Novel approach to study gastropod-mediated innate immune reactions against metastrongyloid parasites

Abstract

The anthropozoonotic metastrongyloid nematodes Angiostrongylus cantonensis and Angiostrongylus costaricensis, as well as Angiostrongylus vasorum, Crenosoma vulpis, Aelurostrongylus abstrusus and Troglostrongylus brevior are currently considered as emerging gastropod-borne parasites and have gained growing scientific attention in the last years. However, the knowledge on invertebrate immune responses and on how metastrongyloid larvae are attacked by gastropod immune cells is still limited. This work aims to describe an in vitro system to investigate haemocyte-derived innate immune responses of terrestrial gastropods induced by vital axenic metastrongyloid larvae. We also provide protocols on slug/snail management and breeding under standardized climate conditions (circadian cycle, temperature and humidity) for the generation of parasite-free F0 stages which are essential for immune-related investigations. Adult slug species (Arion lusitanicus, Limax maximus) and giant snails (Achatina fulica) were maintained in fully automated climate chambers until mating and production of fertilized eggs. Newly hatched F0 juvenile specimens were kept under parasite-free conditions before experimental use. An improved protocol for gastropod haemolymph collection and haemocyte isolation was established. Giemsa-stained haemolymph preparations showed adequate haemocyte isolation in all three gastropod species. Additionally, a protocol for the production of axenic first and third stage larvae (L1, L3) was established. Haemocyte functionality was tested in haemocyte-nematode-co-cultures. Scanning electron microscopy (SEM) and light microscopy analyses revealed that gastropod-derived haemocytes formed clusters as well as DNA-rich extracellular aggregates catching larvae and decreasing their motility. These data confirm the usefulness of the presented methods to study haemocyte-mediated gastropod immune responses to better understand the complex biology of gastropod-borne diseases.

The invasive giant African snail Lissachatina fulica as natural intermediate host of Aelurostrongylus abstrusus, Angiostrongylus vasorum, Troglostrongylus brevior, and Crenosoma vulpis in Colombia

“…It is possible to speculate, therefore, that low larval burdens in gastropods may relate to their innate immune system, with effective formation of so-called invertebrate extracellular phagocyte traps (InEPTs) by gastropod haemocytes in response to metastrongyloid lungworm larvae, which has recently been shown [71]. This question requires, however, further in-depth investigation of how gastropods defend themselves against invading metastrongyloid parasites [72]. The maximal metastrongyloid larval burden found in this survey was 314 Ael .…”

Physiology and immunity of the invasive giant African snail, Achatina (Lissachatina) fulica, intermediate host of Angiostrongylus cantonensis

“…It is of note that immunological studies of A. fulica have employed mostly cell biology and biochemical-type analyses that did not yield a detailed characterization of the factors involved as is now common in the "omics"-era. In light of resurging interest in A. fulica, also for the reasons provided in this review (Penagos-Tabares et al, 2018), such data may be anticipated in the near future.…”

Canine Angiostrongylus vasorum-Induced Early Innate Immune Reactions Based on NETs Formation and Canine Vascular Endothelial Cell Activation In Vitro

“…The supernatant was discarded and larval numbers were determined microscopically (Olympus CX41). Three leopard slugs ( L. maximus ) were orally infected with 2000 vital larvae each and according to [ 8 , 17 ].…”

“…Over the last decades, canine angiostrongylosis has geographically spread into previously non-endemic areas and life-threatening cases have increasingly been reported [ 11 , 12 , 13 , 14 ]. Nowadays, canine angiostrongylosis is considered an emerging disease not only in Europe but also in North and South America [ 9 , 13 , 15 , 16 , 17 ]. A. vasorum -infected dogs show a wide range of clinical signs from mild coughing to neurological disorders, along with the presence of respiratory symptoms, coagulopathies (e.g., vascular thrombosis, diathesis, hemorrhages), gastrointestinal disorders, leukophilia and hypercalcemia representing common findings [ 18 , 19 , 20 ].…”