AbstractAscaridoid nematodes comprise a wide range of heteroxenous parasites infecting top fish predators and marine mammals as definitive hosts, with crustaceans, squids, and fishes acting as intermediate/paratenic hosts. Limited data exist on the species and role of several intermediate and paratenic hosts in the life cycle of these parasites. In the aim of adding knowledge on the role of squid species in their life cycle, we have here investigated the larval ascaridoid nematodes collected from the deep-sea umbrella squid Histioteuthis bonnelli and the reverse jewel squid Histioteuthis reversa captured in the Central Mediterranean Sea (Tyrrhenian Sea). Morphological study and sequence analysis of the internal transcribed spacer (ITS) regions of the ribosomal DNA (rDNA) and the mitochondrial cytochrome c oxidase subunit 2 (mtDNA cox2) gene locus revealed the occurrence of Anisakis physeteris and of an unidentified species of the genus Lappetascaris. Sequence analysis revealed that specimens of Lappetascaris from both squid species matched at 100% sequences previously deposited in GenBank from larval ascaridoids collected in octopuses of the genus Eledone of the Mediterranean Sea. The Bayesian inference tree topology obtained from the analysis of the fragments amplified showed that Lappetascaris specimens were included in a major clade comprising Hysterothylacium species collected in fishes of the families Xiphiidae and Istiophoridae. As regards the site of infection in the squid host species, A. physeteris larvae predominated (60.7%) in the gonads, while those of Lappetascaris (76.3%) were found infecting the mantle musculature. The overall high values of parasitic load suggest both squid species as transmitting hosts of third stage larvae of Lappetascaris to top predator fishes, as well as the umbrella squid as an intermediate/paratenic host in the life cycle of A. physeteris in the Mediterranean Sea.