Host-Parasite Incongruences in Rodent Eimeria Suggest Significant Role of Adaptation Rather than Cophylogeny in Maintenance of Host Specificity

The degree of host specificity, its phylogenetic conservativeness and origin are virtually unknown in Eimeria. This situation is largely due to the inadequate sample of eimerian molecular data available for reliable phylogenetic analyses. In this study, we extend the data set by adding 71 new sequences of coccidia infecting 16 small-mammal genera, mostly rodents. According to the respective feasibility of PCR gene amplification, the new samples are represented by one or more of the following genes: nuclear 18S rRNA, plastid ORF 470, and mitochondrial COI. Phylogenetic analyses of these sequences confirm the previous hypothesis that Eimeria, in its current morphology-based delimitation, is not a monophyletic group. Several samples of coccidia corresponding morphologically to other genera are scattered among the Eimeria lineages. More importantly, the distribution of eimerians from different hosts indicates that the clustering of eimerian species is influenced by their host specificity, but does not arise from a cophylogenetic/cospeciation process; while several clusters are specific to a particular host group, inner topologies within these clusters do not reflect host phylogeny. This observation suggests that the host specificity of Eimeria is caused by adaptive rather than cophylogenetic processes.     

Generalist Eimeria species in rodents: Multilocus analyses indicate inadequate resolution of established markers

• Intracellular parasites of the genus Eimeria are described as tissue/host‐specific. Phylogenetic classification of rodent Eimeria suggested that some species have a broader host range than previously assumed. We explore whether Eimeria spp. infecting house mice are misclassified by the most widely used molecular markers due to a lack of resolution, or whether, instead, these parasite species are indeed infecting multiple host species.
• With the commonly used markers (18S/COI), we recovered monophyletic clades of E. falciformis and E. vermiformis from Mus that included E. apionodes identified in other rodent host species (Apodemus spp., Myodes glareolus, and Microtus arvalis). A lack of internal resolution in these clades could suggest the existence of a species complex with a wide host range infecting murid and cricetid rodents. We question, however, the power of COI and 18S markers to provide adequate resolution for assessing host specificity. In addition to the rarely used marker ORF470 from the apicoplast genome, we present multilocus genotyping as an alternative approach. Phylogenetic analysis of 35 nuclear markers differentiated E. falciformis from house mice from isolates from Apodemus hosts. Isolates of E. vermiformis from Mus are still found in clusters interspersed with non‐Mus isolates, even with this high‐resolution data.
• In conclusion, we show that species‐level resolution should not be assumed for COI and 18S markers in coccidia. Host–parasite cospeciation at shallow phylogenetic nodes, as well as contemporary coccidian host ranges more generally, is still open questions that need to be addressed using novel genetic markers with higher resolution.

     

Phylogeny of the reptilian Eimeria: are Choleoeimeria and Acroeimeria valid generic names?

Reptiles are the animals with the most described coccidian species among all vertebrates. However, the co‐evolutionary relationships in this host–parasite system have been scarcely studied. Paperna & Landsberg (South African Journal of Zoology, 24, 1989, 345) proposed the independent evolutionary origin of the Eimeria‐like species isolated from reptiles based on morphological and developmental characteristics of their oocysts. Accordingly, they suggested the reclassification of these parasites in two new genera, Choleoeimeria and Acroeimeria. The validity of the genera proposed to classify reptilian Eimeria species remained unresolved due to the lack of species genetically characterized. In this study, we included 18S rRNA gene sequences from seven Eimeria‐like species isolated from five different lizard host families. The phylogenetic analyses confirmed the independent evolutionary origin of the Eimeria‐like species infecting lizards. Within this group, most species were placed into two monophyletic clades. One of them included the species with ellipsoidal oocysts (i.e. Choleoeimeria‐like oocysts), whereas the species with more spheroidal oocysts (i.e. Acroeimeria‐like oocysts) were included in the second one. This result supports the taxonomic validity of the genera Acroeimeria and Choleoeimeria.     

Purification of Eimeria Sporozoites by DE-52 Anion Exchange Chromatography

An anion exchange column of DE-52 has been used to purify Eimeria sporozoites from a post-excystation mixture of oocysts, oocyst shells, sporocysts, sporocyst shells, and sporozoites. The mean recovery from several experiments was 94% and virtually all non-sporozoite material was removed. Infectivity studies in vitro with sporozoites showed that they were viable after purification and were at least as infectious as the unpurified sporozoites; furthermore, oocysts in the crude preparation could be recovered from the DE-52 cellulose by resuspending them in a 20% (w/v) sodium chloride solution.     

True Intermediate Hosts for Eimeria funduli (Apicomplexa) from Estuarine Fishes1

Grass shrimp (Palaemonetes pugio) fed liver containing sporulated oocysts of Eimeria funduli permitted development of sporozoites that became infective to a variety of killifishes. The shrimp's gastric mill mechanically ruptured the oocysts. Sporozoites then excysted through an opening in the sporocyst, and by 12 and 13 h postinfection (p.i.) numerous empty sporocysts and free sporozoites occurred extracellularly in the intestine of the grass shrimp. Even at 5, 7, 8, 11, 46, 79, and 83 days p.i., and presumably for many months, numerous sporozoites still occurred free in the alimentary tract or between intestinal cells. The coccidium did not infect killifish at either 2 or 4 days p.i., but did at 5 days; after release from the sporocyst, it became more elongate with a distinct nucleus and two relatively large refractile bodies. Infections of E. funduli resulted in about one half of the fish that were fed either entire hepatopancreas or tips of hepatopancreas from experimentally infected shrimp. Feeding either the entire alimentary tract proximal to the first abdominal segment or any portion of that section from experimentally infected shrimp produced infections in nearly all tested fish. Feeding portions of the cephalothorax without any attached hepatopancreas or alimentary tract failed to produce an infection. Feeding killifish with wild grass shrimp from an enzootic area produced infections in only a fourth of the fish sample; however, feeding experimentally infected wild, laboratory-reared, and juvenile grass shrimp produced infections in nearly all fish. Palaemonid shrimps other than P. pugio also can serve as intermediate hosts for E. funduli, and these shrimps include Palaemonetes vulgaris, P. paludosus, P. kadiakensis, and Macrobrachium ohione. In contrast, a penaeid shrimp, mysidacean, amphipod, and crab fed liver with sporulated oocysts did not produce infections when fed to killifish.     

The Coccidian Genus Calyptospora n. g. and Family Calyptosporidae n. fam. (Apicomplexa), with Members Infecting Primarily Fishes1

Calyptospora n. g. was erected for Eimeria funduli because the sporocyst of that species lacks Stieda and sub-Stieda bodies, has a veil supported by sporopodia, and has an anterior apical opening. A suture may be present, but it does not completely divide the sporocyst into two valves. Because C. funduli has an obligatory invertebrate intermediate host, we established Calyptosporidae n. fam. to accommodate Calyptospora and tentatively to accept Goussia. A new subgenus, Plagula, is erected for species of Goussia with a sporocystic veil not supported by sporopodia. We consider the family more primitive than Eimeriidae, Sarcocystidae, and possibly Lankesterellidae.     

Optimized excystation protocol for ruminant Eimeria bovis- and Eimeria arloingi-sporulated oocysts and first 3D holotomographic microscopy analysis of differing sporozoite egress

Successful excystation of sporulated Eimeria spp. oocysts is an important step to acquire large numbers of viable sporozoites for molecular, biochemical, immunological and in vitro experiments for detailed studies on complex host cell-parasite interactions. An improved method for excystation of sporulated oocysts and collection of infective E. bovis- and E. arloingi-sporozoites is here described. Eimeria spp. oocysts were treated for at least 20 h with sterile 0.02 M _L-cysteine HCl/0.2 M NaHCO₃ solution at 37 °C in 100% CO₂ atmosphere. The last oocyst treatment was performed with a 0.4% trypsin 8% sterile bovine bile excystation solution, which disrupted oocyst walls with consequent activation of sporozoites within oocyst circumplasm, thereby releasing up to 90% of sporozoites in approximately 2 h of incubation (37 °C) with a 1:3 (oocysts:sporozoites) ratio. Free-released sporozoites were filtered in order to remove rests of oocysts, sporocysts and non-sporulated oocysts. Furthermore, live cell imaging 3D holotomographic microscopy (Nanolive®) analysis allowed visualization of differing sporozoite egress strategies. Sporozoites of both species were up to 99% viable, highly motile, capable of active host cell invasion and further development into trophozoite- as well as macroment-development in primary bovine umbilical vein endothelial cells (BUVEC). Sporozoites obtained by this new excystation protocol were cleaner at the time point of exposure of BUVEC monolayers and thus benefiting from the non-activation status of these highly immunocompetent cells through debris. Alongside, this protocol improved former described methods by being is less expensive, faster, accessible for all labs with minimum equipment, and without requirement of neither expensive buffer solutions nor sophisticated instruments such as ultracentrifuges.     

The viability and survival of sporozoites of Eimeria in vitro

Some factors affecting excystation and viability of sporozoites of several species of Eimeria from chickens were examined in vitro. Chicken embryos or cultured kidney cells were inoculated with sporozoites in order to assess viability.Sporozoites of E. tenella survived in phosphate buffer (P.B.S.) containing 0·9 per cent NaCl for 14 days. Some sporozoites survived in solutions containing up to 16 per cent NaCl for 3 days at +4°C. Sporozoites of E. maxima and E. acervulina survived for only 27 h in phosphate buffer containing 1 or 2 per cent NaCl.Sporozoites of E. brunetti, E. maxima, and E. acervulina var: mivati were released rapidly from sporocysts in vitro, but survived for relatively short periods in PBS at 4°C. However, the addition of serum or gelatine to these solutions increased survival to at least 96 h.The viability of sporozoites after freezing and storing in liquid nitrogen was best when 12 per cent dimethyl sulphoxide (DMSO) was added to the sporozoite suspensions. P.B.S. with DMSO was less suitable than the other solutions used and serum or gelatine with the DMSO, was needed to increase survival. Increasing the density of sporozoites in the frozen stabilates did not increase survival.     

Morphological and molecular characterization of two Trichodina (Ciliophora,Peritrichia) species from freshwater fishes in China

In the present study, we provide morphological and molecular characterization of two Trichodina species, T. acuta Lom, 1970 and T. funduli Wellborn, 1967, isolated from koi (Cyprinus carpio) and loach (Paramisgurnus dabryanus), respectively. Morphological characters of the two Trichodina species were mainly investigated on the basis of dry silver nitrate-impregnated specimens. Both species are medium-sized and possess well-developed denticles comprising strongly sickle-shaped blades, well-developed central parts, and straight rays. Trichodina acuta can be easily distinguished from the other Trichodina species that possess a clear central circle by the well-developed sharp blade apophysis, and the gap between ray tip and central circle. Trichodina funduli is a poorly known species that is easily confused with T. heterodentata Duncan, 1977, however the latter species has thinner denticles. The small subunit ribosomal RNA gene sequences of Trichodina acuta and T. funduli were incorporated into phylogenetic analyses. Our findings suggest that the phylogenetic lineage of trichodinids might not correspond with their living environments, host species or even some morphological characteristics.     

Switch,disperse, repeat: host specificity is highly flexible in rodent-associated Eimeria

Interplay between conserved host specificity and occasional host switches is an important process determining the evolution of host-parasite systems. Here, we address the dynamics of host switches at the population level in rodent-associated Eimeria. Focusing mainly on two ecologically similar host groups, Murinae and Arvicolinae, we show that the Eimeria infecting those hosts form a complex system of many genetic lineages with different host specificities. The broad geographic distribution of lineages indicates that they are well-established genetic forms which retained their host specificities while spreading across large geographic areas. We also demonstrate that genetic structure is only partially reflected by morphological traits.     

Life cycle of Calyptospora funduli (Apicomplexa: Calyptosporidae)

The taxonomic status of the extraintestinal piscine coccidium Calyptospora funduli is based in part on its requirement of an intermediate host (the daggerblade grass shrimp Palaemonetes pugio). In the present study, grass shrimp fed livers of Gulf killifish (Fundulus grandis) infected with sporulated oocysts of C. funduli exhibited numerous sporozoites suspended in the intestinal contents when fresh squash preparations were examined by light microscopy. Using this method, sporozoites were not seen in intestinal epithelial cells of the grass shrimp or in any other cell types. Ultrastructural examination, however, revealed sporozoites in the cytoplasm of the gut basal cells. Cross-sections of 1-13 sporozoites were seen within a single cell, and those sporozoites each appeared to be situated in individual membrane-bound vesicles, rather than in a single parasitophorous vacuole. These ultrastructural observations indicate that in the grass shrimp intermediate host, sporozoites that develop into an infective stage probably undergo that development in gut mucosal basal cells. Prior studies revealed that these sporozoites modified their structure over 4-5 days and that before that time, they were not infective to the fish host. Following ingestion of an infected shrimp by a killifish, the infective sporozoites apparently reach the liver of their killifish definitive hosts through the bloodstream. Sporozoites were seen in blood smears from the longnose killifish, Fundulus similis, 4 hr after fish were fed experimentally infected grass shrimp. Additionally, coccidian trophozoites and early meronts were seen in hepatocytes from several longnose killifish at 48, 72, and 96 hr postinfection. This study, in conjunction with previous findings, clearly confirms that a true intermediate host is required in the life cycle of C. funduli, that a developmental period of about 5 days in grass shrimp is necessary for sporozoites to become infective to killifishes, and that sporozoites do occur intracellularly in gut basal cells of the grass shrimp.     

Eimeria species in the Pearson Island Rock Wallaby, Petrogale lateralis pearsoni

Four species of previously described Eimeria were identified from oocysts present in the faeces of Pearson Island rock wallabies, Petrogale lateralis pearsoni from Pearson Island, South Australia. Eimeria petrogale and Eimeria sharmani have been reported in P. lateralis; however, the presence of Eimeria godmani and Eimeria inornata expands the suite of eimerian species known in rock wallabies of the lateralis complex. These observations indicate that the host distribution of Eimeria species in rock wallabies is more diverse than previously thought, and supports the hypothesis that these coccidia have an ancient association with rock wallabies which persisted as the hosts radiated.     

Studies on the Motility of Plasmodium Sporozoites*

Albumin was found to have a striking stimulatory effect on motility of Plasmodium sporozoites, while serum globulins had an inhibitory effect. Albumin also preserved viability of sporozoites in vitro at 4 C for several days. P. berghei, P. cynomolgi, and P. falciparum sporozoites each had a distinct and characteristic type of motility. P. berghei sporozoites from oocysts had a different type of motility from that of salivary gland sporozoites, each type presumably associated with different invasive capacities at different times during the life cycle of the parasite. This change in sporozoite motility during development was also associated with other physiologic developmental changes in the sporozoite. The degree of motility of a given pool of sporozoites was to some degree associated with other parameters of metabolic activity of these sporozoites, i.e. infectivity, immunogenicity, and secretory activity. Secretions of the rhoptry-microneme complex may play a role in sporozoite motility.     

Sensitivity and Specificity of the Indirect Fluorescent Antibody Test in the Study of Four Murine Coccidia1

The sensitivity and specificity of the indirect fluorescent antibody (IFA) test for the detection of serum antibodies were examined in mice that were infected with Eimeria falciformis, E. ferrisi, E. papillata, or E. vermiformis. For the study of each species, five groups of mice were given graded inoculation doses of 10, 10², 10³, 10⁴, or 10⁵ sporulated oocysts in a primary infection. The sixth group was infected with three sequential doses of 1.5 times 10³, 1.5 times 10⁴, and 1.5 times 10⁵ sporulated oocysts per mouse at two- to three-week intervals. All groups of infected mice developed serum antibodies. Sera were titrated by the IFA test with purified sporozoites. Strong fluorescence and high IFA titers were observed with homologous reactions mainly with the sera from mice infected with the higher inoculation dose levels in primary infections and from those given three sequential inoculation doses. Immunological cross reaction among the four species of Eimeria occurred at dilutions of 1:10 to 1:160. Very weak or no fluorescence of free sporozoites was observed with sera from noninfected mice, and there was no fluorescence of sporozoites contained in intact sporocysts.     

Co‐evolutionary patterns in congeneric monogeneans: a review of Dactylogyrus species and their cyprinid hosts

Patterns associated with the evolution of parasite diversity, speciation and diversification were analysed using Dactylogyrus species (gill monogeneans) and their cyprinid hosts as a model. The aim of this study was to use this highly specific host–parasite systems to review: (1) the diversity and distribution of Dactylogyrus species, (2) the patterns of organization and structure of Dactylogyrus communities, (3) the evolution and determinants of host specificity and (4) the mode of Dactylogyrus speciation and co‐evolutionary patterns in this Dactylogyrus–cyprinid systems. Dactylogyrus are a highly diverse group of parasites, with their biogeography and distribution clearly linked to the evolutionary history of their cyprinid hosts. The coexistence of several Dactylogyrus species on one host is facilitated by increasing niche distances and the differing morphology of their reproductive organs. The positive interspecific and intraspecific interactions seem to be the most important factors determining the structure of Dactylogyrus communities. Host specificity is partially constrained by parasite phylogeny. Being a strict specialist is an ancestral character for Dactylogyrus, being the intermediate specialists or generalists are the derived characters. The evolution of attachment organ morphology is associated with both parasite phylogeny and host specificity. Considering larger and long‐lived hosts or hosts with several ecological characters as the measures of resource predictability, specialists with larger anchors occurred on larger or longer‐living fish species. Intra‐host speciation, a mode of speciation not often recorded in parasites, was observed in Dactylogyrus infecting sympatric cyprinids. Sister parasite species coexisting on the same host occupied niches that differed in at least one niche variable. Intra‐host speciation, however, was not observed in Dactylogyrus species of congeneric hosts from geographically isolated areas, which suggested association by descent and host‐switching events.     

Vital role for the Plasmodium actin capping protein (CP) beta‐subunit in motility of malaria sporozoites

Successful malaria transmission from the mosquito vector to the mammalian host depends crucially on active sporozoite motility. Sporozoite locomotion and host cell invasion are driven by the parasite's own actin/myosin motor. A unique feature of this motor machinery is the presence of very short subpellicular actin filaments. Therefore, F‐actin stabilizing proteins likely play a central role in parasite locomotion. Here, we investigated the role of the Plasmodium berghei actin capping protein (PbCP), an orthologue of the heterodimeric regulator of filament barbed end growth, by reverse genetics. Parasites containing a deletion of the CP beta‐subunit developed normally during the pathogenic erythrocytic cycle. However, due to reduced ookinete motility, mutant parasites form fewer oocysts and sporozoites in the Anopheles vector. These sporozoites display a vital deficiency in forward gliding motility and fail to colonize the mosquito salivary glands, resulting in complete attenuation of life cycle progression. Together, our results show that the CP beta‐subunit exerts an essential role in the insect vector before malaria transmission to the mammalian host. The vital role is restricted to fast locomotion, as displayed by Plasmodium sporozoites.     

The Challenge of Fish Coccidia1

Species of Eimeria occurring in fishes show important differences, especially in their life cycles, with respect to what is known for those forms found in mammals or other hosts. In some cases, at least, some small invertebrate may play the role of vector or intermediate host. Present studies of fish coccidia may throw light on the possible evolution of certain aspects of host-parasite interrelationships involving eimerian species in particular: for example, the earliest site (perhaps not the gut) of infection and even the “original” host group (perhaps invertebrates rather than vertebrates).     

Multivariate analyses of morphometrical features from Apatemon gracilis (Rudolphi, 1819) Szidat, 1928 and A. annuligerum (v. Nordmann, 1832) (Digenea: Strigeidae) metacercariae

Only two species of the strigeid subgenus Apatemon (Apatemon) sensu Sudarikov (1959), Apatemon gracilis (Rudolhi, 1819) and A. annuligerum (von Nordmann, 1832), are known to infect British fishes. A. annuligerum is poorly characterised, with few life-cycle data available, since only the adult and metacercarial stages have been described. Both of these life-stages demonstrate only minor morphological differences from those of A. gracilis. The multidimensional approach of principal components analysis (PCA) was employed to determine the intra- and inter-specific morphological variation that exists for metacercariae of these species. Analyses revealed that metacercarial morphometrics cannot confidently discriminate between Apatemon gracilis and A. annuligerum specimens. Indeed, levels of intra-specific variation present between A. gracilis metacercariae originating from different piscine hosts were as marked for certain populations as those between the two nominal species. Individual populations of A. gracilis metacercariae differed most in the relative dimensions of their internal organs, whereas variation between the two nominal species was primarily in length-related variables. A. gracilis specimens originating from Welsh and Scottish stoneloach represented two populations with the most comparable total body lengths, yet clustered separately. Conversely, A. gracilis metacercariae infecting rainbow trout and salmon parr differed markedly in size, but demonstrated minimal composite morphological variation. Much, if not all, of the variation described between populations and species of parasites can be attributed to host or to metacercarial location within a particular host. These findings and the lack of strong life-cycle data for A. annuligerum place some doubt on its validity as a species discrete from A. gracilis.