High-pressure freezing and freeze-substitution fixation reveal the ultrastructure of immature and mature spermatozoa of the plant-parasitic nematode Trichodorus similis (Nematoda; Triplonchida; Trichodoridae)

•High-pressure freezing and freeze-substitution instead of chemical fixation enables the first ultrastructural study of spermatogenesis in Triplonchida (Nematoda).•Our results confirm that cryo-fixation is a promising method to freeze biological samples of 0.2–6mm, such as nematodes.•The absence of a nuclear envelope in the mature and immature spermatozoon of T. similis is a major difference between the subclass Enoplia, i.e., between the order Triplonchida and the order Enoplida.•Asynchronous formation of MO and FB and late formation of MO in the class Enoplea, including Triplonchida and T. similis, suggest a different type of aberrant organelles formation in Enoplea and a distinct difference between the two nematode classes, Enoplea and Chromadorea.