatomaceous earth, on the control of N. buttnerae in tambaqui, and possibly shedding some light for the development of strategies to reduce disease outbreaks in fish farms.

Fish and acclimation period
The experiment was conducted at the Federal University of Rondônia (UNIR), Presidente Médici Campus (Rondônia state, Brazil), in the Animal Health Experimental Center (CESA), and Environmental Science Laboratory (LCA). Four-hundred juvenile C. macropomum (average weight 107.75 g), were purchased from a commercial fish farm (Rolim de Moura, RO, Brazil). Fish were transported to CESA in transportation tanks with an oxygenation system, and randomly distributed into twenty-one 1000-L tanks with constant aeration, in a flow-through system at a density of 18 fish per experimental unit (2 g/L). The experimental protocol was approved by the Ethics Committee on the Use of Animals (CEUA) from UNIR (protocol #035/2016). Fish were acclimated to the experimental conditions for 45 days. During acclimation, fish were fed a commercial extruded feed (36% crude protein, 3-4 mm pellets: Karino, Nutrizon, Brazil) twice a day at 2% biomass. Approximately 50% of the total water volume of the experimental units was renewed daily. Water quality parameters, i.e., temperature, pH, dissolved oxygen, and conductivity, were measured three times a week using a multiparameter probe (AKSO AK87 CE) and a digital oximeter (YSI Pro20), during the acclimation and the experimental periods.

Experimental design
The trial was conducted in a completely randomized design, with seven treatments, three replicates each, and 54 fish per treatment. The fish were fed a control feed (commercial feed coated with vegetable oil only) and six treatment feeds (commercial feed coated with the different active principles, diluted in oil, according to the respective manufacturer's recommendations). Table 1 presents the treatments and dosage (dose, frequency, and duration) of administration.
The products were incorporated to the feed at a rate of 4% by diluting in vegetable oil and spraying onto the commercial feed. The medicated feeds were then identified and stored at 4 °C until used. Fish were fed the medicated feed twice a day (9 a.m. and 4 p.m.) during the experimental period, according to frequency and duration of each treatment (Table 1). Between doses for treatments with trichlorfon, albendazole, piperazine, ivermectin, and calcium hydroxide, and between samplings 3 and 4, fish were fed with the control diet twice a day (9 a.m. and 4 p.m.).
The trial was conducted for 63 days, and fish from all treatments were sampled four times during the experimental period (same day sampling). The first fish sampling was at the beginning of the trial, after the acclimation period (sampling point 1). Twelve fish were sampled for initial weight and mean initial parasite infection intensity. The following fish samplings were done on days 15 (sampling point 2) and 35 (sampling point 3). Day 35 was the last day of medicated feed. Sampling at the end of the 63-day trial (sampling point 4) was conducted to verify any residual effect of the treatments and any effect on fish growth. On sampling points 2, 3, and 4, six fish from each experimental unit were

Fish weight gain, parasite analysis, and efficacy of treatments
Fish weight was taken after anesthesia (eugenol 65 mg/L, Roubach et al. 2005) for weight gain calculation, according to Monteiro et al. (2021). To quantify N. buttnerae, anesthetized fish were euthanized by cranial puncture. After necropsy, the intestines were removed and placed on Petri dishes with 0.65% saline solution. The parasites were separated from the intestinal contents and rinsed for counting the total number of parasites per fish. The parasitic rate of prevalence and mean intensity were calculated (Bush et al. 1997). The efficacy of the treatments was calculated using the formula: Efficacy = (mean number of parasites in the control group − mean number of parasites in the treated group) × 100 / (mean number of parasites in the control group) (Maciel and Affonso 2021).

Statistical analysis
Data on fish weight, weight gain, mean parasitic intensity, and treatment efficacy were submitted to analysis of variance with repeated measures over time considering treatments, time (sampling points), and the interaction between these two factors. All data was considered normal and homoscedastic by the Shapiro-Wilk and Bartlett tests, respectively. In case of significant difference, means were compared by Tukey test (p < 0.05). As for the variable mean parasitic intensity, there was no interaction between the factors, the data were also submitted to analysis of variance (one-way ANOVA), isolating the time factor. Results are expressed as mean ± standard error. Statistical analysis was performed using the statistical software R (R Core Team 2016).

Mean parasitic intensity
The prevalence of N. buttnerae in tambaqui was 100% and the mean intensity in fish before the experiment (sampling point 1) was 70.15 ± 51.94 parasites per fish. No significant differences were found in the mean parasitic intensities of tambaqui fed with the control or medicated feeds and over the sampling times (Table 2). However, when comparing the mean parasitic intensity without the effect of time, a significant decrease (p < 0.05) was observed in fish treated with the non-traditional treatments (calcium hydroxide and diatomaceous earth) compared to albendazole, but not statistically different from the control group (Table 2). No fish mortality occurred during the trial.

Efficacy values
Efficacy values were low for all tested treatments, ranging from 0 to 48.2%. The highest efficacy index was found for the non-traditional treatments, in the three sampling times ( Table 3). Effect of time on efficacy was observed, with statistically lower efficacy at the end of trial (day 63) compared to day 35, indicating a low residual effect of the tested products. On day 15, efficacy of the diatomaceous earth treatment was significantly higher (p < 0.05) than the albendazole and ivermectin treatments, but not different from the treatments with trichlorfon, piperazine, and calcium hydroxide. On day 35, last day of medicated feeds, the effectiveness of calcium hydroxide was statistically higher (p < 0.05) than the treatment with albendazole (Table 3).

Effect on growth (weight and weight gain)
The active ingredients added to the experimental feeds did not affect growth, as significant increase (p < 0.05) in fish weight after 63 days of trial was observed for all treatments, indicating that there was homogeneous growth in treated and untreated (control) fish. However, at the end of the sampling periods, fish fed with feed added with calcium hydroxide and diatomaceous earth presented lower weight (p < 0.05), whereas for the other treatments, there was no significant difference compared to the control (Table 4). Weight gain was significantly reduced (p < 0.05) at the end of the administration of the medicated feeds (sampling 3, day 35), and 30 days later (sampling 4, day 63), compared to the first 15 days of experiment (sampling 2). Weight gain of fish treated with calcium hydroxide was significantly lower (p < 0.05) than in fish treated with albendazole. For both variables, a significant interaction was found between administration period and type of treatment (Table 4).

Discussion
Tambaqui has remained the most farmed native species across the country, especially in the Brazilian Amazon states (Valenti et al. 2021). However, one of the major drawbacks is the occurrence of parasitic diseases, such as acanthocephalosis. In addition to spreading pathogens to the environment and causing economic losses to farmers, the disease represents a risk to public health, especially regarding drug residues in fish muscle Chagas et al. 2019). Laboratory diagnosis and effective treatments are necessary, as inappropriate use of drugs can result in negative impact to the fish and the environment, as well as the emergence of drug-resistant parasite species (Zhang et al. 2014).
In the present study, six chemicals were tested against N. buttnerae (Acanthocephala), namely trichlorfon, albendazole, ivermectin, and piperazine, commonly used as anthelmintics in aquaculture, and calcium hydroxide and diatomaceous earth with other uses in the production systems. The administered doses were based on the manufacturer's recommendations. For the anthelmintic active principles, the recommendation was two administrations with an interval between them. Data from recently published studies on acanthocephalan treatments in tambaqui were also reviewed Costa et al. 2020;Sebastião et al. 2020).
In general, the efficacy of anthelmintics to treat N. buttnerae in tambaqui was low, but the non-traditional chemists, calcium hydroxide and diatomaceous earth showed some promising results (efficacy values of 48.2% and 36.4%, respectively). Diatomaceous earth is a white powder from sedimentary rocks constituted mainly of silicon dioxide. It is used to control agricultural pests, as it affects the cuticle and exoskeleton of insects. Smiderle and Cicero (1999) demonstrated the insecticidal effect of diatomaceous earth in the treatment of corn seeds to control insect pests during grain storage, without causing toxicity to the seeds. The addition of diatomaceous earth to the fish feed promoted the best efficacy in Table 4 Final weight and weight gain of Colossoma macropomum at 14, 35 and 63 days after six treatments in the control of Neoechinorhynchus buttnerae Different uppercase letters indicate significant differences among the collection times, and different lowercase letters indicate significant differences among the treatments by Tukey test (p < 0.05). There was no interaction between treatment and time for the exposed variables Time ( the present study on days 15 and 35 of continuous treatment. This is the first report of diatomaceous earth being used as treatment for acanthocephalosis in tambaqui, so far. Calcium hydroxide or hydrated lime is used as a disinfectant, acid neutralizer, and flocculating agent in agriculture and aquaculture. Singhal et al. (1986) demonstrated the efficacy of hydrated lime (1.0 and 10 mg/L) in the mortality of myxosporidia cysts of Myxobolus in Cirrhinus mrigala and Ophicephalus punctatus. Hader et al. (2016) recommended its use to control parasites in fish, proving it is certified for use in aquaculture. In the present study, calcium hydroxide also showed efficacy of approximately 50% at the end of day 35 of treatment against N. buttnerae. Oral route is considered the best option for treating endoparasites such as the acanthocephalans, as it may be less stressful, lower cost, and easy-tohandle route of administration Valladão et al. 2020). Longer administration times for these compounds, analyzing physiological aspects of the fish should be tested in future studies.
Trichlorfon is commonly used to control a variety of fish ectoparasites, because of the mode of action related to the inhibition of the enzyme acetylcholinesterase, which leads to parasite paralysis . In this study, the efficacy of trichlorfon varied from 20 to 23%. Contrary to what was observed in this study with acanthocephalan, Thing et al. (2016) found that trichlorfon killed isopod parasites Caecognathia coralliophila within 24 h at 0.2 mg/L in farmed tiger grouper Epinephelus fuscoguttatus. Regarding withdrawal period, the analyses of the muscle of grass carp Ctenopharingodon idella revealed that after 5 days of the last administration of 0.25 mg/L of tricloforn, no signs of bioaccumulation of the chemical or of its metabolites were found (Guimarães et al., 2019). However, Guimarães et al. (2019) observed an effect on the growth parameters of C. idella, which was not observed in the present study.
The active principle albendazole belongs to the group of benzimidazoles and is widely used in the treatment of cestodes, nematodes, and acanthocephalans in fish and mammals (Taraschewski et al. 1988;Chagas et al. 2017). The pharmacological target of albendazole is the helminth's β-tubulin, which blocks the formation of microtubules, causing a late paralysis of the parasite. Weber and Junge (2000) reported the efficacy of albendazole in the treatment of the acanthocephalan Moniliformis clarki in a New World monkey Saguinus oedipus with protocols using 50 mg/kg for 16 days or 100 mg/kg for 3 days with bi-weekly boosters, with four treatments. In the present study, contrary to expectations, the efficacy of albendazole was below 10%. Similarly, none of the nine concentrations (250-2250 mg/L) of the drug tested in vitro caused significant N. buttnerae mortality (Oliveira et al. 2019). Likewise, no significant efficacy against N. buttnerae was observed with the use of albendazole in in vivo evaluation at five concentrations (100, 200, 300, 400, and 500 mg/kg of diet) added to the feed and administered for 21 consecutive days to tambaqui . The low solubility of the drug, according to Oliveira et al. (2019) and Farias et al. (2021), results in incomplete product bioavailability, which may also have occurred in the present study.
Ivermectin belongs to the group of avermectins, which pharmacological target is the ion channels of helminths, especially the chlorine channels controlled by glutamate. In the present study, the efficacy of ivermectin was less than 15%, differing from the in vitro results found by Oliveira et al. (2019). The dose of 4500 mg/L of ivermectin was 100% effective in killing in vitro N. buttnerae after 24 h of direct exposure of the parasite to the product added into the water (Oliveira et al. 2019). Taraschewski et al. (1988) did not observe the effect of injected ivermectin (0.1, 0.2, and 10 mg/kg) in eels Anguilla anguilla, for the treatment of Anguillicola crassus (Nematoda). The commercial product used in the present study was for injectable use, and therefore, the dose used in the feed was 600 µg of ivermectin/kg of LW/day for 5 consecutive days. It is possible that the effective dose was not reached in oral administration, thus not being effective in reducing the intensity of acanthocephalan infection in the intestine of tambaqui.
Piperazine belongs to the group of piperazines that act on the GABA receptors of helminths, causing paralysis of the parasite, and has been used to control nematodes, such as the human filarial nematode Wuchereria bancrofti (Bockarie and Deb 2010). In horses, the anthelmintic effect of piperazine in the control of cystotomies was demonstrated in association with mebendazole (Pereira et al. 1989). In this study, the treatment showed efficacy of 25%, 15 days after the first administration of the product, with lower efficacy in the following periods. On the other hand, Santamarina et al. (1991) reported that piperazine was not effective in controlling in vitro or in vivo Gyrodactylus sp. in rainbow trout Oncorhynchus mykiss.
In addition, it was observed that the treatments affected the weight gain of the fish under evaluation, with a significant reduction in weight gain at the end of the protocols and after 63 days of experiment. Goodrich et al. (2022) reported that the increased energy demands of a highly buffered feed, like the one with calcium hydroxide, will lead to increased acid secretion in the stomach, which would produce a greater specific dynamic action response and then result in reduced fish growth efficiency. Stone et al. (2003) stated that diatomaceous earth is a nutrient diluent and does not interfere with feed intake, digestibility, or growth of silver perch, Bidyanus bidyanus. Also, the study informed that the linear reduction in weight gain of fish fed increasing amounts of diatomaceous earth was consistent with previous research (Allan and Rowland 1998;Dias et al. 1998). However, there was an improvement in efficacy against the parasite with the use of the non-traditional products (best results obtained in this study), followed by ivermectin and piperazine. Data reported here constitute important contribution to the tambaqui technology package. Future experiments evaluating the physiological parameters of fish under the influence of the evaluated treatments, longer drug administration periods, and diverse experimental conditions to treat N. buttnerae in tambaqui are warranted to provide more strategies for the disease management.
Author contribution Bruna Rafaela Caetano Nunes, Yuri Vinicius de Andrade Lopes, and Thiago Tetsuo Ushizima contributed to the study conception and design. Material preparation, data collection, and laboratorial analysis were performed by Bruna Rafaela Caetano Nunes, Yuri Vinicius de Andrade Lopes, Ricardo Henrique B. de Souza, Raul Dirceu Pazdiora, Thiago Tetsuo Ushizima, Simone Paiva Medeiros, Lorrayne Tavares Corrêa, Igor Izidoro Cardoso, and Matheus Vitor Vieira Andrade. The statistical analysis was performed by Bruna Rafaela Caetano Nunes and Patricia Oliveira Maciel-Honda. The first draft of the manuscript was written by Bruna Rafaela Caetano Nunes, Patricia Oliveira Maciel-Honda, Edsandra Campos Chagas, and Fernanda de Alexandre Sebastião and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Data availability
The data that support the findings of this study are available upon request from the corresponding author (P.O.Maciel-Honda).

Declarations
Ethics approval By the Ethics Committee on the Use of Animals (CEUA) from UNIR (protocol #035/2016).