Cellular organization and phylogeny of Cryptonaiada gen. n. (Cryptonaiadidae fam. n.) shed light on the evolution of intracellular vertebrate parasitism in Apicomplexa

ABSTRACT Apicomplexan parasites have coevolved with animal hosts for hundreds of millions of years, yet only a handful of their lineages have made the transition to infecting vertebrates. Among the least understood vertebrate-infecting apicomplexans is Nematopsis temporariae , recently rediscovered after nearly a century of neglect. Here, we isolated this parasite from tadpole tissues and examined its host relationship, ultrastructure, and phylogenetic placement. We show that N. temporariae ’s monozoic oocysts are persistently associated with hepatic Kupffer cells in tadpoles of several frog species and undergo maturation within them, while being absent from adult hosts. Sporozoites possess a folded multimembrane pellicle, numerous paraglycogen granules, and an apical complex with polar rings and rhoptries. Exceptionally abundant micronemes are distributed throughout the cell rather than restricted to the apical region. Although several traits superficially resemble Nematopsis eugregarines from marine bivalves, single- and multi-gene phylogenies demonstrate that N. temporariae belongs to a distinct, distantly related lineage of freshwater actinocephaloid eugregarines. This evidence warrants the establishment of the new genus Cryptonaiada gen. n. in the new family Cryptonaiadidae fam. n.—the first known gregarine apicomplexan to have evolved vertebrate parasitism. We propose that Cryptonaiada develops within macrophages upon engulfment, evading digestion, and actively modulating them through sporozoite-mediated secretion. The persistent intracellular interaction between Cryptonaiada and tadpole macrophages may represent an early stage in parasite coevolution with vertebrate adaptive immunity, coinciding with a window of host immunological immaturity. Our findings highlight the value of studying neglected symbionts of amphibians, a group facing a significant global population decline. IMPORTANCE How parasites evolve to infect vertebrate hosts with adaptive immunity remains a major question in parasitology. Apicomplexans provide a unique perspective on this transition, as vertebrate parasitism has arisen in only a handful of ancient lineages. Here, we characterize Cryptonaiada temporaria , an early-branching freshwater apicomplexan infecting tadpoles. The evidence indicates that Cryptonaiada represents a new gregarine genus and family that have evolved to infect tadpoles intracellularly, via oocysts engulfed by and developing within liver macrophages. The ultrastructure of the oocyst containing a single sporozoite reveals a semi-open state combining features consistent with environmental defense and secretion into the host cell. Cryptonaiada is absent from metamorphosed adults, suggesting that it exploits a developmental window of reduced host immune competence. These findings reveal an unusual form of intracellular vertebrate parasitism in gregarines, providing new insight into the evolutionary transition of apicomplexans from invertebrate to vertebrate hosts.