Novel approach to study gastropod-mediated innate immune reactions against metastrongyloid parasites
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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.
KeywordsInvertebrate immunology Metastrongyloidea Haemocytes Arion lusitanicus Limax maximus Achatina fulica
CH, AT and FPT conceived and designed the protocols. FPT and MKL performed haemocyte-related experiments. UG and AS performed the scanning electron microscopy (SEM) analysis. HM contributed with the constant supply of A. vasorum larvae. FPT, MKL and CH drafted the work. CH, AT and HM revised the manuscript. All authors read and approved the final manuscript.
This research was partially financed by the Bayer Animal Health Care, Leverkusen, Germany. MKL was supported by a grant of the Ewald and Hilde Berge Foundation.
Compliance with ethical standards
The study published does not involve living vertebrates or invertebrates of the classes Cephalopoda or Decapoda. Nevertheless, gastropods involved were treated humanely avoiding unnecessary pain, distress, suffering or lasting harm. All applicable international, national (German Animal Welfare of 25.05.1998—BGBL I S.1105—section 5 paragraph 8a) and/or institutional guidelines for the care and use of animals/invertebrates were followed. The isolation of A. vasorum L1 from faeces of experimentally infected red foxes (Vulpes vulpes) were kindly provided by the Department of Veterinary Disease Biology, University of Copenhagen, Denmark, Danish experimental animal license no. 2010/561-1914.
According to the German Animal Welfare Act (Tierschutzgesetz of 25.05.1998—BGBL I S.1105—section 5 paragraph 8a), ethics approval research with invertebrates is only required for experiments where animals of the classes Cephalopoda and Decapoda are used.
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could bear a potential conflict of interest.
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