Exploring the Genetic and Morphological Diversity of Argulus Ectoparasite Infecting the Aquaculture and Ornamental Fish in Tripura

The increasing global demand for fish force farmers to intensify aquaculture practices creating stressful environments, resulting in an epidemic of infectious diseases (Laltlanmawia et al., 2023). Parasitic diseases have emerged as a significant concern within the domain of fish pathogens and are the leading contributors to socio-economic losses in Indian aquaculture (Sahoo et al., 2020). Most wild and farmed fish parasites are protozoa, helminths, myxozoans and crustaceans (Thakur et al., 2023; Saravanan et al., 2021). Among the crustaceans, the most common one affecting Indian major carps (IMCs) is Argulus, which is responsible for argulosis and is a significant barrier to the development of the Indian freshwater aquaculture industry (Sahoo et al., 2013; 2020). 
       
Argulosis has several detrimental effects on freshwater fish, including skin ulceration, osmotic imbalance, immune suppression, irregular swimming behaviour, haemorrhages, growth impairment, anaemia and increased susceptibility to secondary infections (Sahoo et al., 2012; Rahman et al., 2019; Datta et al., 2022). There is a global distribution of approximately 129 recognized species of Argulus, with 85 species inhabiting freshwater environments and 44 species found in marine waters (Poly, 2008, Kismiyati et al., 2023). Argulus coregoni, A. japonicus and A. foliaceus are recognized as the three most extensively investigated species in freshwater ecosystems globally (Steckler and Yanong, 2013). To date, 20 species have been documented in both cultured and wild fish populations in India, as reported by Valarmathi, (2017), Bari (2018) and Sahoo et al., (2020). According to Sahoo et al., (2013), the prevalence of A. japonicus and A. siamensis was evident in the genetic composition of Argulus species obtained from diverse freshwater aquaculture settings in India.
       
Taxonomist uses morphological features alone for identification of Argulus spp., however, many a time due to lack of expertise, erroneous identification of Argulus spp. may occur (Tandel et al., 2021). Several researchers have utilized molecular identification techniques to examine Argulus spp. and its population (Tandel et al., 2021, Wadeh et al., 2010). However, according to Nagasawa, (2021), it is conceivable that molecular data pertaining to misidentified congeneric species and may have been erroneously submitted in GenBank under the classification of Argulus spp. Therefore, the identification and characterization of the Argulus may be accomplished via the use of both morphological and molecular approaches (Patra et al., 2016, Nagasawa, 2021).
       
Morphological identification and 18S rRNA genetic marker sequencing confirm findings of Argulus spp. up to species level in this investigation in a specific geographical region and the spatial distribution of Argulus spp. infecting Indian Major carps (IMCs) and ornamental fishes in Tripura, India. The current research additionally evaluated the host ranges of several Argulus spp. and the pathological alterations caused by Argulus infestations in hosts.

Study site and sample collection
 
A total of 670 fishes were collected from seven different location of Tripura, India viz. farm-fish samples from Udaipur, (23°31'39.7"N,91°30'15.0"E), Amarpur (23°31' 16.5"N 91°39'17.6"E), Taidu (23°43'53.9"N 91°40'20.9"E); Bamutia (23°57'14.2"N 91°17'10.8"E), Lembucherra (23°54'36.6"N 91°18'55.8"E), Bagabasa (23°33'35.6"N 91°25'18.5"E), Gangachhara (23°27'34.3"N  91°27' 24.6"E) and ornamental fish samples were collected form aquarium settings of Agartala, Tripura (23°50'16.7"N 91°16'41.3"E) with sampling duration from February 2022 and June 2023. Parasites were manually removed from from different body parts (operculum, dorsal fin, ventral fin, pectoral fin and tail) of infested fish using blunted forceps and a fine brush. The actively swimming argulus parasites were washed three times (10-15 min each) with phosphate buffer saline (PBS, pH 7.2). Morphological and molecular identification were done on Argulus samples for which they were transferred to RNAse/DNAse-free cryotubes and preserved in a diluent for DNA extraction (Himedia, USA) and stored at -20°C for DNA isolation and subsequent analysis. The research was carried out for a duration of two years at College of Fisheries, Central Agricultural University, Lembucherra, Tripura.
 
Morphological analysis
 
To facilitate morphological identification, the acquired Argulus samples were subjected to an initial washing procedure using PBS (pH 7.2, for 2-5 minutes). Subsequently, the samples were examined at different magnification levels using a Leica S6D stereo zoom microscope and a Zeiss Primostar 3 upright light microscope on the day of collection. To facilitate future morphological investigations, the specimens were preserved in 4% neutral buffered formalin (NBF) and 70% ethanol using the methodology outlined by Sahoo et al., (2012) and Tandel et al., (2021). The A. japonicus specimens were identified morphologically following the key characteristics given by Wadeh et al., (2008); Soes et al., (2010); Mousavi et al., (2011); Sahoo et al., (2012) and Nagasawa (2021). A. foliaceus specimens were identified according to Tokşen (2006); Møller (2011); Soes et al., (2010) and Mousavi et al., (2011). However, specimens of A. coregoni were confirmed based on the key morphological features described by Shimura (1981); Everts and Avenant-Oldewage (2009); Mousavi et al., (2011) and Nagasawa and Yuasa (2019). 
 
Molecular analysis
 
DNA extraction and PCR amplification
 
The genomic DNA was isolated from morphologically distinct Argulus sp. kept in DNA diluent using the phenol: chloroform isolation procedure as reported by Khan et al., (2020) with slight modifications. However, the macerated samples were vortexed and incubated overnight at 54°C in a water bath. The extracted DNA was used as a template for polymerase chain reaction (PCR) and universal primer set-1 amplified the 650 bp fragment of 18S rRNA gene using 18S rRNA F-566:5'- CAG CAG CCG CGG TAA TTC C-3' (forward) and for 18S rRNA R-1200:5'- CCC GTG TTG AGT CAA ATT AAG C-3' (reverse) (Hadziavdic et al., 2014). PCR amplification was performed in a thermal cycler (Applied biosystemTM VeritiTM, Thermofisher Scientific) using the following PCR conditions; 95°C for 15 min, 35 cycles consisting of 95°C for 45 sec, 60°C for 45 sec, 72°C for 1 min and a final extension step of 72°C for 10 min. The other universal eukaryotic primer set-2 for targeting 18S rRNA include, 18S rRNA F-ERIB1, 5'-ACC TGG TTG ATC CTG CCA G-3' and 18S rRNA R-ERIB10, 5' CTT CCG CAG GTT CAC CTA CGG-3' amplified a fragment size of 1800 bp (Patra et al., 2016). Initial denaturation step at 95°C for 5 min was followed by 35 cycles of denaturation for 30 sec, annealing at 51°C for 30 sec, extension at 72°C for 60 sec and final extension at 72°C for 5 min. HiPurA® PCR product purification kit (Himedia, India) was used to purify PCR amplicons before bidirectional DNA sequencing using Sanger sequencing (Bioserve Biotechnologies Pvt. Ltd., India).
 
DNA sequencing and phylogenetic analysis
 
Based on 18S rRNA sequences from the current isolates and other Argulus spp., Molecular Evolutionary Genetics Analysis (MEGA, version 11) was used to align them. The GenBank database was searched for comparable Argulus sp. sequences using Nucleotide BLAST. Phylogenetic analysis and species pairwise distances were performed as stated by Tamura et al., (2007). The maximum likelihood approach with 1000 replicates bootstrap analysis was used for graphical depiction of 18S rRNA divergences across Argulus species. The evolutionary pairwise distances were calculated using the Tamura-Nei substitution model, as described by Tamura et al., (2013).
 
Prevalence analysis
 
The prevalence study was evaluated using the methodology and formula specified by Margolis et al., (1982).
         
The Chi-square tests (χ²) was conducted to compare the difference in parasite prevalence across host species in terms of parasite infection rates.

Host diversity and prevalence analysis
 
Out of 670 fish specimens examined, 395 individuals were found to be infected with Argulus spp. parasites. The most affected species was Goldfish (249 no.), followed by Rohu and Catla species (104 and 21 no., respectively). The remaining fish species had lower rates of infection (Mrigal- 13, Subunkin-4, Milky white koi carp-3 and Tiger Oscar-1). This study revealed that A. japonicus has a propensity to infect a diverse array of fish hosts, infecting 247 goldfish, 104 rohu, 21 catla, 13 mrigal, 4 subunkin and 3 milky koi carp. Additionally, it was observed that A. foliaceus only infected a single tiger oscar (Astronotus ocellatus), whereas A. coregoni infected two goldfish individuals. 
       
In the Indian context, A. japonicus has been documented in many fish species, including Cyprinus carpio (Sahoo et al., 2012), goldfish/Prussian carp (Kumari et al., 2019) and Himalayan snow trout, Schizothorax richardsonii (Tandel et al., 2021). Saha and Bandyopadhyay (2015), were the first to report the infestation of A. coregoni, A. japonicus, and A. foliaceus, in Red Can Oranda Gold Fish (Carassius auratus auratus) in West Bengal. Similarly, in our present study, A. japonicus has been found to infect a wide range of host species with various infestation rate including goldfish (90.20%), milky koi carp (50%), mrigal (46.43%), rohu (38.24%), Catla (36.21%) and subunkin (27%), whereas, A. foliaceus and A. coregoni infected only tiger oscar (50%) and goldfish (13.33%), respectively (Fig 1 and 2), showing low host diversity than A. japonicus. However, there was no significant difference in terms of prevalence rates between the host species and Argulus spp. (P>0.05) (Fig 2). This is the first record of A. japonicus infecting milky koi carp and A. foliaceus infecting tiger oscar in India. This phenomenon could potentially be attributed to the transmission of Argulus spp. infections among fish hosts in the same aquatic system or to the introduction of infected fish into a culture environment.
 

 

 
Pathological changes
 
Acute hemorrhagic inflammation of the epidermis, increased mucosal production, scale spillage and fin corrosion were identified at the sites of Argulus infestation (Alas et al., 2010). These were consistent with the pathological findings in the present study (Fig 3).
 

 
Morphological description of Argulus spp.
 
The morphological characteristics previously described were used to differentiate A. coregoni, A. foliaceus and A. japonicus based on the morphology of the abdominal lobes, respiratory regions and length of the cephalothoracic carapace as described by Noaman et al., (2010) and Wafer et al., (2015). As per Noaman et al., (2010), A. coregoni exhibits pointed abdominal lobes, posterior lobes of the cephalothoracic carapace do not extend beyond the commencement of the abdomen, similar to A. foliaceus. However, in A. foliaceus it has rounded abdominal lobes with the posterior emargination not extending to the midline. Additionally, adults of A. coregoni has a body length of 12 mm. In this context, it is observed that the fully developed female specimen of A. coregoni displays a greater magnitude in terms of physical dimensions and spermathecae are filled with sperms when compared with both A. japonicus and A. foliaceus as per Shimura (1981) (Fig 4-IIA, B). Conversely, the posterior lobes of the cephalothoracic carapace in A. japonicus extend beyond the commencement of the abdomen. Furthermore, it has been observed that the abdominal lobes in A. japonicus have a more pointed shape compared to those in A. foliaceus, as reported by Noaman et al., (2010) and Wafer et al., (2015). These were the best visual cues used for the distinction among A. coregoni, A. foliaceus and A. japonicus in the current study (Fig 4-I and II). The female isolates of A. japonicus, A. foliaceus and A. coregoni reported in the present study have a body length and width of 6-8 and 3-5 mm, 4-8 and 3-5 mm and 7-12 and 4-6 mm respectively.
 

       
In the adult gravid female specimens of A. japonicus, several morphological variations were observed in the body length, width, carapace appearance, shape and appearance of the abdominal lobes (Table 1 and Fig 4-I). Moreover, a shovel shaped peg on the posterior distal end of basis located on the coxa of the 4^th swimming legs is characteristics feature of male specimen in A. japonicus (Sahoo et al., 2012), has also been reported in the present study (Fig 5a-c). All the male isolates of A. japonicus in the present study were in the size of 4-5 (body length) and 3-4 mm (width) in size.
 

 

       
Based on the morphological variations and their sampling sites, three female isolates of A. japonicus were designated as COF_AHE_P (01) (Udaipur), COF_AHE_P (03) (Lembucherra), and COF_AHE_P (06) (Agartala); and one female isolate each of A. coregoni (COF_AHE_P (07)) (Agartala) and A. foliaceus (COF_AHE_P (08)) (Agartala) (Table 2). However, there was no morphological variations in collected male specimens of A. japonicus unlike the female counterparts. But, based on their sampling sites the male specimens have been designated as A. japonicus COF_AHE_P (02) (Udaipur), COF_AHE_P (04) (Lembucherra), COF_AHE_P (05) (Agartala) and COF_AHE_P (09) (Agartala) (Table 2).
 

 
Molecular and phylogenetic analyses
 
The Argulus samples obtained in this investigation was subjected to molecular characterization using the 18S rRNA genetic marker. The sequences of PCR product (650 bp and 1800 bp) of Argulus samples acquired in this study were subsequently analyzed using NCBI Gene Bank BLAST analysis demonstrated a high level of similarity, with six A. japonicus isolates, namely COF_AHE_P(02) (OR418123.1), COF_AHE_P(03) (OR418124.1), COF_AHE_P(04)  (OR418125.1),  COF_AHE_P(05) (OR418126.1), COF_AHE_ P(06) (OR418127.1)  and COF_AHE_P(09) (OR418130.1), exhibited sequence similarities of 99.33%, 99.32%. 99.49%, 99.83%, 97.77% and 99.49% respectively, when compared to A. japonicus isolate A850 (OR687243.1) from Uttarakhand India. A. coregoni isolate COF_AHE_P(07) (OR418128.1) and A. foliaceus COF_AHE_P(08) (OR418129.1) exhibited a 100% and 99.66% sequence identity, respectively, when compared to the A. coregoni (JQ740820.1) and A. foliaceus (JQ740819.1) isolates from Iran. The level of similarity between A. japonicus isolates COF_AHE_P(01) (OR237569.1) and A. japonicus isolate MWS2 (KF747859) from China was found to be 96.70%. Using multiple sequence alignment, the nucleotide sequences were then aligned with the inclusion of 18S rRNA sequences from fifteenth and five different Argulus species and one additional outgroup species. The phylogenetic tree illustrating the evolutionary relationship of the Argulus spp. was depicted in Fig 6 A and 6 B.
 

       
The evolutionary pair-wise distances among the collected specimens of A. japonicus (OR418123, OR418124, OR418125, OR418126, OR418127, OR418130, OR237569) in comparison with specimens from other regions of India (Uttarakhand) (OR687243) and China (KF747859), exhibited a range of 0.01% to 0.78% (Table 3). Similarly, for A. coregoni it ranged from 0.0% (A. coregoni COF_AHE_P (07) OR418128) to 0.01% (A. coregoni Iran JQ740820) (Table 4). In case of A. foliaceus the evolutionary pair-wise distances varied from 0.0% (A. foliaceus COF_AHE_P (08) OR418129) to 0.03 % (Argulus foliaceus isolate MWS7 18S Iran (KF747861) (Table 4) and between Argulus spp. and the outgroup (Lernaea cyprinacea Japan KP235363) exhibited a range of 0.01% to 0.91% (Table 3) and 0.01 % to 0.21% (Table 4). The present findings indicate that the morphological and phylogenetic characteristics of the Argulus specimens under investigation corresponded to those of A. japonicus, A. foliaceus and A. coregoni. Based on genetic diversity studies, A. japonicus specimens showed variations in genetic makeup within individuals and across species, suggesting genetic drift due to stable or unstable environmental factors in each region. These parasites may have come from various nations or locations with different sampling circumstances. Another possible explanation for the variations could be mutations in the mitochondrial DNA of the isolates, as suggested by Wadeh et al., (2010). Genomic and morphological variation among A. japonicus (Fig 4-I) species may be influenced by environmental variables during climatic adversity. However, more research is needed to grasp these issues.
 

 

The comprehensive examination of Argulus spp. in this study yielded valuable insights into their morphological features, genetic composition, diverse fish hosts and pathological changes. The morphological characterization revealed distinct traits among the species for precise identification. Molecular analysis affirmed these identifications, showcasing high sequence similarities among isolates and correlations with known sequences from different geographical locations, establishing the presence of A. japonicus, A. foliaceus and A. coregoni. The prevalence study delineated host preferences, with A. japonicus exhibiting a broader host range compared to A. foliaceus and A. coregoni, highlighting the varied impact on different fish species. However, future study is necessary to get a more comprehensive knowledge of the morphological and molecular variations of Argulus within and between species, as well as across various geographical regions.

The research was conducted with facilities developed under the project “National Surveillance Programme for Aquatic Animal Diseases - Phase II,” Department of Fisheries, Ministry of Fisheries, Animal Husbandry, and Dairying, Government of India. The first author expresses gratitude to the Ministry of Tribal Affairs, Government of India, for the “National Fellowship and Scholarship for Higher Education of ST Students” scholarship. The authors appreciate Dr. Anupam Mishra, Vice-chancellor of CAU, Imphal, for kindly providing all research facilities.
 
Ethical concern
 
The Institutional Animal Ethics Committee (IAEC) of College of Fisheries, CAU (I), Tripura, dated March 1, 2022, conducted a thorough evaluation and granted approval for this work vide approval number CAU-CF/48/IAEC/2018/02 where live fishes were treated in a manner consistent with ethical standards.

All authors declare that they have no conflicts of interest.