Stomatocystis goerresi, a new species of gregarine parasite (Apicomplexa, Monocystidae) from the invasive Japanese earthworm Amynthas tokioensis (Megascolecidae), with a description of the parasite's life cycle.

Stomatocystis goerresi sp. n., a gregarine (phylum Apicomplexa, Monocystidae) parasite of an important invasive earthworm in North America, Amynthas tokioensis (Beddard), is described. This is the second species placed into the genus, and details of its morphology and life cycle support Stomatocystis Bandyopadhyay, Mitra et Göçmen, 2006 as a valid taxon. The new species is described using standard nomenclature, measurements, shape descriptors, and photographs of living cells. The parasite was found only in A. tokioensis, and absent in sympatric earthworm species, suggesting it arrived when the earthworms were introduced from their origin from Japan. The species is distinctive from the type species in the genus, S. indica Bandyopadhyay, Mitra et Göçmen, 2006, in being substantially larger in all stages, found in only the host's seminal vesicles, and found in a different host species from East Asia. The distinctive trophozoites/gamonts develop a large funnel structure ringed with a collar of pronounced ridges, and the funnel appears even in the smallest cells. This funnel varies greatly in relative size (to the cell body) and shape, sometimes forming a large fan. The life cycle of S. goerresi is described including distinctive syzygy in which the funnels fuse and then produce a large cell with local centres of isogamete production (thus sex without gender). Gametes are large ( ~5 µm) spheres with complex tips. Oocyst production is large, > 1,000 per mature gametocyst. The genus Stomatocystis is placed into the Monocystidae, but the life cycle of the new species differs from those of other monocystid taxa, which may mean the Monocystidae are not monophyletic or life cycles are variable within the family. Prevalence of S. goerresi at the type locality was high (~ 90%). The parasites destroy the earthworm's organ of sperm self-storage thus eliminating the male function in the hermaphroditic host which may influence the ability of the earthworm to invade and be successful at new sites.

A New Species of Monocystis (Apicomplexa: Gregarina: Monocystidae) from the Asian Invasive Earthworm Amynthas agrestis (Megascolecidae), with an Improved Standard for Monocystis Species Descriptions.

Monocystis perplexa n. sp., a parasite of an important invasive Japanese earthworm in North America, Amynthas agrestis, is described from a site in Vermont. An improved standard for Monocystis species descriptions is proposed including a standard nomenclature to reduce synonymies, a standard set of biometrics and shape descriptions for living cells, and a DNA genomic sequence for the 18S rRNA (∼1,700 base pairs). Comparing morphologies of Monocystis parasites in sympatric earthworm species indicates that M. perplexa is specific to A. agrestis in the study region. Also, polymerase chain reaction primers specific to M. perplexa amplified samples of A. agrestis earthworms taken from several sites in Japan. This suggests the parasite entered North America from Japan, the origin of the invasive Amynthas earthworm, and thus M. perplexa would be the first Monocystis described from the diverse Japanese Amynthas earthworms and the first from East Asia. Monocystis perplexa was found in every population of A. agrestis surveyed in Vermont, always reaching 100% prevalence by late summer (the host has an annual life cycle in Vermont). The 18S gene sequence differed from that of Monocystis agilis from the sympatric earthworm Lumbricus terrestris (the only other sequence available for Monocystis), and a genetic similarity tree places them closest among other gregarines. Many of the 95 described species of Monocystis are very similar in morphology (based on species descriptions), so the 18S gene can act as a barcode for Monocystis species and thus will help to eliminate both synonymies and reveal cryptic species.

A symbiotic bacterium of shipworms produces a compound with broad spectrum anti-apicomplexan activity.

Apicomplexan parasites cause severe disease in both humans and their domesticated animals. Since these parasites readily develop drug resistance, development of new, effective drugs to treat infection caused by these parasites is an ongoing challenge for the medical and veterinary communities. We hypothesized that invertebrate-bacterial symbioses might be a rich source of anti-apicomplexan compounds because invertebrates are susceptible to infections with gregarines, parasites that are ancestral to all apicomplexans. We chose to explore the therapeutic potential of shipworm symbiotic bacteria as they are bona fide symbionts, are easily grown in axenic culture and have genomes rich in secondary metabolite loci [1,2]. Two strains of the shipworm symbiotic bacterium, Teredinibacter turnerae, were screened for activity against Toxoplasma gondii and one strain, T7901, exhibited activity against intracellular stages of the parasite. Bioassay-guided fractionation identified tartrolon E (trtE) as the source of the activity. TrtE has an EC50 of 3 nM against T. gondii, acts directly on the parasite itself and kills the parasites after two hours of treatment. TrtE exhibits nanomolar to picomolar level activity against Cryptosporidium, Plasmodium, Babesia, Theileria, and Sarcocystis; parasites representing all branches of the apicomplexan phylogenetic tree. The compound also proved effective against Cryptosporidium parvum infection in neonatal mice, indicating that trtE may be a potential lead compound for preclinical development. Identification of a promising new compound after such limited screening strongly encourages further mining of invertebrate symbionts for new anti-parasitic therapeutics.

Apicomplexan-like parasites are polyphyletic and widely but selectively dependent on cryptic plastid organelles.

The phylum Apicomplexa comprises human pathogens such as Plasmodium but is also an under-explored hotspot of evolutionary diversity central to understanding the origins of parasitism and non-photosynthetic plastids. We generated single-cell transcriptomes for all major apicomplexan groups lacking large-scale sequence data. Phylogenetic analysis reveals that apicomplexan-like parasites are polyphyletic and their similar morphologies emerged convergently at least three times. Gregarines and eugregarines are monophyletic, against most expectations, and rhytidocystids and Eleutheroschizon are sister lineages to medically important taxa. Although previously unrecognized, plastids in deep-branching apicomplexans are common, and they contain some of the most divergent and AT-rich genomes ever found. In eugregarines, however, plastids are either abnormally reduced or absent, thus increasing known plastid losses in eukaryotes from two to four. Environmental sequences of ten novel plastid lineages and structural innovations in plastid proteins confirm that plastids in apicomplexans and their relatives are widespread and share a common, photosynthetic origin.

Host food quality and quantity differentially affect Ascogregarina barretti parasite burden, development and within-host competition in the mosquito Aedes triseriatus.

Host condition depends in large part on the quality and quantity of available food and heavily influences the outcome of parasite infection. Although parasite fitness traits such as growth rate and size may depend on host condition, whether host food quality or quantity is more important to parasite fitness and within-host interactions is poorly understood. We provided individual mosquito hosts with a standard dose of a gregarine parasite and reared mosquitoes on two food types of different quality and two quantities. We measured host size, total parasite count and area, and average size of parasites within each treatment. Food quality significantly influenced the number of parasites in a host; hosts fed a low-quality diet were infected with more parasites than those provided a high-quality diet. In addition, we found evidence of within-host competition; there was a negative relationship between parasite size and count though this relationship was dependent on host food quality. Host food quantity significantly affected total parasite area and parasite size; lower food quantity resulted in smaller parasites and reduced overall parasite area inside the host. Thus both food quality and quantity have the potential to influence parasite fitness and population dynamics.

Motility and cytoskeletal organisation in the archigregarine Selenidium pygospionis (Apicomplexa): observations on native and experimentally affected parasites.

Representatives of Apicomplexa perform various kinds of movements that are linked to the different stages of their life cycle. Ancestral apicomplexan lineages, including gregarines, represent organisms suitable for research into the evolution and diversification of motility within the group. The vermiform trophozoites and gamonts of the archigregarine Selenidium pygospionis perform a very active type of bending motility. Experimental assays and subsequent light, electron, and confocal microscopic analyses demonstrated the fundamental role of the cytoskeletal proteins actin and tubulin in S. pygospionis motility and allowed us to compare the mechanism of its movement to the gliding machinery (the so-called glideosome concept) described in apicomplexan zoites. Actin-modifying drugs caused a reduction in the movement speed (cytochalasin D) or stopped the motility of archigregarines completely (jasplakinolide). Microtubule-disrupting drugs (oryzalin and colchicine) had an even more noticeable effect on archigregarine motility. The fading and disappearance of microtubules were documented in ultrathin sections, along with the formation of α-tubulin clusters visible after the immunofluorescent labelling of drug-treated archigregarines. The obtained data indicate that subpellicular microtubules most likely constitute the main motor structure involved in S. pygospionis bending motility, while actin has rather a supportive function.

An Overview of Peripheral Blood Mononuclear Cells as a Model for Immunological Research of Toxoplasma gondii and Other Apicomplexan Parasites.

In biology, models are experimental systems meant to recreate aspects of diseases or human tissue with the goal of generating inferences and approximations that can contribute to the resolution of specific biological problems. Although there are many models for studying intracellular parasites, their data have produced critical contradictions, especially in immunological assays. Peripheral blood mononuclear cells (PBMCs) represent an attractive tissue source in pharmacogenomics and in molecular and immunologic studies, as these cells are easily collected from patients and can serve as sentinel tissue for monitoring physiological perturbations due to disease. However, these cells are a very sensitive model due to variables such as temperature, type of stimulus and time of collection as part of posterior processes. PBMCs have been used to study Toxoplasma gondii and other apicomplexan parasites. For instance, this model is frequently used in new therapies or vaccines that use peptides or recombinant proteins derived from the parasite. The immune response to T. gondii is highly variable, so it may be necessary to refine this cellular model. This mini review highlights the major approaches in which PBMCs are used as a model of study for T. gondii and other apicomplexan parasites. The variables related to this model have significant implications for data interpretation and conclusions related to host-parasite interaction.

Puromycin selection for stable transfectants of the oyster-infecting parasite Perkinsus marinus.

Perkinsus marinus is a marine protozoan parasite that infects natural and farmed oysters, attracting attention from researchers in both fisheries and evolutionary biology. The functions of almost all cellular components and organelles are, however, poorly understood even though a draft genome sequence of P. marinus is publicly available. One of the major obstacles for a functional study of the parasite is limited experimental means for genetic manipulation: a transfection method was established in 2008, and the first drug selection system with bleomycin was reported in 2016. We here introduce the second drug-selectable marker for selection of P. marinus transfectants. The parasite growth is efficiently inhibited by puromycin (IC50 = 4.96 µg/mL), and transfection of its resistance gene, puromycin-N-acetyl-transferase (pac), confers resistance to the drug on the parasite. Stable transfectants can be obtained within 2 months by treating with puromycin at 100 µg/mL. Furthermore, combining puromycin and bleomycin treatment can select transfectants co-expressing two marker genes. This dual-transfection method raises the possibility of using co-localization to identify the cellular localization of novel proteins in P. marinus, thereby contributing to the understanding of cellular functions and pathogenesis.

Lipid metabolism in insect disease vectors.

More than a third of the world population is at constant risk of contracting some insect-transmitted disease, such as Dengue fever, Zika virus disease, malaria, Chagas' disease, African trypanosomiasis, and others. Independent of the life cycle of the pathogen causing the disease, the insect vector hematophagous habit is a common and crucial trait for the transmission of all these diseases. This lifestyle is unique, as hematophagous insects feed on blood, a diet that is rich in protein but relatively poor in lipids and carbohydrates, in huge amounts and low frequency. Another unique feature of these insects is that blood meal triggers essential metabolic processes, as molting and oogenesis and, in this way, regulates the expression of various genes that are involved in these events. In this paper, we review current knowledge of the physiology and biochemistry of lipid metabolism in insect disease vectors, comparing with classical models whenever possible. We address lipid digestion and absorption, hemolymphatic transport, and lipid storage by the fat body and ovary. In this context, both de novo fatty acid and triacylglycerol synthesis are discussed, including the related fatty acid activation process and the intracellular lipid binding proteins. As lipids are stored in order to be mobilized later on, e.g. for flight activity or survivorship, lipolysis and ß-oxidation are also considered. All these events need to be finely regulated, and the role of hormones in this control is summarized. Finally, we also review information about infection, when vector insect physiology is affected, and there is a crosstalk between its immune system and lipid metabolism. There is not abundant information about lipid metabolism in vector insects, and significant current gaps in the field are indicated, as well as questions to be answered in the future.

First Ultrastructural and Molecular Phylogenetic Evidence from the Blastogregarines, an Early Branching Lineage of Plesiomorphic Apicomplexa.

Blastogregarines are poorly studied parasites of polychaetes superficially resembling gregarines, but lacking syzygy and gametocyst stages in the life cycle. Furthermore, their permanent multinuclearity and gametogenesis by means of budding considerably distinguish them from other parasitic Apicomplexa such as coccidians and hematozoans. The affiliation of blastogregarines has been uncertain: different authors considered them highly modified gregarines, an intermediate apicomplexan lineage between gregarines and coccidians, or an isolated group of eukaryotes altogether. Here, we report the ultrastructure of two blastogregarine species, Siedleckia nematoides and Chattonaria mesnili, and provide the first molecular data on their phylogeny based on SSU, 5.8S, and LSU rDNA sequences. Morphological analysis reveals that blastogregarines possess both gregarine and coccidian features. Several traits shared with archigregarines likely represent the ancestral states of the corresponding cell structures for parasitic apicomplexans: a distinctive tegument structure and myzocytotic feeding with a well-developed apical complex. Unlike gregarines but similar to coccidians however, the nuclei of male blastogregarine gametes are associated with two kinetosomes. Molecular phylogenetic analyses reveal that blastogregarines are an independent, early diverging lineage of apicomplexans. Overall, the morphological and molecular evidence congruently suggests that blastogregarines represent a separate class of Apicomplexa.

Advances in the application of genetic manipulation methods to apicomplexan parasites.

Apicomplexan parasites such as Babesia, Theileria, Eimeria, Cryptosporidium and Toxoplasma greatly impact animal health globally, and improved, cost-effective measures to control them are urgently required. These parasites have complex multi-stage life cycles including obligate intracellular stages. Major gaps in our understanding of the biology of these relatively poorly characterised parasites and the diseases they cause severely limit options for designing novel control methods. Here we review potentially important shared aspects of the biology of these parasites, such as cell invasion, host cell modification, and asexual and sexual reproduction, and explore the potential of the application of relatively well-established or newly emerging genetic manipulation methods, such as classical transfection or gene editing, respectively, for closing important gaps in our knowledge of the function of specific genes and proteins, and the biology of these parasites. In addition, genetic manipulation methods impact the development of novel methods of control of the diseases caused by these economically important parasites. Transient and stable transfection methods, in conjunction with whole and deep genome sequencing, were initially instrumental in improving our understanding of the molecular biology of apicomplexan parasites and paved the way for the application of the more recently developed gene editing methods. The increasingly efficient and more recently developed gene editing methods, in particular those based on the CRISPR/Cas9 system and previous conceptually similar techniques, are already contributing to additional gene function discovery using reverse genetics and related approaches. However, gene editing methods are only possible due to the increasing availability of in vitro culture, transfection, and genome sequencing and analysis techniques. We envisage that rapid progress in the development of novel gene editing techniques applied to apicomplexan parasites of veterinary interest will ultimately lead to the development of novel and more efficient methods for disease control.

First record of gregarines (Apicomplexa) in seminal vesicle of insect.

Gregarines (Apicomplexa) are a diverse group of protozoan parasites, which infects gut and other body cavities of invertebrate hosts. In reproductive system of insects, gregarine has been reported only in the accessory glands and spermathecae of females; therefore, this is the first report of a gregarine species in seminal vesicles of insects. Different developmental stages, including sporozoytes, oocysts and trophozoites were described from morphological descriptions using light and electron transmission microscopy. The parasites were described in seminal vesicles of the beetle Tribolium castaneum a model organism and an important insect pest. DNA sequence analysis suggests that the protozoan parasite was an Ascogregarina sp.

Host Diet Affects the Morphology of Monarch Butterfly Parasites.

Understanding host-parasite interactions is essential for ecological research, wildlife conservation, and health management. While most studies focus on numerical traits of parasite groups, such as changes in parasite load, less focus is placed on the traits of individual parasites such as parasite size and shape (parasite morphology). Parasite morphology has significant effects on parasite fitness such as initial colonization of hosts, avoidance of host immune defenses, and the availability of resources for parasite replication. As such, understanding factors that affect parasite morphology is important in predicting the consequences of host-parasite interactions. Here, we studied how host diet affected the spore morphology of a protozoan parasite ( Ophryocystis elektroscirrha ), a specialist parasite of the monarch butterfly ( Danaus plexippus ). We found that different host plant species (milkweeds; Asclepias spp.) significantly affected parasite spore size. Previous studies have found that cardenolides, secondary chemicals in host plants of monarchs, can reduce parasite loads and increase the lifespan of infected butterflies. Adding to this benefit of high cardenolide milkweeds, we found that infected monarchs reared on milkweeds of higher cardenolide concentrations yielded smaller parasites, a potentially hidden characteristic of cardenolides that may have important implications for monarch-parasite interactions.

Cationic amino acid transporters play key roles in the survival and transmission of apicomplexan parasites.

Apicomplexans are obligate intracellular parasites that scavenge essential nutrients from their hosts via transporter proteins on their plasma membrane. The identities of the transporters that mediate amino acid uptake into apicomplexans are unknown. Here we demonstrate that members of an apicomplexan-specific protein family-the Novel Putative Transporters (NPTs)-play key roles in the uptake of cationic amino acids. We show that an NPT from Toxoplasma gondii (TgNPT1) is a selective arginine transporter that is essential for parasite survival and virulence. We also demonstrate that a homologue of TgNPT1 from the malaria parasite Plasmodium berghei (PbNPT1), shown previously to be essential for the sexual gametocyte stage of the parasite, is a cationic amino acid transporter. This reveals a role for cationic amino acid scavenging in gametocyte biology. Our study demonstrates a critical role for amino acid transporters in the survival, virulence and life cycle progression of these parasites.

Temporal Community Structure in Two Gregarines (Rotundula gammari and Heliospora longissima) Co-Infecting the Amphipod Gammarus fasciatus.

This study surveyed gregarine parasites that infect the amphipod, Gammarus fasciatus , to investigate temporal dynamics in infracommunity structure. We sampled a population of hosts for 2 yr from the north branch of the Raritan River in New Jersey. These hosts were infected with 2 direct life cycle gregarine parasites, Rotundula gammari and Heliospora longissima. Infections were separated temporally, with the prevalence of R. gammari peaking within the amphipod population in the fall (prevalence = 78% year 1 and 97% year 2) and H. longissima peaking in early spring (prevalence = 41% year 1 and 52% year 2). Increases in host population density did not significantly correlate with the abundance of these 2 parasites. However, H. longissima abundance was positively correlated with host body weight while R. gammari showed no significant relationship. The mean body mass of amphipods infected with H. longissima was 20.7 ± 1. 2 mg, and with R. gammari 8.1 ± 0.2 mg, which suggests a sized-based infection pattern. Mixed species infections were infrequent with an overall prevalence of 4.6%. When both gregarine species co-infected the same host, the R. gammari but not the H. longissima infrapopulation size was significantly lower when compared to single-species infections, suggesting asymmetric interactions. We conclude that the observed temporal patterns of infection by the 2 parasites are driven by a seasonal change in host demographics and size-dependent infections. We argue that specificity for host developmental stages may have arisen as a mechanism to avoid overlap between these gregarine species.

Investigation into the Physiological Significance of the Phytohormone Abscisic Acid in Perkinsus marinus, an Oyster Parasite Harboring a Nonphotosynthetic Plastid.

Some organisms have retained plastids even after they have lost the ability to photosynthesize. Several studies of nonphotosynthetic plastids in apicomplexan parasites have shown that the isopentenyl pyrophosphate biosynthesis pathway in the organelle is essential for their survival. A phytohormone, abscisic acid, one of several compounds biosynthesized from isopentenyl pyrophosphate, regulates the parasite cell cycle. Thus, it is possible that the phytohormone is universally crucial, even in nonphotosynthetic plastids. Here, we examined this possibility using the oyster parasite Perkinsus marinus, which is a plastid-harboring cousin of apicomplexan parasites and has independently lost photosynthetic ability. Fluridone, an inhibitor of abscisic acid biosynthesis, blocked parasite growth and induced cell clustering. Nevertheless, abscisic acid and its intermediate carotenoids did not affect parasite growth or rescue the parasite from inhibition. Moreover, abscisic acid was not detected from the parasite using liquid chromatography mass spectrometry. Our findings show that abscisic acid does not play any significant roles in P. marinus.

Developmental stages identified in the trophozoite of the free-living Alveolate flagellate Colpodella sp. (Apicomplexa).

In this study we performed light, immunofluorescent and transmission electron microscopy of Colpodella trophozoites to characterize trophozoite morphology and protein distribution. The use of Giemsa staining and antibodies to distinguish Colpodella life cycle stages has not been performed previously. Rhoptry and ß-tubulin antibodies were used in immunofluorescent assays (IFA) to identify protein localization and distribution in the trophozoite stage of Colpodella (ATCC 50594). We report novel data identifying "doughnut-shaped" vesicles in the cytoplasm and apical end of Colpodella trophozoites reactive with antibodies specific to Plasmodium merozoite rhoptry proteins. Giemsa staining and immunofluorescent microscopy identified different developmental stages of Colpodella trophozoites, with the presence or absence of vesicles corresponding to maturity of the trophozoite. These data demonstrate for the first time evidence of rhoptry protein conservation between Plasmodium and Colpodella and provide further evidence that Colpodella trophozoites can be used as a heterologous model to investigate rhoptry biogenesis and function. Staining and antibody reactivity will facilitate phylogenetic, biochemical and molecular investigations of Colpodella sp. Developmental stages can be distinguished by Giemsa staining and antibody reactivity.

Ultrastructure of Selenidium pendula, the Type Species of Archigregarines, and Phylogenetic Relations to Other Marine Apicomplexa.

Archigregarines, an early branching lineage within Apicomplexa, are a poorly-known group of invertebrate parasites. By their phylogenetic position, archigregarines are an important lineage to understand the functional transition that occurred between free-living flagellated predators to obligatory parasites in Apicomplexa. In this study, we provide new ultrastructural data and phylogenies based on SSU rDNA sequences using the type species of archigregarines, the Selenidiidae Selenidium pendulaGiard, 1884. We describe for the first time the syzygy and early gamogony at the ultrastructural level, revealing a characteristic nuclear multiplication with centrocones, cryptomitosis, filamentous network of chromatin, a cyst wall secretion and a 9+0 flagellar axoneme of the male gamete. S. pendula belongs to a monophyletic lineage that includes several other related species, all infecting Sedentaria Polychaeta (Spionidae, Sabellaridae, Sabellidae and Cirratulidae). All of these Selenidium species exhibit similar biological characters: a cell cortex with the plasma membrane - inner membrane complex - subpellicular microtubule sets, an apical complex with the conoid, numerous rhoptries and micronemes, a myzocytosis with large food vacuoles, a nuclear multiplication during syzygy and young gamonts. Two other distantly related Selenidium-like lineages infect Terebellidae and Sipunculida, underlying the ability of archigregarines to parasite a wide range of marine hosts.

The life cycle of Gregarina cuneata in the midgut of Tribolium castaneum and the effects of parasitism on the development of insects.

Tribolium castaneum Herbst 1797 (Coleoptera: Tenebrionidae), an important pest of stored grains and byproducts, is naturally infected by Gregarina cuneata Stein 1848 (Apicomplexa: Gregarinidae). Changes in the life cycle of insects caused by the parasite development in the midgut were studied. Trophozoites, gamonts (solitary and associated), and gametocysts were present in the midgut of the insects. In young trophozoites, the apical region differentiated into an epimerite that firmly attached the parasite to the host epithelial cells. With maturation, trophozoites developed in gamonts that were associated with the initiation of sexual reproduction in the cell cycle, culminating in the formation of the spherical gametocyst. Morpho-functional analyses indicated that gregarines absorb nutrients from infected cells and can occlude the midgut as they develop. Consequently, nutritional depletion may interfere with the host's physiology, causing decreased growth, delayed development, and high mortality rates of the parasitized insects. These results suggest G. cuneata could be an important biological agent for controlling T. castaneum in integrated pest management programs.

Effect of Exposure Dose on Ichthyophonus Prevalence and Infection Intensity in Experimentally Infected Rainbow Trout, Oncorhynchus mykiss.

This study describes the effect of increasing exposure dose on Ichthyophonus prevalence and infection intensity in experimentally infected rainbow trout, Oncorhynchus mykiss. Specific-pathogen free trout were exposed per os to increasing numbers of Ichthyophonus schizonts obtained from naturally infected donor fish, then sampled after 30 and 60 days post-exposure. Both in vitro explant culture and histology revealed that as the number of schizonts per dose increased there was a proportionate increase in the number of infected fish, as well as an increase in the number of infected organs; parasite density in individual infected organs also increased with dose. Explant culture revealed that all fish exposed to the highest dose (≥2,080 schizonts) became infected, while only 67% of those exposed to the intermediate dose (1,040-1,153 schizonts) were Ichthyophonus-positive after 60 days; Ichthyophonus was not detected in fish exposed to the 2 lowest doses (≤280 schizonts). Histologic examination of individual infected organs also revealed increasing infection prevalence and parasite density in response to exposure to increasing numbers of Ichthyophonus schizonts.