Mussel Immune Responses As Indicators Of Fecal Pollution

Marine coastal areas worldwide are impacted by effluent discharges, especially close to big cities, receiving a wide range of pollutants and high microbial diversity. In order to assess bathing conditions and public health safety, Fecal Indicator Bacteria (FIB) can be monitored in water bodies to indicate fecal contamination and the occurrence of other pathogenic bacteria.

FIB inhabit the gastrointestinal tracts of warm-blooded animals, including humans. They are released in large numbers in feces, survive for long periods in the environment, and are relatively quick and easy to detect, making them ideal indicators of fecal pollution. Total and thermotolerant coliforms such as Escherichia coli and enterococci are the main monitoring targets.

Water quality is crucial for aquaculture activities, particularly in terms of bivalve mollusks. These animals are filter-feeding; thus, they can accumulate pathogenic bacteria from the water column. Consumption of shellfish harvested from wastewater polluted areas constitutes a potential public health risk, with direct impacts on the economy. Accordingly, appropriate legislation for safeguarding consumers can minimize the probability of microbial contamination and economic losses via shellfish. For example, the European Union and the United States have strict legislation for sanitary production of bivalves for human consumption and they both use levels of fecal indicator organisms as a proxy for risk assessment of pathogen exposure.

Bivalves have an efficient immune system, which is activated in bacteria-enriched environments. Hemocytes are their defense cells and circulate in the hemolymph, their circulatory fluid. These cells are rapidly activated during defense reactions and inflammatory processes. They produce toxic metabolites such as reactive oxygen species (ROS), release lysosomal enzymes and exhibit phagocytic activity. Bacterial death is mainly induced to phagocytosis by hemocytes, a process that involves recognition, binding, and internalization of foreign bodies. Consequently, hemocytes might be useful for assessing the health of a bivalve that, in turn, can be linked to environmental quality, including contamination by sewage and bacterial load.

The brown mussel Perna perna is considered a key aquaculture species globally but is particularly important in Venezuela, South Africa, and Brazil. To evaluate bivalve immune-related parameters as potential indicators of fecal pollution, the authors assessed hemocyte parameters of the brown mussel collected from four urban beaches from Guanabara Bay, Rio de Janeiro, Brazil, with different degrees of fecal contamination. FIB in mussel hemolymph and seawater was determined by E. coli, enterococci, total and thermotolerant coliform counts, whereas the immune response of mussels was assessed by hemocyte parameters — such as relative cell size and internal complexity, cell density, phagocytic activity and production of ROS.

The results show that beaches with increased water circulation exhibit less bacterial loading in seawater. Consequently, mussels from these beaches presented lower numbers of FIB in their hemolymph compared to those from beaches more sheltered to wave action. In addition, E. coli and enterococci were not found in the hemolymph of mussels, but they were detected in seawater from three beaches. This suggests that despite filtering E. coli and enterococci from seawater, P. perna mussels can eliminate these pathogenic bacteria from their hemolymph via their hemocyte-mediated immune defense system.

Mussels from cleaner beaches presented greater hemocytes internal complexity than those from contaminated beaches. Brown mussels present two hemocyte sub-types, known as hyalinocytes and granulocytes. Thus, these internal complexity differences in circulating hemocytes suggest altered proportions and involvement of hemocyte sub-types as a response to fecal pollution exposure. Moreover, seawater fecal pollution significantly increased the density of circulating hemocytes in P. perna and their phagocytic activity. Mussels from the FIB-richest beach showed hemocyte density and phagocytic activity six and eight times higher, respectively, than mussels from the FIB-poorest beach. These responses indicate a mussel immune strategy to enhance rates of bacterial clearance and defense against bacterial infection.

ROS production by hemocytes was higher in mussels harvested from the beach with the second-highest abundance of FIB in mussel hemolymph. ROS production is closely related to defense against bacteria in hemolymph. Therefore, we had expected greater ROS production by mussel hemocytes from the most polluted beach. However, differences in ROS production have been demonstrated elsewhere depending on the bacterial strain to which shellfish were exposed. Thus, mussels from distinct beaches are subjected to particular bacterial loads, which may explain the different hemocytes ROS production found in this study.

In conclusion, significant brown mussel (P. perna) immune-related parameters response suggests its stimulation under high fecal bacteria loads. Thus, immune parameters — such as density of circulating hemocytes, phagocytic activity, and ROS production — seem to be effective indicators of fecal contamination since they reflect the abundance of FIB detected in mussel hemolymph. In addition, the way that P. perna deals with bacteria in the hemolymph and mussel filtration can be a potential strategy for bioremediation in marine ecosystems.

These findings are described in the article entitled Evaluation of the immune responses of the brown mussel Perna perna as indicators of fecal pollution, recently published in the journal Fish and Shellfish Immunology. This work was conducted by Fernanda S. dos Santos, Raquel A. F. Neves, Wanderson F. de Carvalho, Natascha Krepsky and Mirian A. C. Crapez from the Fluminense Federal University and the Federal University of the State of Rio de Janeiro.