Argulus japonicus has been reported in several freshwater ornamental fish species worldwide, particularly in C. auratus, which is highly susceptible to infestation (Lester and Roubal 1995; Noga 2010; Kumari et al. 2019). In the current study, the presence of A. japonicus was primarily observed over the caudal and dorsal fins with numerous reddish point and haemorrhagic areas. The infected fish were also observed rubbing their bodies against the aquarium glass as reported in earlier studies (Sahoo et al. 2012; Roberts 2012). The mode of attachment and feeding on blood and other body fluids cause haemorrhageic wounds (Noaman et al. 2010), potentially leading to secondary infection with aeromoniasis or pseudomoniasis (Richards 1977; Noaman et al. 2010; Shameena et al. 2021).
In the present study, the identified species of Argulus infesting C. auratus were determined as Argulus japonicus based on morphological attributes. This included rounded abdominal lobe with abdominal incision exceeding half of the abdominal length, hook on the anterior spine of the 1st antenna, legs without pigmentation and flagellated swimming legs. These morphological attributes align with the findings of Rushton-Mellor (1994), Yıldız and Kumantas 2002; Noaman et al. 2010; Soes et al. 2010; Sahoo et al. 2012; Wafer et al. 2015; Saha and Bandyopadhyay 2015; Tandel et al. 2021).
The water quality parameters estimated in the study were within the tolerance limits for goldfish. The temperature ranged from 25.3 to 32.8°C (Winkaler et al. 2007; Chapman 2000; Wedemeyer 1996), pH level ranged from 6.9 to 8 (Winkaler et al. 2007), dissolved oxygen levels ranged from 5.8 to 6.9 mg L− 1 (Chapman 2000). However, during the toxicity study, a decrease in DO levels ranging from 5.38 to 6.0 mg L− 1, was observed. An artificial infection was induced through co-habitation method to achieve a moderate infection intensity of 15 to 25 juvenile Argulus per fish, consistent with the approaches demonstrated by Kumar et al. (2012) and Kumari et al. (2019). Similarly, Parida et al. (2018) reported a comparable infection intensity of 20–25 juvenile Argulus per fish in a group challenged with Argulus eggs. The present study showed in vitro antiparasitic efficacy of 100% for Nootkatone at 80 and 100 ppm against Argulus in 4 and 2h respectively. Similar conclusion was driven by Ekanem et al. (2004); who reported 100% mortality of Argulus parasites at 100 ppm of M. pruriens extract and 200 ppm for C. papaya extract after 6 h of in vitro treatment. The petroleum ether extract of M. pruriens and C. papaya exhibited 35% and 60% antiparasitic effectiveness within 3 h. Similarly, high antiparasitic efficacy of Quillaja saponaria extract was determined against Caligus rogercresseyi (Canon et al. 2021) copepodite stage at 500 ppm in 24 h. Further, Nootkatone based product at 300 ppm was effective in 90% killing of nematodes, trematodes, cestodes, or helminths within 24 h of exposure (Amick et al., 2018). Goldsmith (2017) reported that the application of Nootkatone ≥ 38 mg L− 1 could be able to control sea lice infection. In earlier studies, exposure of termites to 100 ppm Nootkatone for 15 d significantly reduced digging behaviours, survivorship and feeding activities by 83.5, 63.2 and 95.4%, respectively (Ibrahim et al. 2007). Furthermore, the use of hydraulic sprayer to apply Nootkatone formulation at 0.84% resulted in 100% control of I. scapularis within 1 week (Bharadwaj et al. 2014). The aqueous formulation of 5% Nootkatone treatment could result in complete suppression of Ixodes scapularis (deer ticks) and Amblyomma Americanum (lone star tick) nymphs (Dolan et al. 2009).
In the present study, DMSO was used as co-solvent to enhance the solubility of Nootkatone in water, a practice reported to improve solubility of compounds in aqueous solution (Kuiper et al. 1997; Girhard et al. 2009; Li et al. 2021). The findings of acute toxicity test of the present study showed 96 h LC50 of Nootkatone for C. auratus was found at 17.57 ppm, with values of LC50 decreasing with an increase in time. A similar pattern was reported by Sharma et al. (2016) and Kumar et al. (2012) for azadirachtin in goldfish. The 24h, LC50 and LC90 of Nootkatone for 2nd instars of Aedes aegypti were 180.460 ppm and 334.629 ppm, respectively. Further, 24h LC50 and LC90 values for 4th instars were 210.93 and 349.48 ppm, respectively (Ivoke et al. 2013). Bubble froth formation on the tank water surface with increased intensity at higher concentrations of Nootkatone solution in the present study reveal the volatile nature of compound nootkatone, which when escapes (evolves), creating bubbles that may circulate in the liquid surface (Parfitt and Wilson 2009; Chang and Urban 2016) and hence it can be suggested that the Nootkatone is environmentally safe and does not persist very long in the environment (Knox 2011), minimizing the potential for harmful impacts.
The in-vivo study showed that the bath treatment with different Nootkatone solution concentrations resulted in a significant detachment of Argulus from infected fish at 13.8 ppm to 23.6 ppm in 3 h. The observed detachment of Argulus in the test groups could be attributed to the effects of the Nootkatone, as a similar detachment from infected fish was not observed in the control groups. However, it is to note here that after 3 h, the detached Argulus reattached to the fish body again. The reattachment was occurred because Nootkatone is a volatile compound (Knox 2011), and thus lower concentration of chemical may not cause to kill the parasite and resulted in paralysed parasite revive once again. To overcome this problem, 3 sets of experimental tanks were set and at every 3 h of intervals fishes were being replacing to the next set of tanks without disturbing the detached Argulus left with the same tank to assess the number of dead Argulus detached from fish. In this experiment, 18.4 ppm and 23.6 ppm were showing almost 100% parasite detachment at 9 hr. But the 100% mortality was observed only after leaving the detached Argulus for further 3 hrs in the same third tank. Similarly, Kumar et al. (2012) reported that 100% antiparasitic efficacy of azadirachtin solution was found at 15 and 20 ppm for 72 and 48 hrs, respectively. Piperine is used as a potential natural agent to control Argulus infection at 9 ppm in 48 hrs (Kumar et al. 2012). Under in-vivo condition the EC50 value at 72 hrs of aqueous neem leaf extract against A. japonicus is 1.831 gL-1 (Kumari et al. 2019). An immersion or bath treatment with commercial milbemycin oxime and lufenuron at 0.015 and 0.30 mg L-1, respectively for 6h weekly once/two times, could completely eradicate Argulus infestation from freshwater fishes like Prochilodus magdalenae and Potamotrygon orbignyi (Tang et al. 2018). The present in-vivo anti-parasitic efficacy (AE) of Nootkatone against A. japonicus infected goldfish was recorded as 100% at 18.4 ppm in 12 h. Further, the EC50 of different Nootkatone concentrations was recorded as 15.31 ppm at 12 h with therapeutic index (TI) of 1.54. Similarly, the therapeutic index of piperine, azadirachtin and aqueous extract of neem leaf were reported as 5.8, 4.1 and 2.43 respectively, in 48 h of exposure, against Argulus infesting goldfish (Kumar et al. 2012; Kumari et al. 2019). Higher TI value (> 1) signifies the safety of Nootkatone for the treatment of Argulus infected goldfish.
Therefore, the findings of the present study reveal that Nootkatone at a concentration of 18.4 ppm could be a potential natural agent for controlling Argulus infection in C. auratus, provided dose precautions need to be taken during its application.