Histopathological survey of putative parasites and pathogens in non-native slipper limpets Crepidula fornicata.

Two populations of the invasive slipper limpet Crepidula fornicata were sampled in Swansea Bay and Milford Haven, Wales, UK, to determine the presence of putative pathogens and parasites known to affect co-located commercially important shellfish (e.g. oysters). A multi-resource screen, including molecular and histological diagnoses, was used to assess 1800 individuals over 12 mo for microparasites, notably haplosporidians, microsporidians and paramyxids. Although initial PCR-based methods suggested the presence of these microparasites, there was no evidence of infection when assessed histologically, or when all PCR amplicons (n = 294) were sequenced. Whole tissue histology of 305 individuals revealed turbellarians in the lumen of the alimentary canal, in addition to unusual cells of unknown origin in the epithelial lining. In total, 6% of C. fornicata screened histologically harboured turbellarians, and approximately 33% contained the abnormal cells-so named due to their altered cytoplasm and condensed chromatin. A small number of limpets (~1%) also had pathologies in the digestive gland including tubule necrosis, haemocytic infiltration and sloughed cells in the tubule lumen. Overall, these data suggest that C. fornicata are not susceptible to substantive infections by microparasites outside of their native range, which may contribute in part to their invasion success.

Invasive slipper limpets Crepidula fornicata are hosts for sterilizing digenean parasites.

Invasion and spread of alien species can drive ecosystem changes, such as, the dynamics of infectious diseases. The non-native, marine gastropod Crepidula fornicata has become established across European coastlines over the last century, but there remains little insight into its disease carrying capacity and potential role as a source/sink of parasites. To address this knowledge gap, we surveyed limpets from two sites in South Wales, UK for signatures of disease/pathology using polymerase chain reaction-based methods (haemolymph) and histology (solid tissue). We encountered trematode-like parasites in ~1% individuals (5 out of 462). Three limpets displayed gross damage in the gonad, i.e. castration, and encysted metacercariae were found in the muscle of two other individuals. On the basis of 28S rDNA and internal transcribed spacer 2 genomic targets, we identified the gonad-infecting trematodes as members of the family Microphallidae ­ putative novel species related to the genus Longiductotrema. Earlier reports suggest that C. fornicata is not a host for trematode parasites in either its native or alien range but may act as a sink due to its filter feeding lifestyle. We provide clear evidence that C. fornicata is parasitized by at least one trematode species at two sites in Wales, UK, and likely act as a spillback or accidental host among native littorinids.

Laccase and catecholoxidase activities contribute to innate immunity in slipper limpets, Crepidula fornicata.

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.