Development of Eimeria callospermophili and E. bilamellata from the Uinta Ground Squirrel Spermophilus armatus in Cultured Cells*

SYNOPSIS. Cell lines or established cell lines of bovine, ovine or human origin and primary cells from whole embryos of groundsquirrels were used in a study of the in vitro development of Eimeria callospermophili and E. bilamellata from the Uinta ground squirrel, Spermophilus armatus. Monolayers in Leighton tube cultures were inoculated with sporozoites of either of these 2 species and examined with phase-contrast microscopy at various intervals. After such examination, coverslips were fixed in Schaudinn's or Zenker's fluid and variously stained. E. callospermophi sporozoites penetrated cells and underwent development to mature 1st generation schizonts in most cell types. At different times after inoculation, both species formed sporozoite-shaped schizonts, which later became spheroidal. Intracellular movements of sporo zoite-shaped schizonts of E. callospermophili were observed and such schizonts penetrated cells when freed by mechanical disintegration of the host cells. Merozoites were formed at the periphery of the schizont in both species. Mature 1st generation schizonts of E. callospermophili, with 6–14 merozoites, were first seen 15 hr after inoculation; the corresponding values for E. bilamellata were 12–27 merozoites and 4 days. Merozoites of both had anterior and posterior refractile bodies. Exposure to a trypsin-bile solution stimulated motility in merozoites of E. callospermophili. Second generation trophozoites and immature schizonts of E. callospermophili were seen in cultures of primary cells of whole ground-squirrel embryos 20–24 hr and 44–48 hr, respectively, after inoculation of sporozoites.     

Plasmodium (Sauramoeba) pelaezi n. sp., a malaria parasite of the mexican iguanid lizardUrosaurus bicarinatus bicarinatus (Dumeril, 1856) (Sauria: Iguanidae)

A new Mexican species of saurian malaria parasite,Plasmodium (Sauramoeba) pelaezi, is described from the iguanid lizardUrosaurus bicarinatus bicarinatus. Two out of 12 specimens collected at Chila de la Sal (Puebla, México) were found infected. The species is characterized by round and oval gametocytes. Schizonts are mostly round with a single mass of pigment and with 16 merozoites in mature forms. Gametocytes cause shrinkage of infected cells and schizonts render the host cell nuclei spherical.     

Life Cycles of Leucocytozoon dubreuili Mathis and Leger, 1911 and L. fringillinarum Woodcock, 1910 (Haemosporidia: Leucocytozoidae)*

SYNOPSIS The development of Leucocytozoon dubreuili and L. fringillinarum was studied on successive days in simuliid and avian hosts. Sporogony of both parasites is completed in at least 5 species of sylvatic Simuliidae in a minimum of 4-5 days at 21 C. The pattern of development of the 2 species is similar but the size of the oocysts and the number of sporozoites differ. Sporozoites of L. dubreuili and L. fringillinarum were injected into uninfected robins (Turdus m. migratorius) and grackles (Quiscalus quiscula versicolor), respectively. Hepatic biopsies were performed on some of the injected birds. These and others were killed at intervals following inoculation and their tissues examined to detect stages of schizogony. Blood and macerated tissues from birds injected with sporozoites were transferred to uninfected birds to determine whether asexual stages would develop in the latter as a result of the inoculations. The 1st asexual cycle of L. dubreuili is completed in hepatic parenchymal cells in a minimum of 84 hr. Merozoites produced by the hepatic schizonts apparently follow one of 3 courses: invade hepatic parenchymal cells to initiate another cycle; penetrate blood cells and become gametocytes; penetrate tubular cells of the kidneys and grow into renal schizonts. The minimum prepatent period in infections with L. fringillinarum is 76 hr. The 1st asexual cycle occurs in hepatic parenchymal cells and in tubular cells of the kidney. A schizogonic cycle is completed in a minimum of 72 hr in the former and 96 hr in the kidney. Merozoites from the primary hepatic schizonts apparently give rise to (a) gametocytes; (b) secondary hepatic schizonts; (c) renal schizonts. Thus the schizogonic cycles of L. dubreuili and L. fringillinarum differ from each other and from those of L. simondi in ducks.     

TAXONOMIC STUDY OF BIGELOWIELLA LONGIFILA SP. NOV. (CHLORARACHNIOPHYTA) AND A TIME‐LAPSE VIDEO OBSERVATION OF THE UNIQUE MIGRATION OF AMOEBOID CELLS1

A new species of a chlorarachniophyte alga, Bigelowiella longifila sp. nov., is described. It is classified as a member of Bigelowiella as flagellate cells constitute the main stage of the life cycle. However, this alga is different from the only described species of the genus, B. natans Moestrup, in having a unique amoeboid stage in the life cycle. We observed an interesting behavior of amoeboid daughter cells after cell division: One of the two daughter cells inherits the long filopodium of the parental cell, and it subsequently transports its cell contents through the filopodium to develop at its opposite end. The other daughter cell forms a new filopodium. This unequal behavior of daughter cells may have evolved before the chlorarachniophytes and some colorless cercozoans diverged.     

Plasmodium (Huffia) hermani sp. n. from wild turkeys (Meleagris gallopavo) in Florida*

SYNOPSIS. Plasmodium (Huffia) hermani sp. n. is described from wild turkeys (Meleagris gallopavo Linnaeus) in Florida. It produces rounded schizonts with 6–14 nuclei arranged peripherally as a rosette and elongate, slender gametocytes with irregular margins. Asexual stages parasitize all cells in the erythrocyte series and, in heavy infections, predominantly occur in erythroblasts and their precursors. Presence and degree of pigmentation vary with maturity of the host cell. Gametocytes occupy erythrocytes only, with pigment dispersed in black granules throughout the cytoplasm. Cells containing schizonts are often rounded and enlarged and those parasitized by gametocytes may be somewhat distorted in shape by lateral hypertrophy. Host cell nuclei may be displaced, but are not distorted, except slightly by pressure from the parasite. Plasmodium hermani differs from P. (Giovannolaia) durae by producing low level (> 6%), nonlethal parasitemias in turkey poults, an absence of phanerozoites in capillary endothelium of the brain and viscera, and inability to infect chicks. Plasmodium hermani is more like P. (Huffia) elongatum in gametocyte morphology, schizogony in all types of erythrocyte precursors, with gametocytes occurring in erythrocytes only, and concentration of schizonts in heavy infections in bone marrow and spleen. It differs from P. elongatum by its lack of infectivity to passeriform and anseriform hosts and by a strong immune response which develops in infected birds.     

Delineation of the Genera Struvea Bonder and Phyllodictyon J. E. Gray (Cladophorales, Chlorophyta)

Plants of the stalked, net-forming green alga Struvea plumosa Sender, the type species of the genus Struvea, divide segregatively at every stage of their multicellular differentiation. The segregative process results in virtually simultaneous internal cleavage of the cytoplasts of parent axes or laterals into uniseriate series of nearly identically sized daughter cells m which intercalary cross-wall formation never takes place. Several branch orders result through a repeated process by which each daughter cell produces a pair of opposite protrusions at its distal end; the protruded arms subsequently undergo segregative divisions themselves after reaching a sufficient length. Struvea elegans Børgesen is seemingly the only other member of the genus in which the thallus divides by this segregative process. The remaining species appear to lack segregative cell division, their septation resulting from non-synchronous, centripetal wall ingrowths that divide parent cells into more or less equal halves. Intercalary cell divisions are common, this process being easily seen in the most widely distributed member of the genus, Struvea anastomosans (Harv.) Pice, et Grunov ex Pice. Phyllodictyon J. E. Gray, based on Phyllodictyon putcherrimum. is currently considered a synonym of Struvea but should be reinstated to accommodate those former species of Struvea that have Cladophoratype. as opposed to segregative, cell division. Although the two genera thus differ substantially in their modes of cytokinesis and are assumed to represent independent developmental lines, both Struvea and Phyllodictyon are assigned to the Cladophorales on the basis of molecular studies by others showing that recognition of the separate order Siphonocladales renders the Cladophorales paraphyletic.     

Observations on Haemogregarina balli sp. n. from the Common Snapping Turtle,Chelydra serpentina*

SYNOPSIS. Haemogregarina balli sp. n. is described from the blood and organs of the common snapping turtle Chelydra serpentina serpentina and from the gastric and intestinal ceca of the presumed invertebrate hosts, the leeches Placobdella parasitica and Placobdella ornata. In the peripheral blood of the turtle, male and female gametocytes and immature erythrocytic schizonts are found within erythrocytes. The maturation of erythrocytic schizonts containing 6–8 merozoites is recorded from liver imprints. Schizonts with 13–25 merozoites are found in various cells of the liver, lung and spleen. In the gastric ceca of the leeches the host erythrocytes are digested, releasing the gametocytes and immature erythrocytic schizonts. Immature erythrocytic schizonts degenerate. Association of the gametocytes occurs in the intestinal ceca. The microgametocyte apparently gives rise to 4 nonmotile microgametes, one of which fertilizes the macrogamete while the other remain as condensed, residual nuclei on the periphery of the developing oocyst. The oocyst increases in size with maturity. A mature oocyst produces 8 sporozoites from a single germinal center. Sporozoites liberated from the oocyst are found in the tissues of the leech. Transovarial transmission of the parasite does not occur in the turtle. Attempts at experimental transmission failed. Previously unfed (control) leeches were negative for the parasite. Haemogregarina balli is compared with other haemogregarines described from C. serpentina. Features of species of Haemogregarina and Hepatozoon as well as the taxonomy of these genera are discussed.     

A New Method for Intraerythrocytic Cultivation of Malaria Parasites (Plasmodium coatneyi and P. falciparum)

SYNOPSIS. Infected blood (from the rhesus monkey with P. coatneyi or an Aotus trivirgatus monkey with P. falciparum) was placed in a special vial equipped with an overflow tube 2–3 mm above the bottom of the vial. A slow flow (50 ml per day) of culture medium was maintained over the settled layer of red cells. In favorable experiments both species of parasites went thru 2 cycles of development without addition of fresh erythrocytes. so that late schizonts and young rings were present after 4 as well as after 2 days of incubation. There was, however, no increase in total parasite number.     

Segregative cell division and the cytoskeleton in two species of the genus Struvea (Cladophorales,Ulvophyceae, Chlorophyta)

The detailed segregative cell division (SCD) processes and changes in the arrangement of cortical microtubules and actin filaments were examined in two species of Struvea. SCD was initiated by the appearance of annular constrictions along the lateral side of a mother cell. The constrictions decreased in diameter, became thin, tubular in shape, and pinched the protoplasm of the mother cell into several protoplasmic sections. The protoplasmic sections expanded and developed into daughter cells, which appressed each other, and were arranged in a single row. Lateral branches protruded from the upper parts of the daughter cells. The protoplasm of the lateral branches was divided by secondary SCDs and was distributed amongst the new daughter cells. SCD and lateral branch formation were essential for morphogenesis in Struvea. Cortical microtubules were arranged parallel and longitudinally to the cell axis before SCD. When SCD was initiated, there was considerable undulation of the cortical microtubules and several transverse bundles appeared in the cytoplasmic zone where annular constrictions occurred. A microtubule‐disrupting drug (amiprophos methyl) inhibited SCD. Actin filaments maintained reticulate patterns before and during SCD. These results demonstrated that SCD in Struvea species was quite distinct from that in Dictyosphaeria cavernosa reported previously.     

Plasmodium spp. (Apicomplexa: Plasmodiidae) of the flying lizardDraco volans (Agamidae)

The agamid lizardDraco volans from Palawan, Republic of the Philippines, was found to be parasitised by threePlasmodium species:P. draconis n. sp.,P. volans n. sp. andP. vastator, Laird 1960. BothP. draconis andP. volans, but notP. vastator, were also present inD. volans from Sarawak. The species are readily distinguished by schizont size, merozoite number, and gametocyte size and shape.P. volans has schizonts approximately one-half the size of those ofP. draconis and produces 4–6 merozoites in comparison to 4–16 in the larger species. Gametocytes of both new species are predominantly oval in the Palawan sample and elongate in that from Sarawak.P. draconis gametocytes are approximately twice the size ofP. volans gametocytes, but on average no more than one-half the size of gametocytes ofP. vastator.     

Contributions to the Life Cycle of Peneroplis planatus (Fichtel and Moll) Montfort

Direct observations of schizonts and agamonts releasing megalospheres clarified the asexual phase of the life cycle of Peneroplis planatus and made it most probable that this species has a paratrimorphic life cycle. Specimens with maximum lengths between 837 and 3,503 μm released about 500 to 1,500 megalospheric juveniles, which possessed two chambers (proloculi and flexostyles) prior to emergence from the parental shell. The presence of gamonts was not shown and was only implied by the occurrence of the agamonts. Since agamonts and schizonts have been found from December to May and since asexual reproduction occurs in spring in Elat, sexual reproduction probably occurs at another time of year (June to December). More detailed studies of this species need to be conducted throughout the year to improve our knowledge of the life cycle of this species.     

Malaria Parasites of the “Borriguerro'’Lizard,Ameiva ameiva (Sauria: Teiidae) in Panama*

SYNOPSIS. The teiid lizard Ameiva ameiva praesignis in Panama is parasitized by 2 species of Plasmodium: P. cnemidophori Carini and P. diminutivum sp. n. The Panamanian strain of P. cnemidophori has thick macrogametocytes which are larger than the oval microgametocytes, and schizonts which contain 42–119 nuclei. P. diminutivum is characterized by round to oval gametocytes which are usually smaller than the host cell nucleus; fan-shaped schizonts with 4–6 nuclei; and prominently vacuolated young stages, with a thick band of chromatin along the periphery of the vacuole.     

Pre-Erythrocytic Development and Associated Host Responses to Haemoproteus meleagridis (Haemosporina: Haemoproteidae) in Experimentally Infected Domestic Turkeys12

ABSTRACT. Two generations of pre-erythrocytic schizogony occurred in skeletal and cardiac muscle of domestic turkeys infected with sporozoites of Haemoproteus meleagridis. First generation schizonts reached maturity approximately five days post-inoculation (DPI) and developed in capillary endothelial cells and myofibroblasts. The schizonts ranged from 12 to 20 μm in diameter and produced long (5–6 μm), slender merozoites. Early second generation schizonts were first detected in capillary endothelial cells between 5 and 8 DPI. They were cylindrical and ranged in size from 5 to 8 μm in diameter and up to 28 μm in length. Second generation schizonts which reached maturity by 17 DPI were surrounded by a thick, hyaline wall and were packed with numerous spherical merozoites less than 1 μm in diameter. Mature megaloschizonts were fusiform, ranged from 30 to 113 μm in diameter, and extended as much as 465 μm along the long axis of muscle fibers. Merozoites developed as buds from cytomeres that formed between 8 and 14 DPI. Infected turkeys developed a moderate to severe myositis within 5 DPI and were lame in one or both legs. The myositis was associated with the necrosis of scattered groups of muscle fibers. Muscle fibers surrounding mature megaloschizonts were swollen and hyaline. Megaloschizonts were surrounded occasionally by fibroblasts and infiltrates of mononuclear cells. The morphology and site of development of mature megaloschizonts of Haemoproteus meleagridis are contrasted with those of other avian haemosporidians.     

Haustorienbefall und inäquale Teilungen desNostoc-Phycobionten vonLempholemma botryosum (Lichinaceae)

In fully developed parts of thalli ofLempholemma botryosum a relatively high number of algal cells is attacked by haustoria. They differ specifically in form and effect from haustoria of otherLempholemma species. Cells of theNostoc phycobiont attacked by hyphae developing into haustoria may divide to give inequal besides equal daughter cells.

     

Ovicell structure in Callopora dumerilii and C. lineata (Bryozoa: Cheilostomatida)

Anatomical and SEM-studies of the brood-chambers (ovicells) in two bryozoans (Callopora dumerilii and C. lineata) were undertaken to resolve a long-term controversy existing in the literature about the origin of the ovicells. In contrast with the interpretation of Silén (1945 ), both species investigated possess hyperstomial ovicells with the ooecium formed by the distal (daughter) zooid. The ooecial coelomic cavity communicates with the zooidal coelom through a pore-like canal or canals remaining after the closure of an arch-shaped slit. The slit forms during ovicellogenesis. The communication canals are normally plugged by epithelial cells, however incompletely closed canals were also found in Callopora lineata. SEM-studies of noncleaned, air-dried specimens showed a relationship between membranous and calcified parts during early ovicellogenesis. It starts from a transverse wall as the calcification of the proximal part of the daughter zooid frontal wall, and has the shape of two flat rounded plates. There are no knobs or any other outgrowths. Conditions and phenomenology of hyperstomial ovicell formation are discussed.     

In vitro Development of First-Generation Schizonts of Eimeria canadensis (Bruce, 1921)*†

SYNOPSIS. In vitro development of Eimeria canadensis from cattle was studied in monolayer cultures of various bovine cell lines grown on coverslips in Leighton tubes. Excysted sporozoites were used for inoculation of the cell cultures. Sporozoites entered the host cells within a few minutes, but apart from a reduction in the number of refractile bodies, changed little in appearance during the first 9 days. Beginning at 91/2 days postinoculation, sporozoites developed into sporozoite-shaped schizonts or, less frequently, transformed into trophozoites. Sporozoite-shaped schizonts with as many as 8 nuclei were observed transforming into spheroid schizonts. At 111/2 days, intermediate schizonts had a characteristic single mass of refractile granules and 60–80 nuclei. Deep invaginations, which resulted in the formation of several blastophores, usually occurred when schizonts had about 100 nuclei. Merozoites were formed as a result of radial outgrowth from the surface of spheroid schizonts as well as of blastophores. Mature merozoites were seen 1st after 13 days.     

Prosthecobacter fusiformis nov. gen. et sp., the fusiform caulobacter

Four strains of heterotrophic, fusiform caulobacters have been isolated from freshwater sources. A single prostheca extends from one pole of mature cells, and cells attach to various substrata by means of a holdfast located at the distal tip of the appendage. Thus, superficially these bacteria bear a strong resemblance to bacteria in the genus Caulobacter. However, unlike Caulobacter these bacteria do not exhibit a dimorphic life cycle of motile, non-stalked daughter cells and immotile, stalked mother cells. Instead both mother and daughter cells are immotile, and at the time of cell separation the daughter cells are essentially identical mirror-image replicas of the mother cell. In addition, the prosthecae of these fusiform caulobacters do not have crossbands, they are somewhat wider than the stalks of Caulobacter and the pseudostalks of Asticcacaulis, and they terminate in a bulbous tip. The deoxyribonucleic acid (DNA) base composition ranges from 54.6–60.1, well below the 62–67 range for the genus Caulobacter. Based upon these and other differences, a new genus and species, Prosthecobacter fusiformis, is proposed for the fusiform caulobacters.     

The malarial parasites of Anolis species (Sauria: Iguanidae) in Panama

Lizards of the iguanid genus Anolis in Panama are parasitized by four species of Plasmodium. P.floridense occurs in A. limifrons, A. biporcatus, A. pentaprion and A. frenatus. The number of nuclei in mature schizonts is influenced by host species as is gametocyte shape but not gametocyte size. P. tropiduri parasitizes A. limifrons, A. pentaprion, A. biporcatus, A. frenatus, A. lionotus and A. poecilopus. The number of nuclei in schizonts varies according to host and type of blood cell parasitized. Gametocyte size varies slightly by host but shape remains relatively constant. Position of the parasite in the host cell may be affected by host species. Both schizonts and gametocytes are produced in white cells of all host species studied. P. balli occurs in A. limifrons, A. lionotus and A. poecilopus. Schizont and gametocyte size and shape are affected by host and blood cell type, with the parasite exhibiting different preferences for various blood cells in each host. P. minasense parasitizes A. limifrons, A. frenatus and A. capito, but its diagnosis in the last species may not be correct as gametocyte size and distribution of pigment differ considerably from other hosts. The mean number of nuclei in schizonts is not greatly affected by the host species. P. floridense is postulated to be of middle American origin in the genus Anolis, and to have reached Florida through the Caribbean.     

In Vitro Cultivation of the Vascular Phase of Sarcocystis singaporensis

To establish an in vitro culture system for the precystic phase of Sarcocystis singaporensis, we initially tested various excysting fluids for sporocysts. An excysting fluid containing 2.5% bovine taurocholate and 10% bile of the specific intermediate host, Rattus norvegicus, in RPMI medium was the most suitable resulting in excystation of 80% of the sporozoites. Subsequently, we identified brain endothelial cells and pneumonocytes of the rat to promote growth of sporozoites to schizonts. Hepatoma, fibroblastic, or myoblastic cells were not suitable for the parasite's development. First-generation schizonts were seen at days 3-10 postinoculation (PI); a distinct second peak of schizogonic development only occurred in endothelial cells at days 14-18 PI. First-generation schizonts were 26.0 (± 3.8) μm in diameter and contained 32-50 merozoites, second-generation schizonts measured 34.4 (± 10.6) μm and contained 54-72 merozoites. Merozoite yield at large-scale culture conditions (75 cm² flasks) using pneumonocytes as host cells was relatively low. Ultrastructurally, sporozoites and merozoites were quite similar to corresponding stages of other Sarcocystis species. With regard to host cell specificity and developmental kinetics, in vitro cultivation showed close similarities to the situation in vivo.