Live B. dussumieri (n=60) were collected from a brackishwater area located around Pancham Aquaculture Farms, (19°31’32.92’’N and 72°47’57.83’’E), Saphale, Palghar district, Maharashtra, India. The fish (mean length= 11.8 ± 3.17 cm; range= 7.3-17.2 cm) were transported live to the Aquatic Animal Health Laboratory, ICAR-Central Institute of Fisheries Education (CIFE), Mumbai, Maharashtra, India for parasitic examination.
Parasitic examination and identification of microsporidian
Fish were killed by pithing without any tissue damage, after immobilizing them on ice for adequate time. This method was as per the accepted guidelines (https://fisheries.org/docs/policy_useoffishes.pdf.). Initially, gross observations were carried out under a stereomicroscope to find out the presence of any ectoparasites, external lesions, discoloration, haemorrhage or cysts. Subsequently, all the external and internal organs of the fish were examined for the presence of parasites. Microsporidian cysts found in the liver were carefully removed, placed on a clean glass slide in physiological saline, ruptured with fine needles, mounted with a clean cover glass, and observed under a phase-contrast microscope. The spores were treated with 1-2% KOH to observe the polar tube extrusion. Smears of the infected tissues were air-dried, fixed in methanol, and stained with Giemsa stain. Photomicrographs of fresh and stained materials were taken using a research microscope (Nikon eclipse 80i, Japan) with image capture software (NIS elements BR, Nikon, Japan).
The infected tissues were fixed in neutral buffered formalin (NBF) for 24-72 h and washed thoroughly to remove the fixative. The tissues were dehydrated in an ascending series of alcohol followed by acetone and cleared in xylene. Paraffin infiltration and embedding of processed tissues were carried out using a histoembedder (LEICA EG 1140C, Germany). Tissue sections of 3-5 µm thickness were made using a rotary microtome (LEICA RM2125RT, Germany) and stained with Harris haematoxylin and eosin. The sections were dehydrated through different grades of alcohol and acetone. Xylene was used for clearing and sections were mounted in DPX (Sigma-Aldrich, USA).
Scanning electron microscopy
For scanning electron microscopy, microsporidian cysts were fixed in 2.5% glutaraldehyde in 0.2 M sodium cacodylate buffer. The cysts were fixed in 1% osmium tetroxide after washing in cacodylate buffer. Subsequently, after dehydration through graded acetone series, the cysts were transferred to isoamyl acetate and then critical point dried using a Hitachi HCP-2 Critical Point dryer (Hitachi, Japan). The dried cyst was cut open and then mounted on the SEM stub, using an adhesive carbon tape, so as to expose the inner surface of the cyst. Further, it was sputter-coated with gold using Quorum SC76220 mini sputter coater (Quorum Technologies, UK). The processed cyst with microspores was observed and photomicrographs were taken using a TESCAN VEGA 3 scanning electron microscope (TESCAN, Brno, Czech Republic).
The total genomic DNA was extracted from microsporidian-infected liver tissue of fish using DNAzol (Invitrogen®) kit following the manufacturer’s instructions. The small subunit ribosomal DNA (SSU rDNA) of microsporidian spores was amplified using reported primers MicroSSUF: 5’-GGTTGATTCTGCCTGACGT-3’ and MicroSSUR: 5’-GACGGGCGGTGTGTACAAAG-3’ (Baker et al. 1994; Pomport-Castillon et al. 1997). The PCR reaction was carried out in a 25 µL reaction volume with 100 ng of template DNA, 10 mM of dNTP, 10 pmol of each primer, 5 U of Taq DNA polymerase and 1× Taq buffer with 1.5 mM MgCl2, (Invitrogen®, USA). The thermal conditions for PCR were as follows: 95°C for 5 min, 30 cycles of 94°C for 1 min, 62°C for 1.5 min, 72°C for 2 min; followed by 72°C for 10 min final extension. PCR products were visualized on 2% agarose-TAE gel containing 0.5 μg mL¯¹ ethidium bromide. The desired PCR amplicon was extracted from the gel using a gel extraction kit and cloned into PTZ57R/T vector using T4 DNA ligase (Thermo Scientific®, India). The recombinant plasmid was transferred into Escherichia coli (DH5α) and the positive clones were selected by blue-white colony selection. The plasmid was isolated using GeneJET® plasmid extraction kit (Thermo Scientific®, India) and the insert was reconfirmed by PCR amplification with microsporidian-specific primers. The confirmed plasmid DNA was further sequenced in both directions using the same microsporidian-specific primers by a commercial company (Xcelris Labs, Ahmedabad, India). The quality of each sequence was verified by the Phred score (q value) of each nucleotide using Finchtv software. The sequences were subjected to BLAST (Basic Local Alignment Search Tool) analysis with NCBI ‘nr’ database and the sequences with more than 85% similarity were downloaded to estimate the genetic divergence values. Kimura 2 parameter model implemented in MEGAX (Kumar et al. 2018) was used to estimate the genetic distance values. JModeltest was used to assess the best evolutionary model (Posada 2008). Based on the Akaike Information Criterion (AIC), the Transitional model with rate variation among sites (TIM3+G) was selected as the best model to reconstruct the phylogenetic tree using Maximum likelihood and Bayesian Inference. Maximum likelihood and Parsimony methods were used to reconstruct the phylogenetic trees using PAUP software (Swofford 2003). Bayesian inference was also implied to deduce the phylogenetic tree using MrBayes (Ronquist and Huelsenbeck 2003).