Fecundity, in vitro early larval development and karyotype of the zoonotic nematode Anisakis pegreffii

Samantha Moratal; Magda Zrzavá; Jerko Hrabar; María Auxiliadora Dea-Ayuela; Jordi López-Ramon; Ivona Mladineo

doi:10.1016/j.vetpar.2023.110050

Fecundity, in vitro early larval development and karyotype of the zoonotic nematode Anisakis pegreffii

Vet Parasitol. 2023 Oct 12:323:110050. doi: 10.1016/j.vetpar.2023.110050. Online ahead of print.

Authors

Samantha Moratal¹, Magda Zrzavá², Jerko Hrabar³, María Auxiliadora Dea-Ayuela⁴, Jordi López-Ramon⁵, Ivona Mladineo⁶

Affiliations

¹ Laboratory of Functional Helminthology, Institute of Parasitology, Biology Centre Czech Academy of Sciences, Branišovská 1160/31, 37005 České Budějovice, Czechia; Servicio de Análisis, Investigación y Gestión de Animales Silvestres (SAIGAS), Facultad de Veterinaria, Universidad Cardenal Herrera-CEU, CEU Universities, C/ Tirant lo Blanc, Alfara del Patriarca, 46115 Valencia, Spain. Electronic address: samantha.moratal@gmail.com.
² Faculty of Science, University of South Bohemia, Branišovská 1760/31a, 37005, České Budějovice, Czechia; Institute of Entomology, Biology Centre Czech Academy of Sciences, Branišovská 1160/31, 37005, České Budějovice, Czechia.
³ Laboratory of Aquaculture, Institute of Oceanography and Fisheries, 21000 Split, Croatia.
⁴ Departamento de Farmacia, Facultad de Ciencias de la Salud, Universidad Cardenal Herrera-CEU, CEU Universitites, C/ Santiago Ramón y Cajal, Alfara del Patriarca, 46115 Valencia, Spain.
⁵ Servicio de Análisis, Investigación y Gestión de Animales Silvestres (SAIGAS), Facultad de Veterinaria, Universidad Cardenal Herrera-CEU, CEU Universities, C/ Tirant lo Blanc, Alfara del Patriarca, 46115 Valencia, Spain; Wildlife Ecology & Health Group (WE&H), Facultat de Veterinària, Universitat Autònoma de Barcelona (UAB), Travessera dels Turons, Bellaterra, 08193 Barcelona, Spain.
⁶ Laboratory of Functional Helminthology, Institute of Parasitology, Biology Centre Czech Academy of Sciences, Branišovská 1160/31, 37005 České Budějovice, Czechia.

PMID: 37837730
DOI: 10.1016/j.vetpar.2023.110050

Abstract

The in vitro life cycle of zoonotic helminths is an essential tool for -omic translational studies focused on disease control and treatment. Anisakiosis is an emerging zoonosis contracted by the ingestion of raw or undercooked fish infected with the third stage larvae (L3) of two sibling species Anisakis simplex sensu stricto (s.s.) and Anisakis pegreffii, the latter being the predominant species in the Mediterranean basin. Recently, in vitro culture of A. pegreffii has been developed to enable fast and large-scale production of fertile adults. However, the conditions for larval development from hatching to infective L3 were not fulfilled to complete the cycle. Herein, we used a Drosophila medium supplemented with chicken serum and adjusted different osmolarities to maintain the culture of L3 hatched from eggs for up to 17 weeks. The highest survival rate was observed in the medium with the highest osmolarities, which also allowed the highest larval exsheathment rate. Key morphological features of embryogenesis and postembryogenesis studied by transmission electron microscopy revealed that the excretory gland cell is differentiated already up to 48 h post-hatching. Extracellular vesicles and cell-free mitochondria are discharged between the two cuticle sheets of the second stage larvae (L2). Contemporarly cultivated, two populations of adult A. simplex s.s. and A. pegreffii reached an average production of 29,914.05 (± 27,629.36) and 24,370.96 (± 12,564.86) eggs/day/female, respectively. The chromosome spreads of A. pegreffii obtained from mature gonads suggests a diploid karyotype formula of 2n = 18. The development of a reliable protocol for the in vitro culture of a polyxenous nematode such as Anisakis spp. will serve to screen for much needed novel drug targets, but also to study the intricated and unknown ecological and physiological traits of these trophically transmitted marine nematodes.

Keywords: Anisakis pegreffii; Anisakis simplex; Fecundity; Karyotype; Ultrastructure; in vitro.