Asian Seabass juvenile was found infected with a crustacean parasite which was not reported so far in India. The parasite was identified as anchor worm, Lernaea sp. based on the morphological features as reported by earlier workers (Nagasawa et al. 2007; Toksen et al. 2014; Sayyadzadeh et al. 2016; Gervasoni et al. 2018; Hossain et al. 2018). Though the 28S rDNA sequence obtained was 100% matching with the published partial sequences of Lernaea cyprinacea, it could not be ascertained that the sample collected in the present study could be of the same species because of the conspecific nature between L. cyprinacea and L. cruciata (Hua et al. 2019). Morphologically, the collected parasite in the present study had single pair of branched holdfasts similar to L. cruciata against two pairs of holdfasts in L. cyprinacea (Shivaji et al. 2016; USGS 2022). Hua et al. (2019) suggested that the host-induced morphological variations in the holdfasts might lead to misidentification of L. cruciata and L. cyprinacea as different species, but both might be of the same species. Futures in depth molecular characterization studies are required to accept either both are same or different species. The anchor worms were interwoven by vorticellids and diatoms (Gnanamuthu 1951) due to their attachment on the host for long duration. This parasite is found to infect different species of fish opportunistically (Misganaw and Getu 2016; Satyanarayana and Sree Ramulu 2016). The present study showed that pH (7.88 ± 0.1), temperature (28.93 ± 0.5 oC), DO (4.95 ± 0.1 ppm) and salinity (1.03 ± 0.2 ppt) in the pond were within the adoptable range for both host and parasite. In concurrence with the present findings, prevalence and abundance of the parasite were directly proportional up to optimal range of water salinity, temperature and alkalinity (Price et al. 2010). Lernaeasis is usually observed in ponds that are characterized by slow water flow and elevated optimum water temperature of 26-36oC (Berry et al. 1991; Hossain et al. 2013; Toksen et al. 2014; Sayyadzadeh et al. 2016) and reduced development below 20°C (Marcogliese, 1991; Lester and Haywood, 2006). The infected fish were stressed, exhausted and nervous with bleeding spots, dropping scales and stopped feeding. The tongue like-process ending with the holdfast organ induced congestion, skin ulcers, haemorrhages, tissue proliferation and necrosis. This parasite feeds on blood and tissue debris leading to deep ulcers, abscesses or fistulas and skin necrosis. Fish mortality was observed 40% over a period of one week with abnormal behaviors, dullness, sluggish movement, respiratory distress, emaciation and anemia in finfish hatcheries (Hossain et al. 2013) as detected in the current investigation. Lesions, up to 2 mm in diameter, caused by this copepod parasite were noticed in different areas as reported earlier. This parasite infects gills and eyes causing respiratory distress and blindness, respectively (Toksen et al. 2014). Lernaea sp. was also collected and identified from nostrils, fins, gills, operculum, eye, lips and body of Capoeta saadii, Capoeta aculeata, Alburnus mossulensis (all native cyprinids), Carassius auratus and Cyprinus carpio (exotic cyprinids) collected from the Kor River Basin (Kor River and Dorudzan Reservoir), Southwest of Iran in 2010 and 2011 (Sayyadzadeh et al. 2016). In the present study, no parasitic stages could be found neither in gills nor eyes. Lernaea sp. infestation can occur in skin, fins, gills, and oral cavity but in the present outbreak, it was observed only on the skin as the fish juvenile were too small. PM examination of the moribund juveniles shown that there were skin ulcerations, cutaneous haemorrhages, muscle necrosis, intense inflammatory response, anaemic, pallor gill, blanched abdominal viscera and death as reported by the earlier workers (Khalifa and Post 1976; Berry et al. 1991; Daskalov et al. 1999; Hemaprasanth et al. 2008). Lernaea sp. infestation was reported in 3.33% of wild sea bass (Dicentrarchus labrax L.) in the northern Adriatic Sea (Coz-Rakovac et al. 2002). Hossain et al. (2018) recorded highest prevalence 72% with L. cyprinacea in January to March, while the lowest prevalence (8%) was recorded during April-June. Infestations with Lernaea sp. are most prevalent in the summer months and occur more commonly in stagnant or slow-moving water bodies (Marcogliese 1991; Hossain et al. 2013). Similarly, the present study revealed the prevalence of Lernaea sp. was 45.5% with the PI of 8.17 ± 0.15 per fish and mortality of 40% over a period of one week. The prevalence, parasite range and mean abundance of L. cyprinacea in Rainbow Trout (Oncorhynchus mykiss) [ABW, 80–100 g] farmed in Turkey were 100%, 4–9 and 6.2, respectively (Toksen et al. 2014). It is a common parasite all over the world affecting almost 46 species of Cyprinidae (Toksen et al. 2014; Sayyadzadeh et al. 2016). Fish can survive with Lernaea sp. infestation, but chronic infestations lead to lowered productivity in terms of growth retardation with behavioural changes and fish become more vulnerable to secondary bacterial and fungal infections which ultimately kill the fish (Robinson and Avenat-Oldewage 1996). It is a highly adapted crustacean that penetrates the host’s skin to form an extremely strong and damaging attachment. The adult females are usually attached to the surface of the fish as observed in the present study (Gervasoni et al. 2018). The male parasite dies after copulation. The female bores into the host tissue, eventually by means of a large anchor on her anterior (“head”) end to permanently embed into the skin and muscle of the fish. The female matures within 24 hours as a very prolific adult, may begin to release eggs from a pair of sacs on its posterior (“back”) end. In the present study, no developmental stages such as free living nauplii and/or parasitic copepodid could be recovered since treatment was done with EMB.
Lernaea sp. is an immense threat to aquaculture due to the fact that it can infect all freshwater fish and even frog tadpoles and salamanders. This parasite does not have any host specificity (Hossain et al. 2013 & 2018). It can easily get adapted in any water quality conditions and any hosts to complete its life cycle as evidenced from extensive morphological adaptations of the majority of lernaeids (Piasecki et al. 2004). But rarely, reported in Asian Seabass belonging to species of Latidae. Increased human population with enhanced transport facility and globalization have hastened the biological invasions and proportion of introductions of any alien species throughout the world, which is of major environmental issue of public concern (Vitousek et al. 1997; Sakai et al. 2001; Gozlan et al. 2010; Lymbery et al. 2014). Aquatic invasive species (AIS) and bio-invasions (BI) are global issues in marine, brackish and freshwater ecosystems with special reference to alien and invader parasites. Fingerlings become moribund if they are infected by more than six adult female parasites ultimately leading to epizootics and high mortalities in cultured fish (Daskalov et al. 1999; Lester and Haywood 2006; Hossain et al. 2018). In the present study, the PI was 8.17 ± 0.15 per fish with mortality of 40%. In the same pond, Asian Seabass fingerlings of different sizes between 5 and 45 g were maintained in separate cages and found no Lernaea sp. infestation. This may be due to the fact that the scales are not well developed in fish juvenile compared to that of fingerlings and above life stages. A small rigid plate like structures grows out of the skin in fish is called as scale. Though the scales from the bony fish are originated from different tissue, but are considered similar to the structure to teeth. The skin of Asian Seabass is shielded with these defensive scales, which can also afford active camouflage by reflection and coloration. In addition, fingerlings are covered with a layer of mucus (slime) which can protect against pathogenic organisms such as parasites, bacteria, viruses and fungi. The mucus layer eventually increases the swimming speed with reduced surface resistance. Since the scales are typically formed late during the developmental stages of fish, it makes the fish more vulnerable to parasites and the parasitized fish become more susceptible to super infections as well (Bandilla et al. 2006).
Under wet lab EMB experimental condition, intense neutrophilia and lymphocytopenia were noticed as reported in the Lernaea sp. affected fish (Silva-Souza et al. 1999). These conditions are also shown in relationship to the spawning period (Pickering, 1986) or stress due to low water temperature (Bennet and Neville, 1975). Similar deviations can also occur in bacterial, viral (Alvarez-Pellitero and Pinto, 1987) and parasitic infections and treatments (Mahajan et al. 1979; Silva-Souza et al. 1999; Das et al. 2022) as observed in present study. A possible influence of the spawning and/or low temperature, which was shown to cause neutrophilia and lymphocytopenia by other authors, cannot be applied in this present study because the fish infested were neither spawning nor maintained in low temperature. Monocytosis and the high levels of immature neutrophil counts were recorded from the affected groups. Lymphocytes are considered to be the most abundant leucocytes found in the peripheral blood of healthy fish. On the other hand, neutrophils are scarce, and basophils and eosinophils only occasionally seen (Ananda Raja et al. 2020). RBCs collected from the infested fish showed poikilocytosis, anisocytosis and dividing erythrocytes, which are indicative of parasitic anaemia (Martins et al. 2004) as detected in the current investigation. Anchor worm is incriminated as a voracious blood sucker, causing heavy blood loss leading to anaemia in fish as noticed in the present study. Comparative histopathology of gills, skin, muscle, liver and kidney showed that there were marked lesions in the infested untreated groups as compared to that of healthy and recovered fish juvenile. But no specific difference was observed in heart, eye, stomach, intestine and brain tissues of all the groups. Conspicuous lesions such as epidermis degeneration, presence of inflammatory MNCs and hemorrhagic lesion surrounding the parasite anchor in the hypodermis, fatty change with severe congestion and extensive haemorrhage in liver and Bowman’s capsule degeneration were observed in T3 as reported by the earlier workers (Khalifa and Post, 1976; Coz-Rakovac et al. 2002; Hossain et al. 2013).
This study proved that the EMB was 100% effective after 10th day of treatment against a crustacean parasite, Lernaea sp. in Asian Seabass juvenile. The symptoms observed before, disappeared after treatment as noticed by Toksen (2006) against Argulus foliaceus on Oscar, Astronotus ocellatus (Cuvier 1829) and Ananda Raja et al. (2020) against Caligus minimus on Asian Seabass fingerlings. EMB treatment was not attributed to any fish mortalities and/or adverse reactions since the control group was also fed with EMB treated feed (Stone et al. 1999; Ananda Raja et al. 2020). EMB has also been presented effective for controlling many parasitic infestations such as Lepeophtheirus salmonis (Kréyer) in Salmo salar L. (Stone et al. 1999, 2000a, 2000b, 2000c, 2002; Armstrong et al. 2000), Salmincola edwardsii in Salvelinus fontinalis (Duston and Cusack, 2002), Argulus coregoni (Hakalahti et al. 2004) and Salmincola californiensis (Bowker et al. 2012; Gunn et al. 2012) in Oncorhynchus mykiss, Lernanthropus kroyeri in Morone [Dicentrarchus] labrax (Toksen et al. 2006), A. foliaceus in Cyprinus carpio domestica (Braun et al. 2008), Caligus curtus in Gadus morhua (Hamre et al. 2011), Argulus spp. in Carassius auratus (Hanson et al. 2011), Acolpenteron ureteroecetes in Micropterus salmoides (Reimschuessel et al. 2011), and Anguillicoloides crassus in Anguilla rostrata (Larrat et al. 2012). But, reports using EMB against Lernaea sp. was scanty. But there is a similar report in controlling Lernaea sp. using doramectin by incorporating in feed at 1 mg kg− 1 BW of fish for 10 days in Labeo fimbriatus fingerlings and Catla catla yearlings without causing any kind of adverse responses or toxicities to the host fish (Hemaprasanth et al. 2008). However, detailed investigations on the environmental toxicity, pharmacodynamics and pharmacokinetics of doramectin upon their dosing to the aquatic organisms are essentially to be completed before final recommendation for the safe use of this drug to control lernaeosis in Asian Seabass. Since such studies are already reported for EMB (Ananda Raja et al. 2020), which can be scientifically recommended with 100% efficacy and safety to use against lernaeosis in the aquaculture ponds.