Betaine

Four experimental diets were formulated and coded as control (NC), betaine (1%, Solarbio) (BET), high carbohydrate (HC, 20%), and high carbohydrate (20%) + betaine (1%) (HC + BET) diet groups. All diets were isonitrogenous and isolipic with fishmeal and fish oil as the protein and lipid sources, respectively. The diets were formulated to meet the nutritional requirements of mandarin fish, and the carbohydrate content of the HC diet was set based on Zhang et al., [29 (link)]. betaine (with a purity of ≥98.0%) was obtained from Beijing Solarbio Science & Technology Co., Ltd. (IB0150, Solarbio, Beijing, China). The formulation and proximate composition of the experimental diets are shown in Table 1. The ingredients were finely ground and thoroughly mixed before being processed into pellets using a pellet machine. All diets were air-dried and stored at −20 °C until use.
The proximate composition of the experimental diets was determined according to AOAC (2003) methods [30 ]. Briefly, crude protein content was determined using the Kjeldahl nitrogen assay after acid digestion. Crude lipid content was determined using a 4800 Kjeltec Analyzer Unit (FOSS Tecator, Haganas, Sweden). Moisture content was determined by drying the samples to constant weight at 105 °C in an oven. Ash content was determined after calcination in a muffle furnace at 550 °C to constant weight.

A set of specific primers, composed of FIP, BIP, LF, LB, F3, and B3, for LAMP detection of DIV1 was designed to target the gene of the second largest subunit of DNA-directed RNA polymerase II in the DIV1 genomic sequence (GenBank accession No. MF599468), using PrimerExplorerV4 (http://primerexplorer.jp/elamp4.0.0/index.html). These primers compared with the database in NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to analyze sequence similarities. The primers were synthesized by Sangon Biotech (Shanghai, China). Each LAMP mixture contained 1.6 µM each of inner primers FIP and BIP, 0.8 µM each of loop primers LF and LB, 0.2 µM each of outer primers F3 and B3, 1.4 mM of dNTP mix (TaKaRa, Dalian, China), 1.2 M betaine (Solarbio, Beijing, China), 25 µM calcein (Sigma, St. Louis, MO, USA), 500 µM MnCl₂, 6 mM MgCl₂, 8 U Bst 2.0 DNA polymerase (New England Biolabs Inc., Beverly, USA), 1× supplied buffer and the specified amount of template DNA in a final volume of 25 µL. The procedure was 60 cycles for 60 °C, following 5 min at 85 °C on the CFX-96 Quantitative Fluorescence Instrument (BioRad, Hercules, CA, USA) using calcein fluorescent channel. Detection specificity of the LAMP primers were examined using 100 ng of the total DNA extracted from uninfected prawns and shrimp infected with other pathogens, including WSSV, Vp_AHPND, IHHNV, and EHP.