Histopathological evaluation of Wallago attu (Bloch & Schneider, 1801) infested by ectoparasites

Host-parasite interaction studies represent the dependency of the parasites on the host through various models. Gill infesting copepods, monogeneans and isopods served as excellent models to study the details of host-parasite interaction. In the present investigation, Wallago attu (Bloch & Schneider, 1801) was extensively studied for histopathological damage caused by ectoparasites like monogeneans (Thaparocleidus indicus, Thaparocleidus wallagonius and Mizelleus indicus), copepod (Ergasilus malnadensis) and an isopod (Alitropus typus) during August 2017 to February 2018. Both the control (least infested) and severely infested gills were examined through light microscope to approximate the intensity of damage caused by above ectoparasites. Pathological effects of ectoparasites displayed a fusion of secondary lamella, hyperplasia of the gill filaments and the epithelial cells, propagation of bronchial tips, thinning of the central axis, deshaping, shortening and fusion of secondary gill lamellae epithelium, severe degenerative and necrotic changes in gill filaments and secondary lamellae, curling of secondary lamellae and mucous cells proliferation. Ectoparasites cause severe damage to gills of W. attu, thereby diminishing the gill breathing capacity of the fish. Timely pathological studies can reveal the extent of damage caused by the parasites and thus various diagnostic programs and best management practices can be implemented in aquaculture for the better yield.


Background
Ectoparasitic infestation of wild and farmed freshwater and marine fishes caused by crustaceans, monogeneans, protozoans and isopods may severely affect the host health (Johnson et al., 2004;Boxshall, 2005;Shinn et al., 2015;Modi and Vankara, 2021). Branchiuran copepod parasites (Argulus) and ergasilids can cause severe impact on the freshwater fish production (Møller, 2009). Ergasilids are found mainly on the gills of fish which are specialized to perform various functions like gaseous-exchange, hormone production, circulation, ionic and acid-base balance and excretion (Pelster and Bagatto, 2010;Lima et al., 2013;Pádua et al., 2015). The infested gill tissues hinder the water flow and diminish the capability of the fish to sustain normal oxygen uptake (Ojha and Hughes, 2001). Monogeneans do not require any piercing mouthparts to feed on gill blood and need much effort to damage thin walls of the secondary lamellae, perhaps by suction or by applying of digestive secretions. Till date commendable work on gill histopathology of wild and farmed freshwater and marine fish was carried out by Roubal (1999), Campos et al. (2001), Dezfuli et al. (2003), Avenant-Oldewage et al. (2005), Vinobada (2010), Mohammadi et al. (2012), Kaur and Srivastava (2014), Singh and Kaur (2014), Weli et al. (2017), Mathews et al. (2018), and Suliman et al. (2021). Histopathological changes can be extensively used as biomarkers in the appraisal of health condition of fish (Roberts, 2001). The exploitation of these histopathological biomarkers in environmental monitoring permits the inspection of specific organs (lungs, gills, liver, kidney, etc.) accountable for vital functions such as respiration, excretion, accumulation and biotransformation of xenobiotics in the fish (Gernhofer et al., 2001). The thorough information of histology gives a crystal clear idea to differentiate the pathological changes induced by the parasites in infested and control tissues. The type and intensity of mechanical activity exerted by the ectoparasites are the main causes of the tissue damage and severity of the effect, but the secondary infections through bacteria, virus and fungi in the wounds produced by crustacean parasites are considered to be more dangerous.
The freshwater shark, Wallago attu (Bloch & Schneider, 1801) is one among the 10 largest catfish species on earth and are commercially important species harvested across tropical Asia owing to its high nutritional quality and consumer demand (Thella et al., 2018). In the present study, the control gill tissue (least infested) and the infested gill tissue (with severe infection) of the W. attu were considered to note the pathological changes induced by the ectoparasites.

Methods
About 95 freshwater shark (W. attu) (11.52 ± 1.95 cm, mean total length ± standard deviation; total length ranged from 7 to 15 cm and 293.15 ± 100.9gms mean total weight ± standard deviation; total weight ranged from 150 to 500 g) were obtained from the two different sampling stations of YSR Department of Zoology, Yogi Vemana University, India. The temporary slides of monogenetic trematodes were prepared using neutral red (C 15 H 17 N 4 Cl) and ammonium picrate-glycerine mixture by the method of Malmberg (1970) and were identified using the taxonomic volumes of Gusev (1976a, b) and Pandey and Agrawal (2008). The copepod and isopod parasites were fixed in 10% formalin solution and processed according to the method of Pillai (1985). The fragments of both infested and uninfested gill arches were carefully removed and processed according to usual histopathological techniques, embedded in paraffin at 58 °C, sectioned at 4-8 µm with Yorco YSI 115 rotary microtome, and stained in hematoxylin-eosin. The slides were investigated and photomicrographs were obtained using drawing tube. Ocular micrometer measurements were depicted in micrometers (µm) using Image J software (https:// imagej. nih. gov/ ij/ downl oad. html) unless indicated.

Discussion
W. attu infested with ectoparasites (copepods, isopods and monogeneans) demonstrated serious lesions in the gill filaments such as necrosis, hemorrhages, congestion, epithelial and mucus cells proliferation. These alterations may impact the hydromineral balance, gaseous exchange, hormonal production, circulation, nitrogenous waste secretion and making the fish hypoxic or anoxic and finally lead to death and similar pathological results were also observed by Mohammadi et al., (2012) who worked on parasitic infestation of skin and gill on Oscar (Astronotus ocellatus) and discus (Symphysodon discus) and Dias et al. (2021) who worked on Colossoma macropomum (Serrasalmidae), respectively. The severity of respiratory damage is directly related to the number of parasites on the gills. The higher intensity, the more serious the gill damage which can decrease the body weight and condition factor, severe changes in osmoregulation or respiratory dysfunction and ultimately lead to death. Ectoparasites such as monogeneans and copepods append to the primary lamellae with their anchors and legs resulted in cell damage and hemorrhages at the site of adherence. The oozed blood from the hemorrhagic site is fed by the parasite while some parasites secrete exogenous enzymes to digest the gill filaments of the host fish which can be witnessed in the gill histological sections with a high number of cell nuclei within the gut of gill parasites (Vinobada, 2010;Noga, 2010;Seenappa and Venkateshappa, 2000;Pádua et al., 2015). Though the gill epithelial cells are mechanically injured by the hooks of monogenean parasites and copepods (Purivirojkul, 2012), the greatest histopathological damage is induced by scraping and sucking activities of the parasites on host tissues as per the works of Derwa (1995), Endrawes (2001) and Hanna (2001). The histological sections of the infested gills by the parasitic infection revealed the functional impairment of the gill lamellae, attachment of parasite at the tips of the gill filament, and swelling of the host tissue at the site of attachment. Pathological alterations observed in this study were similar to the works of Campos et al. (2001), Fadaei et al. (2001), Barzegar andJalali (2004), Shamsi et al. (2009), Kaur and Shrivastav (2014), Pádua et al. (2015) and Arya and Singh (2020) who also recorded hyperplasia of the epithelial cells and succeeding lamellar fusion to be the distinctive feature of the parasitic infection. Hyperplasia of epithelial cells and the fusion of some secondary lamella are the patterns of the defense mechanisms of gills. Severe stress in fish may result in some alterations in blood vessels and damaged cells cause blood congestion or even an aneurysm (Rosety-Rodriguez et al., 2002). The impact of the diseases caused by ectoparasites especially monogeneans and copepods are not only linked to health status of fish but also to economic and societal impacts in case of culture fish, due to diminished respiratory capacity obstructing the growth leading to high mortality rates (Dezfuli et al., 2011;Shinn et al., 2015). The imbalance in the environment can be detected through host-parasite relationships and the consequences caused by the parasites to its hosts. The present findings stress on the fact that diseases are serious threats to cultivable and wild fishes which can be visualized with naked eyes. Hence, regular diagnostic programs together with exceptional management practices need to be executed from time to time to avoid sudden parasitic outbreaks and economic losses in aquaculture.

Conclusions
In present study we have observed with the histopathological alterations induced by the ectoparasites on the gill tissues. The greatest histopathological damage in the gills of the W. attu was induced by scraping and sucking activities of the parasites on host tissues. Histopathological observations disclose the health status of the host fish, i.e., stunted growth, susceptibility to various diseases, resulting in hypoxia or anoxia and thereby leading to the death. Thus, the findings of the present work accomplished that ectoparasitic infestations are serious threats for fish and can cause extensive damage and yield.