Toll-like receptor of mud crab, Scylla serrata: molecular characterisation, ontogeny and functional expression analysis following ligand exposure, and bacterial and viral infections

Abstract

Toll-like receptors are sentinels of innate immune system, which recognise pathogen-associated molecular patterns, and subsequently activate production of antimicrobial peptides to contain the infection. In the present study, we cloned and characterised a Toll gene from Scylla serrata (SsToll) encoding 1005 amino acids with typical Toll-like receptor domain topology. Phylogenetic analysis revealed that it belongs to insect-type invertebrate Toll family showing 100 % identity with Scylla paramamosain (SpToll). The expression pattern of mRNA in different tissues indicated that SsToll is constitutively expressed in all the tissues examined, with varying expression levels. The expression was also detected in all the life-stages (egg, zoea stages 1-5, megalopa and crab instar) with the highest level observed in zoea 2. In-vitro studies using crab haemocyte culture demonstrated that SsToll transcripts are distinctly modulated in response to ligands such as peptidoglycan and lipopolysaccharide at all time-points. A significant change in SsToll expression was also noticed in haemocytes exposed to poly I:C (3-9 h). On the contrary, the transcription level of SsToll in response to white spot syndrome virus (WSSV) challenge was noticeably different. The change in expression in vitro was not significant at early time-points until 3 h; the transcripts showed a significant up-regulation commencing from 6 h, but not beyond 12 h. However, in vivo expression was unaffected at early time-points of WSSV challenge (until 12 h) and a gradual up-regulation was detected at 24 h. In-vivo challenge with Vibrio parahaemolyticus resulted in delayed up-regulation of the gene. The results obtained in the present study suggest that SsToll might be involved in the innate immunity of mud crab.

Antimicrobial proteins: From old proteins, new tricks

“…For AMPs synthesised and contained within the granules of haemocytes, mature proteins would be released in a regulated way at times of need, with synthesis of new proteins induced by pattern recognition receptors, such as Toll or IMD. Certainly in recent years a growing number of sequence reports of Toll receptors from various decapod species have been published, showing that these genes are expressed across a wide range of tissues and are up-regulated after immune challenge (Arts et al, 2007;Yang et al, 2007Yang et al, , 2008Mekata et al, 2008;Vidya et al, 2014). A very recent study on crayfish has shown that Toll regulates expression of crustin, ALF and lysozyme in P. clarkii, and penaeidins in shrimp with attacin, metchnikowin, and drosomycin over-expressed in Drosophila S2 cells .…”

Immune responses and immunoprotection in crustaceans with special reference to shrimp

“…Most of these reported PRRs are found to be expressed ubiquitously in all the tissues analysed. Immune challenge studies have indicated differential response of the PRRs to different infectious agents such as white spot syndrome virus (WSSV) and bacteria (Vidya et al 2014;Deepika et al 2014;Dechamma et al 2015;Sreedharan et al 2017Sreedharan et al , 2018Li et al 2019b;Deepika et al, 2020aDeepika et al, , 2020b.…”

“…Immune challenge studies have indicated differential response of the PRRs to different infectious agents such as white spot syndrome virus (WSSV) and bacteria (Vidya et al . 2014; Deepika et al . 2014; Dechamma et al .…”

Responses of some innate immune‐genes involved in the toll‐pathway in black tiger shrimp (Penaeus monodon) to Vibrio harveyi infection and on exposure to ligands in vitro

“…For the experimental setup, the cell count was maintained at 2 × 10 6 cells/well. The hemocytes were then exposed to 10 μg/ml LPS or 10 μg/ml PGN (Sigma, USA) prepared in serum-free medium and incubated at 28 C. The concentration of ligands was selected based on a previous study by Vidya et al (2014). Subsequently, at 2, 6, 12, 24, 48, and 72 hr post-exposure, the hemocytes from two wells were harvested and pooled.…”