In April 2022, ten Oncorhynchus mykiss from one of the fish farms were referred to the Chaharmahal and Bakhtiari province aquatic clinics. Infected fish showed heavy infestation with ectoparasites and symptoms like an initial loss of appetite, redness of skin, inflammation, eroded scales, and bloody wounds on the body and buccal cavity. Immediately after the collection of copepods, fish species were evaluated for the presence of copepods. Detached copepods from the various parts of the examined fish, including operculum, gills, and skin, were identified as Lernaea spp. (Fig. 1). Lernaea spp. were counted and meticulously extracted from fish tissues using needles and blades before being stored in 96% ethanol. In the O. mykiss infection experiment, 25 Lernaea specimens were collected from ten fish specimens and morphologically recognized as L. cyprinacea. The presence of Lernaea spp. was determined using a light microscope (4X magnification) (Zhu et al. 2020) (Fig. 2). Histopathology found several lesions in the epidermis, dermis, and muscles. Around the parasite attachment sites, the epidermis was ulcerated with minor acanthosis and spongiosis. A persistent inflammatory response by lymphocytes and macrophages surrounded parasites in the dermis. In certain instances, the implanted parasites stimulated the creation of fibrous tissue. Striated muscle fibers were degenerated and necrotic. Edema, congestion, and rare bleeding were among the other abnormalities (Fig. 3,4).
The molecular method was used to confirm the species and morphological results. DNA was extracted from ten samples using a genomic DNA purification kit (SinaClon Bioscience, Karaj, 3164819711, Iran). Primers 28SF (5' – ACA ACT GTG ATG CCC TTA G – 3') and 28SR (5' – TGG TCC GTG TTT CAA GAC G – 3') targeting partial sequence of 28S rRNA gene were used.11 PCR reactions included a negative control consisting of the reaction mix and 2 µl of Dnase/Rnase-free water instead of DNA and a positive control. All PCRs were performed as a 25 µl reaction containing 12.5 µl Taq DNA polymerase master mix Red (Amplicon, Odense, Denmark), one µM of each primer, and 50 ng DNA templates. The cycles included an initial step at 94°C for 4 min, followed by 30 cycles of denaturing at 94°C for 30 s, annealing at 54°C for 30 s and extension at 72°C for 1 min. PCR elongation was continued at 72°C for 5 min. Amplification products were resolved in 1.5% molecular grade agarose gel (Fischer Biotech, Australia) stained with CYBR Safe stain. Approximately 5 µl of the PCR product from three samples were subsequently purified using the PCR purification kit (Vivantis, Revongen Corporation Center, 47600 Subang Jaya, Selangor Darul Ehsan, Malaysia) and sequenced by specific primers and the Big Dye Terminator V.3.1 Cycle Sequencing kit in an ABI 3130 Genetic Analyzer (Applied Biosystems, 850 lincoln Centre Drive Foster City, CA 94404, USA). The average size fragment of the gene from the amplified Lernaea was 744 bp. The consensus gene sequences were blasted in the NCBI Genbank database, and they showed L. cyprinacea 28S rRNA gene percentage identity ranging from 99.6 to 100.