Laccase and catecholoxidase activities contribute to innate immunity in slipper limpets, Crepidula fornicata. (September 2020)

• Record Type: Journal Article
• Authors: Quinn, Emma A.; Malkin, Sophie H.; Rowley, Andrew F.; Coates, Christopher J.
• Is Part Of: Developmental and comparative immunology. Volume 110(2020)
• Publication Date: 2020-09
• Subjects: Phenoloxidase -- Innate immunity -- Gastropod -- Melanogenesis -- Invasive species -- Lipofuscin -- Haemocyanin; ABTS 2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) -- CO catecholoxidase -- CTAB Cetrimonium bromide -- DHPPA 3, 5-Dihydroxyphenylpropionoic acid -- l-DOPA L-3, 4-Dihydroxyphenylalanine -- EDTA ethylenediaminetetraacetic acid -- 4-HA 4-hydroxyanisole -- 4-HR 4-hexylresorcinol -- PO phenoloxidase -- PPD para-Phenylenediamine -- PTU phenylthiourea -- Syringaldazine 4-Hydroxy-3, 5-dimethoxybenzaldehyde azine -- TY tyrosinase; Immunology -- Periodicals; Developmental immunology -- Periodicals; 616.079
• DOI: 10.1016/j.dci.2020.103724
• Languages: English
• ISSNs: 0145-305X

Abstract

Abstract: The slipper limpet Crepidula fornicata is an invasive, non-native, marine species found throughout the coastal waters of southern England and Wales, UK. These limpets are considered to blight commercial shellfish banks, notably oysters, yet little is known about their disease-carrying capacity or their immunobiology. To address the latter, we isolated haemolymph (blood) from limpets and tested for the presence of the immune-enzyme phenoloxidase. Invertebrate phenoloxidases produce melanic polymers from simple phenolic substrates, which are deployed in the presence of pathogens because of their potent microbicidal and microbiostatic properties. We used a series of established substrates (e.g., tyrosine, hydroquinone) and inhibitors (e.g., 4-hexylresorcinol, benzoic acid) to target three distinct enzymes: laccase ( para -diphenoloxidase), catecholoxidase ( ortho -diphenoloxidase) and tyrosinase (monophenoloxidase). We confirmed laccase and catecholoxidase activities and characterised their kinetic properties across temperature and pH gradients (5–70 °C and 5–10, respectively). Crucially, we demonstrated that products derived from such laccase and catecholoxidase activities reduced significantly the numbers of colony-forming units of both Gram-positive and Gram-negative bacteria in vitro. We further screened limpet tissues for signs of melanin using wax histology, and found cells replete with eumelanin-like pigments and lipofuscin in the digestive gland, connective tissues, barrier epithelia and gills. Our data represent the first account of enzyme-based antibacterial defences, notably laccase, in C. fornicata . Highlights: Characterised laccase and catecholoxidase activities in the haemolymph. Melanin and lipofuscin-like pigments are found in diverse tissue types. Laccase-derived reaction products are potent antiseptics. No mono-phenolase or meta -diphenolase activities detectable.