Transcriptional and Translational Analysis of the Expression of CHH, MIH, and GIH in PmLyO-Sf9 Cell Line Towards Developing a Novel In Vitro Model for Shrimp Endocrinology.

Shrimp maturation is governed by the hormones secreted by neurosecretory structures in the eyestalk known as X-organ sinus gland complex (XOSG). The X-organ consists of a cluster of neurosecretory cells responsible for synthesizing crustacean hyperglycemic family hormones, including crustacean hyperglycemic hormone (CHH), molt inhibiting hormone (MIH), and gonad inhibiting hormone (GIH). CHH family neuropeptides have gained attention for three decades due to their endocrinological role in aquaculture. One of the most challenging tools in crustacean endocrinology research was the unavailability of a crustacean cell line. Recently, a novel hybrid cell line, the PmLyO-Sf9, was developed by fusing Penaeus monodon lymphoid organ cells with Sf9 cells. Focusing on this cell line, we undertook a comprehensive analysis of the transcriptional and translational expression profiling of CHH/MIH/GIH neuropeptides in the PmLyO-Sf9. In the transcriptional expression studies, the cDNA-based gene profiling of CHH (235 bp), MIH (243 bp), and GIH (247 bp) was determined. A comparative gene expression of CHH/MIH/GIH in PmLyO-Sf9 cell line and Lymphoid organ in Penaeus monodon revealed consistent expression in both. Immunofluorescence confirmed the translational expression of CHH/MIH/GIH with immune-positive cells exhibiting neuropeptides localized in the cytoplasm of PmLyO-Sf9 cells. This is the first study that proved the presence of CHH family neuropeptides in PmLyO-Sf9 cell line and in the Lymphoid organ of Penaeus monodon, hitherto not reported. Accordingly, the cell line has been identified as a suitable platform for endocrinological expression, with potential applications in lieu of animal model.