First report of Ichthyobodo necator, a flagellate protozoan parasite, in Schizothorax niger from the River Jhelum in the Kashmir Himalayas, India

Ichthyobodo necator was obtained from skin during a parasitological survey conducted for the detection of ectoparasites in Schizothorax fish species from the River Jhelum, Kashmir, India, over a period of six months. Out of 290 collected fish samples, only one fish was found to be infected with I. necator (flagellate protozoan parasite). The primary pathological changes observed in the skin tissue during this study were the destruction of the epidermis, formation of vacuoles in the epidermal cells, and significant hyperplasia of the goblet cells. Epidermal cells were observed to have lost their interlinking cell membranes, and the outer surface layers of the epidermis occasionally seemed to slough. In addition, scanning electron microscopy (SEM) was used to provide a detailed morphological examination and deeper exploration of the intricate relationship between parasite and host. The SEM images clearly reveal comma-shaped I. necator organisms attached to the host cell through an attachment disc. These observations are based on histopathological observations of a single individual. The findings presented here contribute not only to the knowledge of parasite diversity in the Kashmir Himalayas but also serve as a foundation for future research on the ecology, biology, and management of freshwater ecosystems. This is the first record of the flagellate protozoan fauna of the Schizothoracines from the Kashmir Valley, India.

The Kashmir Valley, located in the northern region of the Indian subcontinent, is rich in water bodies, which play a significant role in regulating its climate and making it more moderate than the surrounding areas, and these water bodies contain a wide variety of freshwater life, including fish (Ahmed et al., 2017). The Jhelum River, the largest and longest in Kashmir, is home to a variety of fish species, including the native Schizothorax species and the exotic Cyprinus carpio. It originates from the Verinag spring, located in Anantnag district at the base of the Panjal Mountains. An average annual fish yield of approximately 20.5 kg ha -1 has been recorded from the Jehlum River (Bhat and Hussein, 2020). However, this river is facing many challenges due to human encroachment and increased pollution, and stress caused by pollution may have a direct or indirect impact on the prevalence of parasites (Wali et al., 2021). Over the past decade, the fish population in the River Jhelum, in particular the native fish Schizothorax, has become susceptible to a number of parasites belonging to the phyla Protozoans, Platyhelminthes, Nematodes, and Acanthocephalans (Ali, 1993), and the fish population is steadily declining (Rukhsana et al., 2008). Pathogenic invasion is more likely when water quality changes (Qayoom and Jaies, 2019). The Protozoan parasites are common in both wild and cultured fish species, and a few of those belonging to the genus Trichodina and Ichthyophthirius have been reported from the lacustrine ecosystem of Kashmir (Dar et al., 2016). However, the presence of Ichthyobodo has not been reported in riverine species from the Kashmir Valley to date.

A flagellate protozoan parasite known as Ichthyobodo infects a variety of fish species worldwide. I. necator, formerly known as Costia necatrix, is widely distributed in freshwater environments and causes serious disease in freshwater species. It has also been documented to survive and cause disease in marine fish (Stoskopf, 2015). It was originally described from the alevins of diseased brown trout (Salmo trutta) cultured in tanks at the College de France, Paris (Henneguy, 1884). In Asia, Ichthyobodo has been reported from different fish species in Iran, Japan, Singapore, the Philippines, and Sri lanka (Isaksen, 2013; Bonham, 2022). I. necator causes ichthyobodosis or costiosis, primarily infecting the skin and gills of fish, and can result in heavy mortality, representing a common problem in fish farms. In India, an assessment of the prevalence of fish diseases in Sambalpur, western Orissa, revealed the occurrence of C. necatrix in 16% of fish farms in this region. The most affected fish species were Labeo rohita, Catla catla, and Cirrhinus mrigala (Khatri et al., 2009). Ichthyobodo spp. possess an attachment disc by which they attach to the host cell, penetrating the host cell cytoplasm through a cytosome canal supported by microtubules. In seawater systems, I. necator infections were first discovered in freshwater salmonids that had been transported to the sea, indicating that the parasite is native to freshwater but can survive and flourish in the marine environment (Needham and Wootten, 1978). In freshwater, the parasite is more common on the skin than on the gills, whereas it occurs almost exclusively on the gills in seawater (Todal et al., 2004). Fish affected with Ichthyobodo often show signs of lethargy, lack of appetite, and respiratory distress. A whitish film caused by excess mucus production is commonly observed on the body surface. Some fish die without visible external signs (Stoskopf, 2015).

The present work was conducted as a random study on the prevalence of ectoparasites among the fish fauna of the River Jhelum. The occurrence of Ichthyobodo infection was an unexpected finding, which motivated us to investigate this parasite further in the economically important local fish Schizothorax niger, which is not only widely distributed but also highly valued as a food fish in Kashmir. To the best of our knowledge, no study has been done on the flagellate protozoan parasite Ichthyobodo in freshwater fish from the Kashmir Valley.

Ethical approval

Before the start of the experiment, the Aquatic Animal Ethics Committee of the Faculty of Fisheries, SKUAST-K, reviewed the protocols that were to be employed throughout the study to ensure that they followed the Aquatic Animal Ethical guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and the Institutional Animal Ethics Committee (IAEC).

Study site

A total of three study sites along the River Jhelum (32°58’42” to 35°08’02” N latitude and 73°23’32” to 75°35’57” E longitude), representing different demographic features of Kashmir, were selected for the sampling. Site I was located at Chatabal in the Srinagar district, Site II at Guree in the Anantnag district, and Site III at Sopore in the Baramulla district. This stretch of the River Jhelum is characterized by dense human population and moderate agricultural activity.

Collection of fish samples

A total of 290 samples of Schizothorax were collected from different sites along the River Jhelum during the spring season (February–July 2023), over a period of six months. Of the 290 samples, 91 samples were collected from Guree, 101 from Chatabal, and 98 from Sopore. These samples were transported aseptically to the laboratory of Aquatic Animal Health Management, Faculty of Fisheries, SKUAST- K. The total and standard lengths of each fish were measured in centimeters (cm) using a measuring tape, while the weight of each fish was measured in grams using a weighing balance. The collected fish were subjected to various gross parasitological investigations at Levels I and II.

Fish examination for ectoparasites

Fish were examined for ectoparasites using a modified version of the methodology described by Kabata (1985). The fish were sacrificed by striking the head, and immediately thereafter, wet mounts of skin smears and gills were prepared and observed under a low-power microscope for the analysis of ectoparasites. Samples of the wet mount were made from each specimen to measure the intensity of parasites in Schizothorax species.

Prevalence, mean intensity, and relative density of parasites

The prevalence, mean intensity, and relative density of parasites were calculated according to the methods described by Qayoom et al. (2015, 2022).

Histopathological analysis

Histopathological analysis was carried out according to the methodology described by Roberts (1989). The skin of the infected fish was used to study the histopathological effect of I. necator using a light microscope. Infected skin samples were fixed in neutral buffered formalin solution (10%), dehydrated using different concentrations of ethanol, and finally cleared with xylene. Tissue infiltration was performed using molten paraffin wax, and blocking of tissue was done by using L (Leuckhart’s) molds. Sections of paraffin, along with the sample (5µ thick), were prepared and stained with hematoxylin-eosin (H&E). The slides were then permanently mounted using Canada balsam and DPX mountant. Prepared mounts were observed under a light microscope (10X and 40X), and images were captured.

Scanning electron microscope

SEM analysis was carried out according to the methodology described by Shah et al. (2015). Infected skin samples were fixed in 2.5% glutaraldehyde and then in 1% osmium tetroxide. The tissues were then dehydrated with ethanol of different concentrations and critical-point dried. The resulting material was mounted on aluminum stubs, sputter-coated with gold, and examined using the Gemini SEM 500.

The study was conducted during the spring of 2023 on the indigenous fish species (S. niger, S. esocinus, and S. curvifrons) from the lower areas of the River Jhelum, Kashmir, India. The main objective of the study was to investigate ectoparasite prevalence in a sample of 290 grow-out fish specimens. One fish sample was found to be positive for the flagellate protozoan parasite (I. necator), which was recorded for the first time in this geographical region.

Clinical cues and microscopic examination

Clinical signs observed in fish infected with I. necator included lethargy and excessive mucus secretion, with some reddish areas indicative of hemorrhage. Microscopic examination of wet mount preparations revealed actively motile, small, and somewhat comma-shaped trophozoites. A pair of flagella of I. necator was clearly observed under the microscope.

Prevalence of Ichthyobodo in Schizothorax species

Table 1 shows the prevalence, mean intensity, and relative density of Ichthyobodo in different Schizothorax species. Overall, of the 290 Schizothorax fish specimens examined, only one specimen (0.34%) was infected with I. necator. The infected specimen was S. niger, with its prevalence recorded equal to 0.34%. The prevalence among S. niger specimens was 0.95%. The mean intensity of I. necator in S. niger, S esocinus, and S. curvifrons at the Chatabal site on the River Jhelum was 8.00, 0.00, and 0.00, respectively, with relative abundance of 0.08, 0.00, and 0.00, respectively. No infections were recorded in specimens collected from the Guree or Sopore sites.

Histopathological examination

Microscopic examination revealed comma-shaped parasites attached to the epithelial cells of the skin. Histopathological examination showed an inflammatory reaction at the site of parasite attachment in the epidermis. The results also revealed destruction of the epidermis and vacuoles in the epidermal cells. There was also significant hyperplasia of the goblet cells. In addition, epidermal cells were seen to have lost their interlinking cell membranes, and the outer surface layers of the epidermis occasionally appeared to slough off (Figure 1).

Scanning electron microscopy

Scanning electron microscopy (SEM) of infected skin revealed numerous comma-shaped microorganisms present on the infected skin. These parasites attached to the host using the flagella and penetrated the epithelial cells (Figure 2). Some of the sessile parasites were clustered together at the same attachment site (Figure 2a). The SEM images clearly reveal that I. necator organisms attach to the host cell through an attachment disc, allowing a cytostome canal, which is supported by microtubules, to enter the cytoplasm of the host cell. The cytostome of I. necator was clearly visible by SEM (scale bar = 1 micrometer). Flagellated trophozoites were clearly visible by SEM, with one or two pairs of flagella of unequal length. The short flagella ended inside the groove, while the long flagella extended past the posterior tip of the trophozoite (Figures 2b–d).

Ichthyobodo necator, formerly known as Costia necatrix, is an important protozoan ectoparasite of cultured fish and wild fish throughout the world. Ichthyobodo spp. are widely studied and well-documented flagellate protozoan ectoparasites of fish, and their importance is reflected in the extensive literature describing various aspects of their biology, including ultrastructure and pathogenesis of Ichthyobodo spp (Diamant, 1987; Fuentes-Valencia et al., 2022; Fadel et al., 2025; Rasha et al., 2025). Costiasis of Atlantic salmon, Salmo salar in seawater (Ellis and wootten, 1978), Molecular taxonomy (Callahan et al., 2002), Ichthyobodo infections of salmon in a hatchery: infection dynamics, morphology and sources of infection (Isaksen et al., 2002). Ichthyobodo necator (I. necator) is an important and highly pathogenic fish ectoparasite. Previous studies have studied the host–parasite interface of I. necator on the skin and gills of salmonids, its host range and geographical distribution, and the effects of I. necator infections on the seawater survival of juvenile chum salmon (Buchmann, 2022; Fadel et al., 2025).

In the Pacific Ocean, Atlantic Ocean, and nearby seas ranging from the subarctic to tropical zones, the ectoparasitic flagellate Ichthyobodo has been found in 25 species of marine fish and three species of anadromous salmon. The genus Ichthyobodo consists of three species: I. necator (Henneguy, 1883), I. nitschei (Nitsche and Weltner, 1894), and I. hippoglossi (Isaksen et al., 2007). A fourth species, Costia pyriformis Davis, 1943, most likely refers to a different genus. In freshwater, I. necator has been found on three-spined sticklebacks, brown trout, rainbow trout, and Atlantic salmon (Isaksen, 2013). The incompletely described Ichthyobodo species I. nitschei from goldfish may represent one of the many unnamed Ichthyobodo species identified through small subunit (SSU) rDNA sequencing in infected goldfish and other cyprinids (Carassius auratus) (Isaksen et al., 2007). The only identified marine species is the parasite I. hippoglossi, which infects the Atlantic halibut (Hippoglossus hippoglossus). I. necator and marine Ichthyobodo share many morphological similarities. Both parasites have a small body with two flagella of unequal length. The shape of parasites changes between the free-swimming and attached forms. In freshwater, the free-swimming form of I. necator is almost round, but the attached form is pyriform. A few morphological changes have been reported in I. necator following transfer from freshwater to seawater (Urawa and Kusakari, 1990; Fadel et al., 2025). It is well known that salmonids infected with Ichthyobodo exhibit extensive epidermal skin destruction and necrosis (Robertson et al., 1981; Urawa, 1992a; Ewers and Dunthorn, 2026). The present study also confirmed that the infected fish suffered severe skin erosion. These histopathological changes resemble those in freshwater salmonid juveniles (Roubal et al., 1987b). The skin of infected fish showed extensive epidermal hyperplasia and vacuolation, followed by degeneration and sloughing of epidermal skin. These histopathological changes resemble those in freshwater salmonid juveniles (Roubal et al., 1987b) and in Japanese flounder infected with Ichthyobodo spp (Urawa et al., 1991, 1998).

The identification of I. necator, a flagellate protozoan parasite, in S. niger from the River Jhelum in the Kashmir Himalayas represents a significant milestone. This discovery underscores the importance of comprehensive ecological studies in relatively unexplored regions and raises questions regarding the potential effects of this newly reported parasite on the health and stability of the host fish population and the broader river ecosystem. Qayoom and Shah (2017) assessed the parasitic bioload of Schizothoracine fishes in the Kashmir Valley in relation to the trophic status of Anchar Lake. The study found that the primary cause of fish parasite infections is an increase in the eutrophication of the lake over time. Combining SEM and histopathology allowed correlating the structural features of parasites with the pathological changes observed in host tissues. This helped in unravelling the complex interactions between host and parasite, including how parasites enter host tissues, evade the host immune system, and cause disease. SEM was particularly valuable for the detailed morphological examination of the parasite at high magnification, providing important information on the external structure and penetration of the parasite into the host. Such observations can aid in the accurate identification of the parasite species, which is crucial for understanding its biology and behavior. The SEM images clearly revealed comma-shaped pathogens attached to the host cell by an attachment disc through which a cytostome canal, supported by microtubules, penetrated the host cell cytoplasm. Flagellated trophozoites were seen with SEM, with one pair of flagella of unequal length. These ultrastructural observations are consistent with previous studies of hatchery-reared parr (Isaksen et al., 2011) and skin cells of juvenile salmon (Salmo salar) (Isaksen et al., 2007).

Earlier studies by Schubert (1966) and Joyon and Lom (1969) reported that I. necator infecting freshwater fish possesses a smooth cytostome process. The findings of the present study are consistent with these observations, confirming that freshwater I. necator infecting S. niger exhibits a smooth attachment disc and cytostome. However, because only a single infected specimen was found during the survey, the clinical and pathological observations reported here should be considered preliminary and descriptive rather than broadly representative of the pathogenicity of I. necator in S. niger species. Another limitation of the present study is the absence of molecular confirmation of the parasite identity through a barcoding approach, and all observations are based on histopathological examination of a single individual. Nevertheless, the morphological characteristics observed in the present study, particularly the distinctive comma-shaped body and smooth attachment disc revealed by SEM examination, are consistent with published descriptions of I. necator. Future studies incorporating molecular markers will be necessary to confirm species identity and further resolve the taxonomic status of Ichthyobodo spp.

To the best of our knowledge, our study represents the first documented report of Ichthyobodo necator, a flagellate protozoan parasite, in Schizothorax niger from the River Jhelum in the Kashmir Himalayas. This study provides a valuable baseline for future research on parasitic infections in freshwater fish of the Kashmir Himalayas and emphasizes the need for comprehensive ecological and conservation efforts to protect the biodiversity of this unique and ecologically important region. These observations are based on histopathological observations of a single individual. Further studies are needed to explore the life cycle, transmission dynamics, and potential host–parasite interactions of I. necator in S. niger. This knowledge will not only enhance our understanding of parasite biology but also contribute to the development of effective strategies for the prevention and control of parasitic infections in freshwater fish populations. Additionally, monitoring and surveillance programs should be established to assess the long-term implications of I. necator infection on the health and sustainability of S. niger populations in the River Jhelum.