Identification of a phenoloxidase- and melanin-dependent defence mechanism in Achatina fulica infected with Angiostrongylus vasorum

Aytube Lucas Coaglio, Mônica Alves Neves Diniz Ferreira, Walter dos Santos Lima, Cíntia Aparecida de Jesus Pereira

Angiostrongylus vasorum has different freshwater aquatic and terrestrial gastropod molluscs as an intermediate host, e.g. Arion spp. The mollusc Achatina fulica is a danger to public health, given the large diversity of nematodes utilizing it as an intermediate host, such as the parasites of the genus Angiostrongylus, of importance in human and veterinary medicine. Achatina fulica has been shown to have an excellent capacity for maintaining outbreaks and natural infections with A. cantonensis in Asia. Within the mollusc, the nematode parasites activate haemocytes and/or haemolymph factors and in some invertebrates, phenoloxidase (PO), that induces the release of toxic elements and eliminates the parasites. Despite the importance of A. fulica in the life-cycle of nematodes, little is known regarding the defence mechanisms involving PO in molluscs infected with nematodes. Here, the presence of PO and nitric oxide (NO) in the haemolymph and haemocytes of A. fulica infected with first-stage (L1) larvae of Angiostrongylus vasorum was evaluated, together with the presence of melanin in the cephalopod mollusc tissue. An increase in PO at one day post infection (dpi), in comparison with the control using the substrates L-tyrosine (F(4,90) = 6.73, P = 0.00006), L-DOPA (F(4,90) = 22.67, P = 0.02) and p-phenylenediamine (PPD) (F(4,90) = 27.58, P = 0.0019), was observed. PO increase coincided with the presence of melanin in the cephalopodal tissue. At 8 dpi, PO activity, compared to L-DOPA (F(4,90) = 22.67, P = 0.00002) and PPD (F(4,90) = 27.58, P = 0.079) decreased, while melanin increased. At 13 dpi, PO decreased with PPD (F(4,90) = 27.58, P = 0.000015) and also the amount of melanin observed in histology. At 30 dpi, PO increased along with the substrates L-DOPA and PPD, while melanin decreased. NO levels increased until 8 dpi, and decreased after 13 dpi. To our knowledge, this is the first study that illustrates PO activity in a helminth-infected A. fulica and provides the first observation of an L-tyrosine dependent PO activity in molluscs infected with A. vasorum. This work suggests that PO pathway may help to control A. vasorum infection in A. fulica.