Description and prevalence of gregarines infecting the amphipod Gammarus pulex, in the Water of Leith, Scotland, UK.

Gregarines are symbiotic protists that are found in a broad spectrum of invertebrates, including insects, crustaceans, and annelids. Among these the globally distributed amphipod Gammarus pulex is one of the earliest recognized hosts for aquatic gregarines and is prevalent among macroinvertebrates in freshwater environments. In this study, samples of G. pulex were collected in the Water of Leith river, Scotland, UK. Gregarines were identified using light and scanning electron microscopy as well as standard molecular techniques. We identified three septate eugregarine symbionts-Heliospora longissima, Cephaloidophora gammari, and the here newly characterized Cephaloidophora conus n. sp. (formerly Cephaloidophora sp.) associated with Gammarus pulex in the Water of Leith. Prevalences for identified gregarine species were calculated and seasonal dynamics of gregarine infections/colonization were analyzed. Prevalences were highest in autumn and spring reaching almost 50 %. While the two Cephaloidophora species showed similar colonization patterns, the prevalence of Heliospora showed an opposite trend. Identifying gregarine infection/colonization patterns is one step towards better understanding the gregarine-host relationship, as well as possible impacts of the gregarines on their hosts.

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.

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.

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.

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.

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.

Extreme heat reduces host and parasite performance in a butterfly-parasite interaction.

Environmental temperature fundamentally shapes insect physiology, fitness and interactions with parasites. Differential climate warming effects on host versus parasite biology could exacerbate or inhibit parasite transmission, with far-reaching implications for pollination services, biocontrol and human health. Here, we experimentally test how controlled temperatures influence multiple components of host and parasite fitness in monarch butterflies (Danaus plexippus) and their protozoan parasites Ophryocystis elektroscirrha. Using five constant-temperature treatments spanning 18-34°C, we measured monarch development, survival, size, immune function and parasite infection status and intensity. Monarch size and survival declined sharply at the hottest temperature (34°C), as did infection probability, suggesting that extreme heat decreases both host and parasite performance. The lack of infection at 34°C was not due to greater host immunity or faster host development but could instead reflect the thermal limits of parasite invasion and within-host replication. In the context of ongoing climate change, temperature increases above current thermal maxima could reduce the fitness of both monarchs and their parasites, with lower infection rates potentially balancing negative impacts of extreme heat on future monarch abundance and distribution.

Histopathological screening of Pontogammarus robustoides (Amphipoda), an invader on route to the United Kingdom.

Biological invasions may act as conduits for pathogen introduction. To determine which invasive non-native species pose the biggest threat, we must first determine the symbionts (pathogens, parasites, commensals, mutualists) they carry, via pathological surveys that can be conducted in multiple ways (i.e., molecular, pathological, and histological). Whole animal histopathology allows for the observation of pathogenic agents (virus to Metazoa), based on their pathological effect upon host tissue. Where the technique cannot accurately predict pathogen taxonomy, it does highlight pathogen groups of importance. This study provides a histopathological survey of Pontogammarus robustoides (invasive amphipod in Europe) as a baseline for symbiont groups that may translocate to other areas/hosts in future invasions. Pontogammarus robustoides (n = 1,141) collected throughout Poland (seven sites), were noted to include a total of 13 symbiotic groups: a putative gut epithelia virus (overall prevalence = 0.6%), a putative hepatopancreatic cytoplasmic virus (1.4%), a hepatopancreatic bacilliform virus (15.7%), systemic bacteria (0.7%), fouling ciliates (62.0%), gut gregarines (39.5%), hepatopancreatic gregarines (0.4%), haplosporidians (0.4%), muscle infecting microsporidians (6.4%), digeneans (3.5%), external rotifers (3.0%), an endoparasitic arthropod (putatively: Isopoda) (0.1%), and Gregarines with putative microsporidian infections (1.4%). Parasite assemblages partially differed across collection sites. Co-infection patterns revealed strong positive and negative associations between five parasites. Microsporidians were common across sites and could easily spread to other areas following the invasion of P. robustoides. By providing this initial histopathological survey, we hope to provide a concise list of symbiont groups for risk-assessment in the case of a novel invasion by this highly invasive amphipod.

Flying on empty: reduced mitochondrial function and flight capacity in food-deprived monarch butterflies.

Mitochondrial function is fundamental to organismal performance, health and fitness - especially during energetically challenging events, such as migration. With this investigation, we evaluated mitochondrial sensitivity to ecologically relevant stressors. We focused on an iconic migrant, the North American monarch butterfly (Danaus plexippus), and examined the effects of two stressors: 7 days of food deprivation and infection by the protozoan parasite Ophryocystis elektroscirrha (known to reduce survival and flight performance). We measured whole-animal resting metabolic rate (RMR) and peak flight metabolic rate, and mitochondrial respiration of isolated mitochondria from the flight muscles. Food deprivation reduced mass-independent RMR and peak flight metabolic rate, whereas infection did not. Fed monarchs used mainly lipids in flight (respiratory quotient 0.73), but the respiratory quotient dropped in food-deprived individuals, possibly indicating switching to alternative energy sources, such as ketone bodies. Food deprivation decreased mitochondrial maximum oxygen consumption but not basal respiration, resulting in lower respiratory control ratio (RCR). Furthermore, food deprivation decreased mitochondrial complex III activity, but increased complex IV activity. Infection did not result in any changes in these mitochondrial variables. Mitochondrial maximum respiration rate correlated positively with mass-independent RMR and flight metabolic rate, suggesting a link between mitochondria and whole-animal performance. In conclusion, low food availability negatively affects mitochondrial function and flight performance, with potential implications for migration, fitness and population dynamics. Although previous studies have reported poor flight performance in infected monarchs, we found no differences in physiological performance, suggesting that reduced flight capacity may be due to structural differences or low energy stores.

Unraveling the Elusive Rhoptry Exocytic Mechanism of Apicomplexa.

Apicomplexan parasites are unicellular eukaryotes that invade the cells in which they proliferate. The development of genetic tools in Toxoplasma, and then in Plasmodium, in the 1990s allowed the first description of the molecular machinery used for motility and invasion, revealing a crucial role for two different secretory organelles, micronemes and rhoptries. Rhoptry proteins are injected directly into the host cytoplasm not only to promote invasion but also to manipulate host functions. Nonetheless, the injection machinery has remained mysterious, a major conundrum in the field. Here we review recent progress in uncovering structural components and proteins implicated in rhoptry exocytosis and explain how revisiting early findings and considering the evolutionary origins of Apicomplexa contributed to some of these discoveries.

New gregarine species (Apicomplexa) from tunicates show an evolutionary history of host switching and suggest a problem with the systematics of Lankesteria and Lecudina.

Apicomplexa (sensu stricto) are a diverse group of obligate parasites to a variety of animal species. Gregarines have been the subject of particular interest due to their diversity, phylogenetically basal position, and more recently, their symbiotic relationships with their hosts. In the present study, four new species of marine eugregarines infecting ascidian hosts (Lankesteria kaiteriteriensis sp. nov., L. dolabra sp. nov., L. savignyii sp. nov., and L. pollywoga sp. nov.) were described using a combination of morphological and molecular data. Phylogenetic analysis using small subunit rDNA sequences suggested that gregarines that parasitize ascidians and polychaetes share a common origin as traditionally hypothesized by predecessors in the discipline. However, Lankesteria and Lecudina species did not form clades as expected, but were instead intermixed amongst each other and their respective type species in the phylogeny. These two major genera are therefore taxonomically problematic. We hypothesize that the continued addition of new species from polychaete and tunicate hosts as well as the construction of multigene phylogenies that include type-material will further dissolve the currently accepted distinction between Lankesteria and Lecudina. The species discovered and described in the current study add new phylogenetic and taxonomic data to the knowledge of marine gregarine parasitism in ascidian hosts.

The sexual side of parasitic protists.

Much of the vast evolutionary landscape occupied by Eukaryotes is dominated by protists. Though parasitism has arisen in many lineages, there are three main groups of parasitic protists of relevance to human and livestock health: the Apicomplexa, including the malaria parasite Plasmodium and coccidian pathogens of livestock such as Eimeria; the excavate flagellates, encompassing a diverse range of protist pathogens including trypanosomes, Leishmania, Giardia and Trichomonas; and the Amoebozoa, including pathogenic amoebae such as Entamoeba. These three groups represent separate, deep branches of the eukaryote tree, underlining their divergent evolutionary histories. Here, I explore what is known about sex in these three main groups of parasitic protists.

Managed bumble bees acquire parasites from their foraging environment: A case study on parasite spillback.

The use of commercially reared bumble bees in agricultural environments has been recognized as a potential threat to wild pollinators due to competition, genetic contamination, and most notably, disease transmission. Higher parasite prevalence near greenhouses where managed bumble bees are used has been linked to parasite spillover from managed to wild bees. However, pathogen transmission is not unidirectional, and can also flow from wild to managed bees. These newly infected managed bees can subsequently re-infect (other) wild bees, in a process known as spillback, which is an alternative explanation for the increased parasite prevalence near greenhouses. Reducing parasite prevalence in managed bees is key to controlling host-parasite dynamics in cases of spillover; in spillback, producing managed bees that are resilient to infection is important. Here we establish that the managed bumble bee Bombus terrestris can acquire parasites from their foraging environment, which is the major infection route for Apicystis spp. and Crithidia spp., but not for Nosema spp.. Managed B. terrestris were found to have a higher prevalence of Crithdia and a higher load of Apicystis than local wild conspecifics, showing that for these parasites, spillback is a possible risk scenario.

Field Evidence of Mosquito Population Regulation by a Gregarine Parasite.

Although parasites are by definition costly to their host, demonstrating that a parasite is regulating its host abundance in the field can be difficult. Here we present an example of a gregarine parasite, Ascogregarina taiwanensis Lien and Levine (Apicomplexa: Lecudinidae), regulating its mosquito host, Aedes albopictus Skuse (Diptera: Culicidae), in Bermuda. We sampled larvae from container habitats over 2 yr, assessed parasite prevalence, and estimated host abundance from egg counts obtained in neighboring ovitraps. We regressed change in average egg count from 1 yr to the next on parasite prevalence and found a significant negative effect of parasite prevalence. We found no evidence of host density affecting parasite prevalence. Our results demonstrate that even for a parasite with moderate virulence, host regulation can occur in the field.

Thermal tolerance and environmental persistence of a protozoan parasite in monarch butterflies.

Many parasites have external transmission stages that persist in the environment prior to infecting a new host. Understanding how long these stages can persist, and how abiotic conditions such as temperature affect parasite persistence, is important for predicting infection dynamics and parasite responses to future environmental change. In this study, we explored environmental persistence and thermal tolerance of a debilitating protozoan parasite that infects monarch butterflies. Parasite transmission occurs when dormant spores, shed by adult butterflies onto host plants and other surfaces, are later consumed by caterpillars. We exposed parasite spores to a gradient of ecologically-relevant temperatures for 2, 35, or 93 weeks. We tested spore viability by feeding controlled spore doses to susceptible monarch larvae, and examined relationships between temperature, time, and resulting infection metrics. We also examined whether distinct parasite genotypes derived from replicate migratory and resident monarch populations differed in their thermal tolerance. Finally, we examined evidence for a trade-off between short-term within-host replication and long-term persistence ability. Parasite viability decreased in response to warmer temperatures over moderate-to-long time scales. Individual parasite genotypes showed high heterogeneity in viability, but differences did not cluster by migratory vs. resident monarch populations. We found no support for a negative relationship between environmental persistence and within-host replication, as might be expected if parasites invest in short-term reproduction at the cost of longer-term survival. Findings here indicate that dormant spores can survive for many months under cooler conditions, and that heat dramatically shortens the window of transmission for this widespread and virulent butterfly parasite.

Biogenesis and maintenance of the apicoplast in model apicomplexan parasites.

The apicoplast is a non-photosynthetic relict plastid of Apicomplexa that evolved from a secondary symbiotic system. During its evolution, most of the genes derived from its alga ancestor were lost. Only genes involved in several valuable metabolic pathways, such as the synthesis of isoprenoid precursors, heme, and fatty acids, have been transferred to the host genome and retained to help these parasites adapt to a complex life cycle and various living environments. The biological function of an apicoplast is essential for most apicomplexan parasites. Considering their potential as drug targets, the metabolic functions of this symbiotic organelle have been intensively investigated through computational and biological means. Moreover, we know that not only organellar metabolic functions are linked with other organelles, but also their biogenesis processes have developed and evolved to tailor their biological functions and proper inheritance. Several distinct features have been found in the biogenesis process of apicoplasts. For example, the apicoplast borrows a dynamin-related protein (DrpA) from its host to implement organelle division. The autophagy system has also been repurposed for linking the apicoplast and centrosome during replication and the division process. However, many vital questions remain to be answered about how these parasites maintain and properly inherit this symbiotic organelle. Here we review our current knowledge about its biogenesis process and discuss several critical questions remaining to be answered in this field.

Effects of Ascogregarina culicis (Eugregarinorida: Lecudinidae) on Mosquito Size and Dengue Virus Infection in Aedes aegypti (Diptera: Culicidae).

Dengue virus infection, transmitted via mosquito bites, poses a substantial risk to global public health. Studies suggest that the mosquito's microbial community can profoundly influence vector-borne pathogen transmissions, including dengue virus. Ascogregarina culicis (Ross) of the phylum Apicomplexa is among the most common parasites of Aedes aegypti (Linnaeus), the principal vector of dengue. Despite a high prevalence worldwide, including in the areas where dengue is endemic, the impact of A. culicis on Ae. aegypti vector competence for dengue virus is unknown. This study aimed to investigate the effects of A. culicis infection on mosquito size and fitness, as measured by wing length, and the susceptibility to dengue virus infection in Ae. aegypti. Our results showed that there was no statistically significant difference in wing lengths between Ae. aegypti infected and not infected with A. culicis. Furthermore, A. culicis infection did not significantly affect dengue virus infection or disseminated infection rate. However, there was a significant association between shorter wings and higher dengue virus infection rate, whereby a 0.1-mm increase in wing length decreased the odds of the mosquito being infected by 32%. Thus, based on our result, A. culicis infection does not influence the body size and dengue virus infection in Ae. aegypti. This study helps to shed light on a common but neglected eukaryotic mosquito parasite.

Genomic and transcriptomic evidence for descent from Plasmodium and loss of blood schizogony in Hepatocystis parasites from naturally infected red colobus monkeys.

Hepatocystis is a genus of single-celled parasites infecting, amongst other hosts, monkeys, bats and squirrels. Although thought to have descended from malaria parasites (Plasmodium spp.), Hepatocystis spp. are thought not to undergo replication in the blood-the part of the Plasmodium life cycle which causes the symptoms of malaria. Furthermore, Hepatocystis is transmitted by biting midges, not mosquitoes. Comparative genomics of Hepatocystis and Plasmodium species therefore presents an opportunity to better understand some of the most important aspects of malaria parasite biology. We were able to generate a draft genome for Hepatocystis sp. using DNA sequencing reads from the blood of a naturally infected red colobus monkey. We provide robust phylogenetic support for Hepatocystis sp. as a sister group to Plasmodium parasites infecting rodents. We show transcriptomic support for a lack of replication in the blood and genomic support for a complete loss of a family of genes involved in red blood cell invasion. Our analyses highlight the rapid evolution of genes involved in parasite vector stages, revealing genes that may be critical for interactions between malaria parasites and mosquitoes.

Photoparasitism as an Intermediate State in the Evolution of Apicomplexan Parasites.

Despite the benefits of phototrophy, many algae have lost photosynthesis and have converted back to heterotrophy. Parasitism is a heterotrophic strategy, with apicomplexans being among the most devastating parasites for humans. The presence of a nonphotosynthetic plastid in apicomplexan parasites suggests their phototrophic ancestry. The discovery of related phototrophic chromerids has unlocked the possibility to study the transition between phototrophy and parasitism in the Apicomplexa. The chromerid Chromera velia can live as an intracellular parasite in coral larvae as well as a free-living phototroph, combining phototrophy and parasitism in what I call photoparasitism. Since early-branching apicomplexans live extracellularly, their evolution from an intracellular symbiont is unlikely. In this opinion article I discuss possible evolutionary trajectories from an extracellular photoparasite to an obligatory apicomplexan parasite.