Plasmodium LCCL domain-containing modular proteins have their origins in the ancestral alveolate.

Plasmodium species encode a unique set of six modular proteins named LCCL lectin domain adhesive-like proteins (LAPs) that operate as a complex and that are essential for malaria parasite transmission from mosquito to vertebrate. LAPs possess complex architectures obtained through unique assemblies of conserved domains associated with lipid, protein and carbohydrate interactions, including the name-defining LCCL domain. Here, we assessed the prevalence of Plasmodium LAP orthologues across eukaryotic life. Our findings show orthologous conservation in all apicomplexans, with lineage-specific repertoires acquired through differential lap gene loss and duplication. Besides Apicomplexa, LAPs are found in their closest relatives: the photosynthetic chromerids, which encode the broadest repertoire including a novel membrane-bound LCCL protein. LAPs are notably absent from other alveolate lineages (dinoflagellates, perkinsids and ciliates), but are encoded by predatory colponemids, a sister group to the alveolates. These results reveal that the LAPs are much older than previously thought and pre-date not only the Apicomplexa but the Alveolata altogether.

Proteomic approaches for protein kinase substrate identification in Apicomplexa.

Apicomplexa is a phylum of protist parasites, notable for causing life-threatening diseases including malaria, toxoplasmosis, cryptosporidiosis, and babesiosis. Apicomplexan pathogenesis is generally a function of lytic replication, dissemination, persistence, host cell modification, and immune subversion. Decades of research have revealed essential roles for apicomplexan protein kinases in establishing infections and promoting pathogenesis. Protein kinases modify their substrates by phosphorylating serine, threonine, tyrosine, or other residues, resulting in rapid functional changes in the target protein. Post-translational modification by phosphorylation can activate or inhibit a substrate, alter its localization, or promote interactions with other proteins or ligands. Deciphering direct kinase substrates is crucial to understand mechanisms of kinase signaling, yet can be challenging due to the transient nature of kinase phosphorylation and potential for downstream indirect phosphorylation events. However, with recent advances in proteomic approaches, our understanding of kinase function in Apicomplexa has improved dramatically. Here, we discuss methods that have been used to identify kinase substrates in apicomplexan parasites, classifying them into three main categories: i) kinase interactome, ii) indirect phosphoproteomics and iii) direct labeling. We briefly discuss each approach, including their advantages and limitations, and highlight representative examples from the Apicomplexa literature. Finally, we conclude each main category by introducing prospective approaches from other fields that would benefit kinase substrate identification in Apicomplexa.

A new and widespread group of fish apicomplexan parasites.

Apicomplexans are obligate intracellular parasites that have evolved from a free-living, phototrophic ancestor. They have been reported from marine environmental samples in high numbers,1 with several clades of apicomplexan-related lineages (ARLs) having been described from environmental sequencing data (16S rRNA gene metabarcoding).2 The most notable of these are the corallicolids (previously ARL-V), which possess chlorophyll-biosynthesis genes in their relic chloroplast (apicoplast) and are geographically widespread and abundant symbionts of anthozoans.3 Corallicolids are related to the Eimeriorina, a suborder of apicomplexan coccidians that include other notable members such as Toxoplasma gondii.4Ophioblennius macclurei, the redlip blenny, along with other tropical reef fishes, is known to be infected by Haemogregarina-like and Haemohormidium-like parasites5 supposedly belonging to the Adeleorina; however, phylogenetics shows that these parasites are instead related to the Eimeriorina.6,7 Hybrid genomic sequencing of apicomplexan-infected O. macclurei blood recovered the entire rRNA operon of this apicomplexan parasite along with the complete mitochondrion and apicoplast genomes. Phylogenetic analyses using this new genomic information consistently place these fish-infecting apicomplexans, hereby informally named ichthyocolids, sister to the corallicolids within Coccidia. The apicoplast genome did not contain chlorophyll biosynthesis genes, providing evidence for another independent loss of this pathway within Apicomplexa. Based on the 16S rRNA gene found in the apicoplast, this group corresponds to the previously described ARL-VI. Screening of fish microbiome studies using the plastid 16S rRNA gene shows these parasites to be geographically and taxonomically widespread in fish species across the globe with implications for commercial fisheries and oceanic food webs.

TKL family kinases in human apicomplexan pathogens.

Apicomplexan parasites are the primary causative agents of many human diseases, including malaria, toxoplasmosis, and cryptosporidiosis. These opportunistic pathogens undergo complex life cycles with multiple developmental stages, wherein many key steps are regulated by phosphorylation mechanisms. The genomes of apicomplexan pathogens contain protein kinases from different groups including tyrosine kinase-like (TKL) family proteins. Although information on the role of TKL kinases in apicomplexans is quite limited, recent studies have revealed the important role of this family of proteins in apicomplexan biology. TKL kinases in these protozoan pathogens show unique organization with many novel domains thus making them attractive candidates for drug development. In this mini review, we summarize the current understanding of the role of TKL kinases in human apicomplexan pathogens' (Toxoplasma gondii, Plasmodium falciparum and Cryptosporidium parvum) biology and pathogenesis.

Description of an intramonocytic haemoparasite, Hepatozoon lainsoni sp. nov. (Apicomplexa: Adeleorina: Hepatozoidae), infecting Ameiva ameiva lizard (Reptilia: Squamata: Teiidae) in northern Brazil.

Haemogregarine (Apicomplexa: Adeleorina) parasites are considered to be the most common and widespread haemoparasites in reptiles. The genus Hepatozoon (Apicomplexa: Adeleorina: Hepatozoidae) can be found parasitizing a broad range of species and, in reptiles, they infect mainly peripheral blood erythrocytes. The present study detected and characterized a haemogregarine isolated from the lizard species, Ameiva ameiva, collected from the municipality of Capanema, Pará state, north Brazil. Blood smears and imprints from lungs, brain, heart, kidney, liver, bone marrow and spleen were observed using light microscopy and the parasite was genetically identified by molecular analysis. Morphological, morphometric and molecular data were obtained. Parasite gamonts were found in 49.5% (55/111) of the blood smears from A. ameiva, and were characterized as oval, averaging 12.0 ± 0.8 × 5.9 ± 0.6 µm2 in size, which displaced the nuclei of parasitized monocytes laterally. Parasite forms resembling immature gamonts were observed in the spleen and bone marrow of the lizards. Furthermore, phylogenetic analyses of 18S rRNA sequences did not reveal gene similarity with other Hepatozoon spp. sequences from reptiles. Thus, morphological and molecular analyses have identified a new species of Hepatozoon parasite, Hepatozoon lainsoni sp. nov., which infects monocytes of the A. ameiva lizard.

Apicomplexa micropore: history, function, and formation.

The micropore, a mysterious structure found in apicomplexan species, was recently shown to be essential for nutrient acquisition in Plasmodium falciparum and Toxoplasma gondii. However, the differences between the micropores of these two parasites questions the nature of a general apicomplexan micropore structure and whether the formation process model from Plasmodium can be applied to other apicomplexans. We analyzed the literature on different apicomplexan micropores and found that T. gondii probably harbors a more representative micropore type than the more widely studied ones in Plasmodium. Using recent knowledge of the Kelch 13 (K13) protein interactome and gene depletion phenotypes in the T. gondii micropore, we propose a model of micropore formation, thus enriching our wider understanding of micropore protein function.

Time to switch gears: how long noncoding RNAs function as epigenetic regulators in Apicomplexan parasites.

Long noncoding RNAs (lncRNA) are emerging as important regulators of gene expression in eukaryotes. In recent years, a large repertoire of lncRNA were discovered in Apicomplexan parasites and were implicated in several mechanisms of gene expression, including marking genes for activation, contributing to the formation of subnuclear compartments and organization, regulating the deposition of epigenetic modifications, influencing chromatin and chromosomal structure and manipulating host gene expression. Here, we aim to update recent knowledge on the role of lncRNAs as regulators in Apicomplexan parasites and highlight the possible molecular mechanisms by which they function. We hope that some of the hypotheses raised here will contribute to further investigation and lead to new mechanistic insight and better understanding of the role of lncRNA in parasite's biology.

Protozoan parasites of birds from the Tremembé formation (Oligocene of the Taubaté Basin), São Paulo, Brazil.

OBJECTIVE: To analyze the presence of protozoan parasites in bird coprolites from the Tremembé Formation (Oligocene of the Taubaté Basin). MATERIALS: Twenty avian coprolites embedded in pyrobituminous shale matrices. METHODS: Samples were rehydrated and subjected to spontaneous sedimentation. RESULTS: Paleoparasitological analyses revealed oocysts compatible with the Eimeriidae family (Apicomplexa) and one single Archamoebae (Amoebozoa) cyst. CONCLUSIONS: The present work increases the amount of information about the spread of infections throughout the Cenozoic Era and reveals that the Brazilian paleoavifauna played an important role in the Apicomplexa and Amoebozoa life cycles. SIGNIFICANCE: This is the first record of protozoans in avian coprolites from the Oligocene of Brazil. These findings can help in the interpretation of phylogenies of coccidian parasites of modern birds, as certain taxonomic characters observed in the Oligocene Protozoa characterize monophyletic groups in current molecular phylogenetic analyses. LIMITATIONS: None of the oocysts were sporulated; therefore, it is not possible to identify the morphotypes to genus or species. SUGGESTIONS FOR FURTHER RESEARCH: Our results create new perspectives related to biogeographic studies of the parasitic groups described and may improve the understanding of the temporal amplitude of parasitic evolutionary relationships between Protozoans and birds.

Histone code: a common language and multiple dialects to meet the different developmental requirements of apicomplexan parasites.

Apicomplexan parasites have complex life cycles, often requiring transmission between two different hosts, facing periods of dormancy within the host or in the environment to maximize chances of transmission. To support survival in these different conditions, tightly regulated and correctly timed gene expression is critical. The modification of histones and nucleosome composition makes a significant contribution to this regulation, and as eukaryotes, the fundamental mechanisms underlying this process in apicomplexans are similar to those in model eukaryotic organisms. However, single-celled intracellular parasites face unique challenges, and regulation of gene expression at the epigenetic level provides tight control for responses that must often be rapid and robust. Here, we discuss the recent advances in understanding the dynamics of histone modifications across Apicomplexan life cycles and the molecular mechanisms that underlie epigenetic regulation of gene expression to promote parasite life cycle progression, dormancy, and transmission.

Pacific marine gregarines (Apicomplexa) shed light on biogeographic speciation patterns and novel diversity among early apicomplexans.

Gregarines are the most biodiverse group of apicomplexan parasites. This group specializes on invertebrate hosts (e.g., ascidians, crustaceans, and polychaetes). Marine gregarines are of particular interest because they are considered to be the earliest evolving apicomplexan lineage, having subsequently speciated (and radiated) through virtually all existing animal groups. Still, mechanisms governing the broad (global) distribution and speciation patterns of apicomplexans are not well understood. The present study examines Pacific lecudinids, one of the most species-rich and diverse groups of marine gregarines. Here, marine polychaetes were collected from intertidal zones. Single trophozoite cells were isolated for light and electron microscopy, as well as molecular phylogenetic analyses using the partial 18S rRNA gene. The cytochrome c oxidase subunit 1 gene was used to confirm morphology-based host identification. This study introduces Undularius glycerae n. gen., n. sp. and Lecudina kitase n. sp. (Hokkaido, Japan), as well as Difficilina fasoliformis n. sp. (California, USA). Occurrences of Lecudina cf. longissima and Lecudina cf. tuzetae (California, USA) are also reported. Phylogenetic analysis revealed a close relationship between L. pellucida, L. tuzetae, and L. kitase n. sp. Additionally, clustering among North Atlantic and Pacific L. tuzetae formed a species complex, likely influenced by biogeography.

Transcript tinkering: RNA modifications in protozoan parasites.

Apicomplexan and trypanosomatid parasites have evolved a wide range of post-transcriptional processes that allow them to replicate, differentiate, and transmit within and among multiple different tissue, host, and vector environments. In this review, we highlight the recent advances that point toward the regulatory potential of RNA modifications in mediating these processes on the coding and noncoding transcriptome throughout the life cycle of protozoan parasites. We discuss the recent technical advancements enabling the study of the 'epitranscriptome' and how parasites evolved RNA modification-mediated mechanisms adapted to their unique lifestyles.

Coral-infecting parasites in cold marine ecosystems.

Coral reefs are a biodiversity hotspot,1,2 and the association between coral and intracellular dinoflagellates is a model for endosymbiosis.3,4 Recently, corals and related anthozoans have also been found to harbor another kind of endosymbiont, apicomplexans called corallicolids.5 Apicomplexans are a diverse lineage of obligate intracellular parasites6 that include human pathogens such as the malaria parasite, Plasmodium.7 Global environmental sequencing shows corallicolids are tightly associated with tropical and subtropical reef environments,5,8,9 where they infect diverse corals across a range of depths in many reef systems, and correlate with host mortality during bleaching events.10 All of this points to corallicolids being ecologically significant to coral reefs, but it is also possible they are even more widely distributed because most environmental sampling is biased against parasites that maintain a tight association with their hosts throughout their life cycle. We tested the global distribution of corallicolids using a more direct approach, by specifically targeting potential anthozoan host animals from cold/temperate marine waters outside the coral reef context. We found that corallicolids are in fact common in such hosts, in some cases at high frequency, and that they infect the same tissue as parasites from topical coral reefs. Parasite phylogeny suggests corallicolids move between hosts and habitats relatively frequently, but that biogeography is more conserved. Overall, these results greatly expand the range of corallicolids beyond coral reefs, suggesting they are globally distributed parasites of marine anthozoans, which also illustrates significant blind spots that result from strategies commonly used to sample microbial biodiversity.

Parallel functional reduction in the mitochondria of apicomplexan parasites.

Extreme functional reduction of mitochondria has taken place in parallel in many distantly related lineages of eukaryotes, leading to a number of recurring metabolic states with variously lost electron transport chain (ETC) complexes, loss of the tricarboxylic acid (TCA) cycle, and/or loss of the mitochondrial genome. The resulting mitochondria-related organelles (MROs) are generally structurally reduced and in the most extreme cases barely recognizable features of the cell with no role in energy metabolism whatsoever (e.g., mitosomes, which generally only make iron-sulfur clusters). Recently, a wide diversity of MROs were discovered to be hiding in plain sight: in gregarine apicomplexans. This diverse group of invertebrate parasites has been known and observed for centuries, but until recent applications of culture-free genomics, their mitochondria were unremarkable. The genomics, however, showed that mitochondrial function has reduced in parallel in multiple gregarine lineages to several different endpoints, including the most reduced mitosomes. Here we review this remarkable case of parallel evolution of MROs, and some of the interesting questions this work raises.

Phylogenomics reveals Adeleorina are an ancient and distinct subgroup of Apicomplexa.

Apicomplexans are a diverse phylum of unicellular eukaryotes that share obligate relationships with terrestrial and aquatic animal hosts. Many well-studied apicomplexans are responsible for several deadly zoonotic and human diseases, most notably malaria caused by Plasmodium. Interest in the evolutionary origin of apicomplexans has also spurred recent work on other more deeply-branching lineages, especially gregarines and sister groups like squirmids and chrompodellids. But a full picture of apicomplexan evolution is still lacking several lineages, and one major, diverse lineage that is notably absent is the adeleorinids. Adeleorina apicomplexans comprises hundreds of described species that infect invertebrate and vertebrate hosts across the globe. Although historically considered coccidians, phylogenetic trees based on limited data have shown conflicting branch positions for this subgroup, leaving this question unresolved. Phylogenomic trees and large-scale analyses comparing cellular functions and metabolism between major subgroups of apicomplexans have not incorporated Adeleorina because only a handful of molecular markers and a couple organellar genomes are available, ultimately excluding this group from contributing to our understanding of apicomplexan evolution and biology. To address this gap, we have generated complete genomes from mitochondria and plastids, as well as multiple deep-coverage single-cell transcriptomes of nuclear genes from two Adeleorina species, Klossia helicina and Legerella nova, and inferred a 206-protein phylogenomic tree of Apicomplexa. We observed distinct structures reported in species descriptions as remnant host structures surrounding adeleorinid oocysts. Klossia helicina and L. nova branched, as expected, with monoxenous adeleorinids within the Adeleorina and their mitochondrial and plastid genomes exhibited similarity to published organellar adeleorinid genomes. We show with a phylogeneomic tree and subsequent phylogenomic analyses that Adeleorina are not closely related to any of the currently sampled apicomplexan subgroups, and instead fall as a sister to a large clade encompassing Coccidia, Protococcidia, Hematozoa, and Nephromycida, collectively. This resolves Adeleorina as a key independently-branching group, separate from coccidians, on the tree of Apicomplexa, which now has all known major lineages sampled.

Coinfection of slime feather duster worms (Annelida, Myxicola) by different gregarine apicomplexans (Selenidium) and astome ciliates reflects spatial niche partitioning and host specificity.

Individual organisms can host multiple species of parasites (or symbionts), and one species of parasite can infect different host species, creating complex interactions among multiple hosts and parasites. When multiple parasite species coexist in a host, they may compete or use strategies, such as spatial niche partitioning, to reduce competition. Here, we present a host­symbiont system with two species of Selenidium (Apicomplexa, Gregarinida) and one species of astome ciliate co-infecting two different species of slime feather duster worms (Annelida, Sabellidae, Myxicola) living in neighbouring habitats. We examined the morphology of the endosymbionts with light and scanning electron microscopy (SEM) and inferred their phylogenetic interrelationships using small subunit (SSU) rDNA sequences. In the host 'Myxicola sp. Quadra', we found two distinct species of Selenidium; S. cf. mesnili exclusively inhabited the foregut, and S. elongatum n. sp. inhabited the mid to hindgut, reflecting spatial niche partitioning. Selenidium elongatum n. sp. was also present in the host M. aesthetica, which harboured the astome ciliate Pennarella elegantia n. gen. et sp. Selenidium cf. mesnili and P. elegantia n. gen. et sp. were absent in the other host species, indicating host specificity. This system offers an intriguing opportunity to explore diverse aspects of host­endosymbiont interactions and competition among endosymbionts.

Identification of a new gregarine parasite associated with mass mortality events of freshwater pearl mussels (Margaritifera margaritifera) in Sweden.

Freshwater bivalves play key ecological roles in lakes and rivers, largely contributing to healthy ecosystems. The freshwater pearl mussel, Margaritifera margaritifera, is found in Europe and on the East coast of North America. Once common in oxygenated streams, M. margaritifera is rapidly declining and consequently assessed as a threatened species worldwide. Deterioration of water quality has been considered the main factor for the mass mortality events affecting this species. Yet, the role of parasitic infections has not been investigated. Here, we report the discovery of three novel protist lineages found in Swedish populations of M. margaritifera belonging to one of the terrestrial groups of gregarines (Eugregarinorida, Apicomplexa). These lineages are closely related-but clearly separated-from the tadpole parasite Nematopsis temporariae. In one lineage, which is specifically associated with mortality events of M. margaritifera, we found cysts containing single vermiform zoites in the gills and other organs of diseased individuals using microscopy and in situ hybridization. This represents the first report of a parasitic infection in M. margaritifera that may be linked to the decline of this mussel species. We propose a tentative life cycle with the distribution of different developmental stages and potential exit from the host into the environment.

Novel cytoskeletal traits in the intestinal parasites (Squirmida, Platyproteum vivax) of Pacific peanut worms (Sipuncula, Phascolosoma agassizii).

The cytoskeletal organization of a squirmid, namely Platyproteum vivax, was investigated with confocal laser scanning microscopy (CLSM) to refine inferences about convergent evolution among intestinal parasites of marine invertebrates. Platyproteum inhabits Pacific peanut worms (Phascolosoma agassizii) and has traits that are similar to other lineages of myzozoan parasites, namely gregarine apicomplexans within Selenidium, such as conspicuous feeding stages, called "trophozoites," capable of dynamic undulations. SEM and CLSM of P. vivax revealed an inconspicuous flagellar apparatus and a uniform array of longitudinal microtubules organized in bundles (LMBs). Extreme flattening of the trophozoites and a consistently oblique morphology of the anterior end provided a reliable way to distinguish dorsal and ventral surfaces. CLSM revealed a novel system of microtubules oriented in the flattened dorsoventral plane. Most of these dorsoventral microtubule bundles (DVMBs) had a punctate distribution and were evenly spaced along a curved line spanning the longitudinal axis of the trophozoites. This configuration of microtubules is inferred to function in maintaining the flattened shape of the trophozoites and facilitate dynamic undulations. The novel traits in Platyproteum are consistent with phylogenomic data showing that this lineage is only distantly related to Selenidium and other marine gregarine apicomplexans with dynamic intestinal trophozoites.

First molecular detection and genetic diversity of Hepatozoon sp. (Apicomplexa) and Brugia sp. (Nematoda) in a crocodile monitor in Nakhon Pathom, Thailand.

The crocodile monitor (Varanus salvator) is the most common monitor lizard in Thailand. Based on habitat and food, they have the potential to transmit zoonoses, with a high possibility of infecting ectoparasites and endoparasites. Diseases that could infect crocodile monitors and be transmitted to other animals, including humans. This research aims to identify and evaluate the phylogenetic relationships of Hepatozoon sp. and sheathed microfilaria in crocodile monitors. The phylogenetic analyses of Hepatozoon, based on 18S rRNA, and sheathed microfilaria, based on the COX1 gene, revealed that the Hepatozoon sp. were grouped with H. caimani, while sheathed microfilaria were grouped together with B. timori. This study provides insights into the genetic diversity and host-parasite interactions of hemoparasites in crocodile monitors in Thailand.

Whole-genome CRISPR screens to understand Apicomplexan-host interactions.

Apicomplexan parasites are aetiological agents of numerous diseases in humans and livestock. Functional genomics studies in these parasites enable the identification of biological mechanisms and protein functions that can be targeted for therapeutic intervention. Recent improvements in forward genetics and whole-genome screens utilising CRISPR/Cas technology have revolutionised the functional analysis of genes during Apicomplexan infection of host cells. Here, we highlight key discoveries from CRISPR/Cas9 screens in Apicomplexa or their infected host cells and discuss remaining challenges to maximise this technology that may help answer fundamental questions about parasite-host interactions.

Lizard host abundances and climatic factors explain phylogenetic diversity and prevalence of blood parasites on an oceanic island.

Host abundance might favour the maintenance of a high phylogenetic diversity of some parasites via rapid transmission rates. Blood parasites of insular lizards represent a good model to test this hypothesis because these parasites can be particularly prevalent in islands and host lizards highly abundant. We applied deep amplicon sequencing and analysed environmental predictors of blood parasite prevalence and phylogenetic diversity in the endemic lizard Gallotia galloti across 24 localities on Tenerife, an island in the Canary archipelago that has experienced increasing warming and drought in recent years. Parasite prevalence assessed by microscopy was over 94%, and a higher proportion of infected lizards was found in warmer and drier locations. A total of 33 different 18s rRNA parasite haplotypes were identified, and the phylogenetic analyses indicated that they belong to two genera of Adeleorina (Apicomplexa: Coccidia), with Karyolysus as the dominant genus. The most important predictor of between-locality variation in parasite phylogenetic diversity was the abundance of lizard hosts. We conclude that a combination of climatic and host demographic factors associated with an insular syndrome may be favouring a rapid transmission of blood parasites among lizards on Tenerife, which may favour the maintenance of a high phylogenetic diversity of parasites.