Albendazole is effective for controlling monogenean parasites of the gills of Piaractus brachypomus (Serrasalmidae) and Megaleporinus macrocephalus (Anostomidae)

Abstract Monogenean infestations can cause high mortality in farmed fish and therefore significant economic losses. The present study investigated the efficacy of albendazole in therapeutic baths against monogeneans of Piaractus brachypomus Cuvier, 1818 and Megaleporinus macrocephalus (Garavello & Britski, 1988). For both fish, a 24 hours therapeutic bath with albendazole concentrations of 150, 300 and 500 mg/L were tested against monogeneans from the gills. The baths had an efficacy from 61.4 ± 32.9 (95%CI=64.5) against monogeneans of P. brachypomus, and from 95.4 ± 5.6 (95%CI=10.9) against monogeneans of M. macrocephalus. In P. brachypomus, the hepatosomatic index (HSI) in fish exposed to 150 mg/L of albendazole was higher than in fish exposed to 300 mg/L. The splenosomatic index (SSI) values in fish exposed to 150 mg/L of albendazole were lower than in fish exposed to 300 mg/L. In M. macrocephalus, the HSI and SSI values decreased in treatments with 150, 300 and 500 mg/L of albendazole to control and treat infestations by monogeneans. For M. macrocephalus, 150 mg/L of albendazole can be used to control and treat infestations by monogeneans, while for P. brachypomus 500 mg/L of albendazole can be used in a 24 hours bath.


Introduction
Over the last two decades, the global aquaculture industry has been highly successful, and continues to grow while achieving the critical objective of environmental, economic, and societal sustainability. Aquaculture is the fastestgrowing food production sector in the world, and globally accounts for more fish biomass than capture fisheries, if non-edible amounts are included, and more total biomass than beef (Boyd et al., 2020;Tavares-Dias, 2021). Due to a decline in wild capture fisheries, aquaculture is a crucial component of future food security, and is essential to meet the demands of a human population expected to grow to nearly 10 billion by 2050 (Huston et al., 2020). Aquaculture is often regulated by national agencies, which represent a legal interpretation of local environmental sustainability. In Brazil, the total freshwater fish production of fish farming in 2021 was 841,005, representing an increase of 4.7% compared to 2020. In the last eight years, this production activity has grown 45%, equivalent to an average of 5.6% per year (PeixeBR, 2022). Hence, Brazil occupies 13th place in global aquaculture fish production, and is eighth in the global inland aquaculture production of finfish (Cavalli et al., 2021).
Among the native fishes that have been cultured in Brazil are Piaractus brachypomus Cuvier, 1818 (Serrasalmidae) and Megaleporinus macrocephalus (Garavello & Britski, 1988) (Anostomidae). An easy acceptance of commercial food and their fast growth have encouraged the increased production of these fish species in some regions of Brazil. Farmed in commercial fish farms, these fish are routinely stressed by intensive production management practices, leading to the emergence of parasitic diseases that naturally occur in the aquatic environment. Hence, parasitic diseases are most common in the farming of these fish species and are among the factors that strongly interfere with their growth (Martins et al., 2017;Negreiros & Tavares-Dias, 2019;Negreiros et al., 2021). Among the main taxa of disease-causing parasites in P. brachypomus (Negreiros & Tavares-Dias, 2019) and M. macrocephalus (Martins et al., 2002(Martins et al., , 2017Negreiros et al., 2021Negreiros et al., , 2022 are helminths monogeneans. Monogeneans are parasitic helminths with a short and direct life cycle, the vertical transmission of which facilitates infestation levels in intensive fish farming that cause several disorders in the animal (Alves et al., 2019;Negreiros et al., 2022); such as the excessive production of mucus in the skin and gills, as well as hyperplasia, edema, the fusion of the secondary lamellae, and branchial necrosis . Furthermore, secondary lesions caused by other pathogens, such as fungi and bacteria can result. Therefore, such problems show that although Brazil has an important role in the production of food from aquatic animals, the industry of fish farming yet faces many challenges.
The management and control of infestations caused by monogeneans poses a constant challenge for fish farming, as it is greatly complicated by the limited availability of licensed anthelmintic drugs, with varying degrees of effectiveness (Alves et al., 2019;Negreiros et al., 2022). In addition, the various chemical drugs that have been used against these parasites present certain problems, such as low efficacy, solubility and specificity, toxicity to host, and resistance (Santamarina et al., 1991;Tojo et al., 1992;Buchmann et al., 1992;Forwood et al., 2013;Alves et al., 2019;Tavares-Dias, 2021). Despite the broad-spectrum and tolerance of benzimidazole albendazole, to our knowledge, it has only been employed for the control and treatment of infestations caused by monogeneans in Anguilla anguilla Linnaeus, 1758 (Buchmann & Bjerregaard, 1990), Onchorhynchus mykiss (Walbaum, 1792) (Tojo et al., 1992), Piaractus mesopotamicus Holmberg, 1887 (Onaka et al., 2003) and Colossoma macropomum (Cuvier, 1816) (Alves et al., 2019). Albendazole is a versatile anthelminthic that is rapidly oxidized to its pharmacologically active metabolite, albendazole sulfoxide, after administration. Since it is widely used around the world, despite it has not yet been regulated for application in fish farming, this anthelmintic has been studied (Cordeiro et al., 2022). However, it has not been assayed to determine its efficacy in controlling monogeneans of P. brachypomus and M. macrocephalus, both fish of great economic importance to aquaculture in Brazil. Thus, the purpose of this study was to investigate the efficacy of albendazole in therapeutic baths against monogeneans of P. brachypomus and M. macrocephalus.

Fish and monogenean parasites
Two hundred fingerlings of P. brachypomus and two hundred fingerlings of M. macrocephalus were obtained from a commercial fish farm in Rio Branco, in the state of Acre, Brazil, and were kept at the laboratory of the Instituto Federal do Acre (IFAC), in Rio Branco (Brazil). The fish were acclimatized for 15 days in 500 L tanks with constant water flow and aeration, and were fed twice a day with commercial feed containing 35% crude protein (Guabi, Brazil). The following water parameters were maintained in the tanks: temperature at 29.1 ± 0.1 °C, dissolved oxygen at 5.6 ± 0.2 mg/L, pH at 5.4 ± 0.2, total ammonia at 0.4 ± 0.01 mg/L, alkalinity at 12.0 ± 0.1 mg/L and water hardness at 12.0 ± 0.1 mg/L. The organic matter that accumulated in the bottom of the tanks was removed once every two days. This stock of fish was used in the in vivo assays described below. These naturally infested fish by monogeneans were used in the experiments.
This study was developed in accordance with the principles adopted by the Brazilian College of Animal Experimentation (COBEA) and with authorization from the Ethics Committee in the Use of Animals of Embrapa Amapá (Protocol N o 013-CEUA/CPAFAP).

Anthelmintic drugs
A solution of Agebendazol® (Agener União, Brazil) with a concentration of 15% albendazole sulfoxide was used.
Therapeutic baths against monogeneans of P. brachypomus and M. macrocephalus One hundred and twenty fingerlings of P. brachypomus (9.0 ± 2.5 cm and 10.8 ± 9.1 g) and one hundred and twenty fingerlings M. macrocephalus (11.5 ± 3.1 cm and 33.4 ± 19.4 g), naturally parasitized by monogeneans, were randomly distributed in twelve 250 L tanks, which were kept in a static water system under constant aeration for 24 hours. The mean temperature in the tanks was 30.4 ± 0.1ºC, the dissolved oxygen content was 5.5 ± 0.2 mg/L, the pH was 5.3 ± 0.2, total ammonia was 0.5 ± 0.2 mg/L, alkalinity was 10.0 ± 0.1 mg/L, and hardness was 10.0 ± 0.1 mg/L.
One 24 hours therapeutic bath consisted of four treatments: 0, 150, 300 and 500 mg/L of albendazole (Onaka et al., 2003;Alves et al., 2019) with three replicates each, and 10 fish in each replicate, making a total of 30 fish per treatment. All treatments were performed simultaneously with the control group for each fish species. During the 24 hours bath, the behavior of the fish was observed, and they were not fed.
After the therapeutic bath with concentrations of albendazole, the fish were euthanized by medullary section, weighed (g) and measured (cm). Their gills were excised, fixed in 5% formalin and examined under a stereomicroscope to identify and quantify the monogenean parasites. The parasites were prepared for identification as recommended by Eiras et al. (2006). After parasite quantification, the prevalence and mean intensity of infection were calculated as described by Bush et al. (1997) and the efficacy of each treatment as described by Onaka et al. (2003). The monogeneans were identified according to recommendations of Cohen et al. (2013). Liver and spleen weight was measured for each fish species and used to determine the splenosomatic index (SSI) and hepatosomatic index (HSI) for each fish species (Tavares-Dias et al., 2000).

Statistical analyses
The data were evaluated based on the Shapiro-Wilk normality test and Bartlett's test of homoscedasticity. Because the intensity and abundance data were not normally distributed, they were analyzed by the Kruskal-Wallis test, followed by Dunn's test for comparison among medians (Zar, 2010).  (Figure 1). Prevalence of monogeneans was lower in fish exposed to 500 mg/L of albendazole, while the mean intensity decreased in treatments with 150, 300 and 500 mg/L, when compared to the control fish. Hepatosomatic index values in fish exposed to 150 mg/L of albendazole were higher than in fish exposed to 300 mg/L. Splenosomatic index values in fish exposed to 150 mg/L of albendazole were lower than in fish exposed to 0 and 300 mg/L ( Table 1).     During the therapeutic baths, P. brachypomus and M. macrocephalus showed behavioral change such as agitation, but no mortality was observed.

Discussion
Fish organ indices such as the hepatosomatic index is an indirect measure of glycogen and carbohydrate levels, and can be used to indicate the nutritional state of the fish. Splenosomatic index is a measure of both the immune status and hematopoietic capacity of the fish (Tavares-Dias et al., 2000;Voorhees et al., 2019;Negreiros et al., 2022). The differences in hepatosomatic index values between treatments with albendazole in P. brachypomus and M. macrocephalus indicate a variation in the consumption of stored energy. The differences in splenosomatic index values of P. brachypomus and M. macrocephalus indicate that hematopoietic capacity may had been affected by the treatment with albendazole.
In fish aquaculture, the drugs are administered by baths or via oral. Safe anthelmintic drugs that can be administered in therapeutic baths are suitable for use in fish farming. No concentration of albendazole caused mortality of P. brachypomus and M. macrocephalus or significant changes in behavior after 24 hours of exposure. Similarly, a therapeutic bath of 30 minutes with 50-500 mg/L of albendazole did not cause mortality in P. mesopotamicus (Onaka et al., 2003), nor in A. anguilla exposed to 24 hours baths with 10 and 100 mg/L of albendazole (Buchmann & Bjerregaard, 1990). However, a therapeutic bath containing 500 mg/L of albendazole caused a mortality of 6.6% of C. macropomum within 24 hours of exposure, but did not change the behavior of exposed fish. Therefore, these results indicate that the toxicity of albendazole is dependent on concentration and time of exposure.
Brazilian legislative framework establishes that medicinal pharmaceutical products for veterinary use must be specifically registered by MAPA, so that they can be prescribed for use in animals. In addition, to register a veterinary drug as antiparasitic, its efficacy must be greater than or equal to 90% (Cordeiro et al., 2022). Therefore, the albendazole treatments evaluated in this study achieved the required MAPA efficacy.
In conclusion, both M. macrocephalus and P. brachypomus were not sensitive to the concentrations of albendazole used. There were differences in the splenosomatic index and hepatosomatic index of M. macrocephalus and P. brachypomus after 24 hours baths with albendazole, indicating that the spleen and liver were affected by the treatment. Furthermore, it was found that 150 mg/L de albendazole can be used to control and treat infestation by monogeneans in M. macrocephalus, while 500 mg/L of albendazole can be used for P. brachypomus, in 24 hours baths.

Ethics declaration
This study was developed in accordance with the principles adopted by the Brazilian College of Animal Experimentation (COBEA) and with authorization from the Ethics Committee in the Use of Animals of Embrapa Amapá (Protocol Nº 013-CEUA/CPAFAP).