Ultrastructural and biochemical characterization of promastigote and cystic forms of Leptomonas wallacei n. sp. isolated from the intestine of its natural host Oncopeltus fasciatus (Hemiptera: Lygaeidae)

Promastigote forms of a trypanosomatid were isolated from the third and fourth ventricles of the midgut and from the hindgut of the phytophagous hemipteran Oncopeltus fasciatus. Some individuals had adhered to its anterior region, close to the flagellar pocket, or to the flagellum up to four rounded aflagellated forms known as straphangers cysts. Scanning electron microscopy revealed that the flagellated forms presented a twisted cell body and a long flagellum, and the cysts, smaller than the parental promastigote, had a nascent flagellum. Transmission electron microscopy showed that promastigotes were typical, while cystic forms were ovoid dense cells devoid of a cyst wall, but presenting a cell coat, a special subpellicular region limited by a membrane unit, and a condensed cytoplasm. The kinetoplast-DNA fibrils appeared as dense spots and the condensed chromatin was arranged in a labyrinthic structure. Desmosome-like structures, observed in the region of adhesion of the precystic forms to the parental promastigote, could explain how cysts remain attached to the mother cell during the encystation process. Release of membranes from the surface of promastigotes and cysts seems to be correlated with the condensation of the cytoplasm during encystment. Morphological and isozyme analyses indicated that this trypanosomatid belongs to the genus Leptomonas. The molecular karyotype of this isolate was compared with that of a strain of Leptomonas oncopelti obtained from Oncopeltus varicolor by contour-clamped homogeneous electric field (CHEF) electrophoresis and revealed similar DNA banding patterns between 2,200-825 Kb, but not in lower bands (825-225 Kb). This suggested that the isolate from O. fasciatus and that from O. varicolor were not identical. Based on our findings we are describing Leptomonas wallacei n. sp. for our isolate from O. fasciatus.     

An integrated morphological and molecular approach to a new species description in the Trypanosomatidae: the case of Leptomonas podlipaevi n. sp., a parasite of Boisea rubrolineata (Hemiptera: Rhopalidae)

Leptomonas podlipaevi n. sp., a new trypanosomatid species, is described herein based on light microscopic, ultrastructural, and molecular phylogenetic data. The organism is pleomorphic both in host and culture, with two predominant forms-a typical promastigote with a long flagellum and a shorter promastigote with a small or barely extending flagellum. Several spliced leader RNA repeat sequences obtained from the original cultures and the clonal lines representing two types of cells were all nearly identical. These sequences formed a tight cluster in the neighbor-joining tree well separated from other trypanosomatid species. Glyceraldehyde phosphate dehydrogenase gene sequences were determined for L. podlipaevi and 10 previously described trypanosomatid species. Molecular phylogenetic analysis has demonstrated that the new species is most closely related to Leptomonas seymouri and Leptomonas pyrrhocoris. The analysis has also highlighted the polyphyly of the genus Leptomonas.     

Interaction of Leptomonas wallacei with the intestinal tract of its natural host Oncopeltus fasciatus (Hemiptera: Lygaeidae)

While investigating the distribution of Leptomonas wallacei in the intestine of the insect host Oncopeltus fasciatus, promastigotes and cyst-like forms of L. wallacei were observed only in the midgut ventricles V(3) and V(4) and the hindgut. In video-microscopy, once contact had occurred, the parasites remained attached to the midgut epithelium. Scanning electron microscopy revealed the adhesion of flagellates and cyst-like forms to the midgut wall and to the rectal pads of the hindgut. Using transmission electron microscopy, we observed that adhesion occurred mainly between the flagellum and the perimicrovillar membranes secreted by the midgut epithelium. No modifications were observed either in the parasite or in the epithelial cells. In the hindgut, adhesion to the superficial wax layer of the epithelial cells of the rectal pads was via flagellum. Host cell morphology appeared unaffected by L. wallacei.     

Leptomonas jaculum (Leger, 1902) Woodcock 1914: a leptomonas or a blastocrithidia?

Flagellates Leptomonas jaculum, inhabiting the intestine of the water scorpion Nepa cinerea posses promastigote organization, typical of the genus Leptomonas. Nevertheless phylogenetic analysis of the 18S rRNA gene revealed that these trypanosomatids form a common phylogenetic clade with cyst-forming representatives of the genus Blastocrithidia. Morphological characters supporting the unity of the group Blastocrithidia + L. jaculum and the probability of including L. oncopelti in it are discussed.     

Ultrastructural study of the relations between Leptomonas oncopelti (Noguchi and Tilden), protozoa trypanosomatidae,and the rectal wall of adults of Oncopeltus fasciatus dallas,hemiptera lygaeidae

A laboratory colony of Oncopeltus fasciatus was found to be infected by Leptomonas oncopelti. The flagellates form a carpet attached to the cuticular intima of the rectal glands of adult bugs. The epithelial cells of these glands are characterized by infolded apical plasma membranes associated with mitochondria; the overlying cuticular intima shows endocuticular canals. The Leptomonas are attached by hemidesmosomes, located most often at the tip of the flagella. The Protozoa multiply by budding and the resultant straphangers cling to the parental flagellum. Adhesion of the flagellates to the cuticular lining is so strong that detaching flagellates carry with them the outer part of the epicuticle. Epicuticle repair presumably occurs through the endocuticular canals.     

Flagellar wave reversal in the kinetoplastid flagellate Crithidia oncopelti

Living Crithidia oncopelti cells swim through their environment by means of tip-to-base waves on their single flagellum. The cells are able to re-orient themselves by using a short burst of asymmetrical base-to-tip waves. All points on a flagellum are capable of initiating waves. Placing a population of cells in a medium of high viscosity initially produces a large number of organisms beating in the reverse mode. An individual cell has a random "switching" behavior. Viscosity affects the frequency of forward and reverse waves in different ways. The concentration of free Ca++ ions determines the direction of wave propagation in reactivated axonemes. Calmodulin may play a role in mediating the Ca++ dependence of wave direction.     

Cytochemical analysis at the fine-structural level of trypanosomatids stained with phosphotungstic acid.

The ethanolic phosphotungstic acid (PTA) technic was used to detect, at the fine-structural level, basic proteins in various developmental stages of pathogenic Trypanosoma cruzi, and nonpathogenic Herpetomonas samuelpessoai, Leptomonas samueli, and Crithidia deanei, trypanosomatids. Reactions were observed in the nucleus of all stages. In the kinetoplast of epimastigote and promastigote forms reactions were noted mainly at the periphery. In trypomastigotes and choanomastigotes forms, however, an intense reacion was observed thorughout the kinetoplast. Reactions were present in cytoplasmic vesicles related to protein storage in T. cruzi and in membrane-bounded peroxisome-like organelles of H. samuelpessoai, L. samueli and C. deanei. The network of filaments which forms the paraxial rod did not react. In the flagellum, reaction was noted only at the peripheral doublet microtubules. PTA reacts also with structures related to the junction between the flagellar and cell body membranes.     

Demonstration that Leptomonas pessoai Galvão,Oliveira, Carvalho & Veiga, 1970, is a Herpetomonas*

SYNOPSIS. Leptomonas pessoai from the reduviid hemipteron Zelus leucogrammus, was cloned. The original strain (ATCC 30252) and clones can differentiate from promastigote to opisthomastigote. Differentiation is faster at high temperature (37 C) and in a defined medium as compared with a complex medium. It is suggested that Leptomonas pessoai be designated Herpetomonas samuelpessoai.     

An electron microscopic study of the ultrastructure of microbial cells in extreme biotopes in situ

The ultrastructure of microbial cells was studied in situ in natural biotopes by high-resolution transmission electron microscopy using the known methods of cryofractography, thin sectioning, and the negative staining of total cell specimens, as well as new methods of the low-temperature fractionation of microbial cells (providing for the recovery of cells from natural sources and their concentration), the preparation of micromonoliths, and aimed electron microscopy. Among the natural biotopes studied were permafrost ground and oil sludge. Most of the microorganisms found in the 1- to 3-million-year-old permafrost ground represented resting forms (spores, cysts, and cyst-like cells with specific organo-mineral envelopes). Oil sludge older than 35 years contained bacteria of atypical morphology and ultrastructure, including various resting forms and ultramicrobacteria. The data obtained is indicative of considerable promise of high-resolution electron microscopy in studying microbial communities in situ.     

Cytochemical Analysis at the Fine-Structural Level of Trypanosomatids Stained with Phosphotungstic Acid*

SYNOPSIS The ethanolic phosphotungstic acid (PTA) technic was used to detect, at the fine-structural level, basic proteins in various developmental stages of pathogenic Trypanosoma cruzi, and nonpathogenic Herpetomonas samuelpessoai, Leptomonas samueli, and Crithidia deanei, trypanosomatids. Reactions were observed in the nucleus of all stages. In the kinetoplast of epimastigote and promastigote forms reactions were noted mainly at the periphery. In trypomastigotes and choanomastigotes forms, however, an intense reaction was observed throughout the kinetoplast. Reactions were present in cytoplasmic vesicles related to protein storage in T. cruzi and in membrane-bounded peroxisome-like organelles of H. samuelpessoai, L. samueli and C. deanei. The network of filaments which forms the paraxial rod did not react. In the flagellum, reaction was noted only at the peripheral doublet microtubules. PTA reacts also with structures related to the junction between the flagellar and cell body membranes.     

Electrophoretic Analysis of Endonuclease-Generated Fragments of k-DNA,of Esterase Isoenzymes,and of Surface Proteins as Aids for Species Identification of Insect Trypanosomatids1

In order to verify the applicability of biochemical methods for species identification of Trypanosomatidae, 13 species of monoxenic trypanosomatids plus the heteroxenous Trypanosoma cruzi were comparatively analyzed by three different biochemical methods. Insect trypanosomatids examined were: Crithidia acanthocephali, C. fasciculata (three varieties), C. luciliae luciliae, C. luciliae thermophila, C. deanei, C. oncopelti, Herpetomonas muscarum muscarum, H. megaseliae, H. samuelpessoai, H. mariadeanei, Leptomonas seymouri, L. collosoma, L. samueli, and Blastocrithidia culicis. Also included in the survey were aposymbiotic strains of C. deanei and C. oncopelti. Methods used were: electrophoretic profiling of endonuclease-generated fragments of k-DNA, esterase isoenzymes profiling, and polyacrylamide-gel electrophoresis (SDS-PAGE) of radioiodinated cell surface proteins. Interspecific but not intraspecific differences were detected by all three methods among the 13 monoxenic species examined. Thus, it is concluded that these methods can be successfully used, in addition to classical criteria, for species identification of insect trypanosomatids.     

Redescription of Flagellar Arrangement in the Duck Intestinal Flagellate, Cochlosoma anatis and Description of a New Species, Cochlosoma soricis N. Sp. from Shrews

Cochlosoma anatis Kotlán (Zoomastigophorea, Retortamonadida, Cochlosomidae), isolated from the large intestines of domestic Rouen ducks, and Cochlosoma soricis n. sp., isolated from the small intestines of shrews, were observed by light and scanning electron microscopy. In both organisms, a single flagellum inserted on the dorsal surface at the same level as the insertion of 4 other flagella on the ventral surface. The 4 ventro-lateral flagella emerged from the left side of the anterior attachment disk below the margin and just above the lateral groove which extended the length of the organism. A 6th flagellum emerged from the margin of the attachment disk. The proximal ends of the flagella formed a bundle with the distal ends becoming unraveled like a rope. During motility, the bundle portion extended straight out from the cell and the free ends of the flagella produced a whipping motion. In C. anatis , the dorsal surface was covered with knob-like lumps and small pits and the cells had an axostyle that emerged slightly to the right of the midline in the posterior 1/3 of the body. The axostylar tip was shorter and thicker than the flagella and in most cells it also had an irregular, knobby appearance. The irregular cell surface and axostyle were absent from C. soricis. The margin of the attachment disk curved toward the center and terminated in C. anatis as a straight edge while in C. soricis it continued as a spiral. Indentations in the mucosal brush border similar to those produced by Giardia , but distinctly belonging to Cochlosoma , were interpreted as points of attachment to the host.     

Redescription of flagellar arrangement in the duck intestinal flagellate, Cochlosoma anatis and description of a new species, Cochlosoma soricis n. sp. from shrews

Cochlosoma anatis Kotlán (Zoomastigophorea, Retortamonadida, Cochlosomidae), isolated from the large intestines of domestic Rouen ducks, and Cochlosoma soricis n. sp., isolated from the small intestines of shrews, were observed by light and scanning electron microscopy. In both organisms, a single flagellum inserted on the dorsal surface at the same level as the insertion of 4 other flagella on the ventral surface. The 4 ventro-lateral flagella emerged from the left side of the anterior attachment disk below the margin and just above the lateral groove which extended the length of the organism. A 6th flagellum emerged from the margin of the attachment disk. The proximal ends of the flagella formed a bundle with the distal ends becoming unraveled like a rope. During motility, the bundle portion extended straight out from the cell and the free ends of the flagella produced a whipping motion. In C. anatis, the dorsal surface was covered with knob-like lumps and small pits and the cells had an axostyle that emerged slightly to the right of the midline in the posterior 1/3 of the body. The axostylar tip was shorter and thicker than the flagella and in most cells it also had an irregular, knobby appearance. The irregular cell surface and axostyle were absent from C. soricis. The margin of the attachment disk curved toward the center and terminated in C. anatis as a straight edge while in C. soricis it continued as a spiral. Indentations in the mucosal brush border similar to those produced by Giardia, but distinctly belonging to Cochlosoma, were interpreted as points of attachment to the host.     

Morphology of Leishmania braziliensis: changes during reversible heat-induced transformation from promastigote to an ellipsoidal form

Leishmania braziliensis, growing axenically at 26 C and transferred to 34 C, changes within 3 hr from the long slender motile promastigote form to an ellipsoidal form with a nonmotile flagellum. This transformation is reversible for heat treatments of up to 12 hr. In this study we show by light microscopic measurements that the cells decrease in length and increase in diameter at constant volume. Quantitative morphometry of electron micrographs further demonstrates that: the distance between nucleus and kinetoplast decreases; the kinetoplast enlarges slightly; the distance between adjacent subpellicular microtubules decreases; and that after 3 hr of heat treatment there is no change in mitochondrial morphology, but after 6 hr of heat treatment the mitochondria lose their cristae and no longer possess a clearly defined double membrane. These observations are compared with the morphological changes that occur normally in the gut of a sandfly and in the in vivo transformation occurring during infection of the mammalian host and of macrophage cultures.     

Ultrastructural Differences Between Species of Trypanosomatids With and Without Endosymbionts1

Species of trypanosomatids without endosymbionts (Leptomonas seymouri, L. collosoma, L. samueli, Crithidia fasciculata, C. luciliae, C. acanthocephali, Herpetomonas megaseliae, H. mariadeanei, H. samuelpessoai, H. muscarum muscarum, Trypanosoma cruzi) and species of trypanosomatids with endosymbionts (Crithidia deanei, C. oncopelti, Blastocrithidia culicis) were comparatively studied by means of electron microscopy. Artificially aposymbiotic strains derived from species with symbiont were also included in the survey. Species with symbiont were found to differ in some ultrastructural aspects from the group of species without symbiont. Paraxial rods of flagella or intraflagellar structure were found exclusively in species without symbiont. Peripheral branching of mitochondria, accompanied by absence of subpellicular microtubules in sites where the mitochondrial branches are appressed to the cell membrane, were found exclusively in species with symbiont. Networks of kinetoplast DNA fibrils were found to be larger and looser in species with symbiont. Symbiont-free strains of species with symbiont retained the same morphological characteristics of their parental species.     

Extracellular cultivation and morphological characterization of amastigote-like forms of Leishmania panamensis and L. braziliensis

Two strains of the Leishmania braziliensis complex have been adapted to grow extracellularly at elevated temperature as amastigote-like forms in a cell-free medium. These parasites can be serially cultivated and maintained at 32 degrees C for L. panamensis (WR442; L. braziliensis panamensis) and at 28 degrees C for L. braziliensis (M5052; L. braziliensis braziliensis). Several observations are presented that the forms adapted at elevated temperature are amastigote-like. Morphologically, the amastigote-like organisms appear rounded to ovoid and are immotile and smaller than promastigotes; the flagellum of the amastigote-like forms does not extend beyond the flagellar pocket. In comparison, the promastigotes are very elongated, with a nucleus at mid-cell length and a very long flagellum. By electron microscopy, the short flagellum of the amastigote-like form is within a distended flagellar pocket; the 9 + 2 axonemal configuration is present but the paraxial rod is not observed. By contrast, the flagellum of the promastigote has a paraxial rod which extends from the axosome level. In addition, these amastigote-like forms of Leishmania are able to infect, to survive and to divide within the macrophage cell line J774.     

I. Extracellular Cultivation and Morphological Characterization of Amastigote-Like Forms of Leishmania panamensis and L. braziliensis

. Two strains of the Leishmania braziliensis complex have been adapted to grow extracellularly at elevated temperature as amastigote-like forms in a cell-free medium. These parasites can be serially cultivated and maintained at 32°C for L. panamensis (WR442; L. braziliensis panamensis ) and at 28°C for L. braziliensis (M5052; L. braziliensis braziliensis ). Several observations are presented that the forms adapted at elevated temperature are amastigote-like. Morphologically, the amastigote-like organisms appear rounded to ovoid and are immotile and smaller than promastigotes; the flagellum of the amastigote-like forms does not extend beyond the flagellar pocket. In comparison, the promastigotes are very elongated, with a nucleus at mid-cell length and a very long flagellum. By electron microscopy, the short flagellum of the amastigote-like form is within a distended flagellar pocket; the 9 + 2 axonemal configuration is present but the paraxial rod is not observed. By contrast, the flagellum of the promastigote has a paraxial rod which extends from the axosome level. In addition, these amastigote-like forms of Leishmania are able to infect, to survive and to divide within the macrophage cell line J774.     

Growth and Reproduction of Leptomonas oncopelti in the Milkweed Bug, Oncopeltus fasciatus

SYNOPSIS. A laboratory colony of Oncopeltus fasciatus was found to be infected with Leptomonas oncopelti. Inasmuch as the parasite is transmitted from parent to offspring an opportunity presented itself to study the biology and transmission of this parasite under controlled laboratory conditions. An apparatus for observing individual bugs was designed and the presence or absence of flagellates in the feces determined. Flagellates were not shed until the bugs became adults after which they appeared in every defecation. Dissection of infected bugs revealed that flagellates were not present in the rectum until adulthood. Further studies indicated that in the midgut of the insect there is a departure from binary fission to budding. The nucleus divides and one of the newly formed nuclei migrates toward a newly formed kinetoplast. Rarely there is still another kinetoplast/nucleus division. In the event the new axoneme grows within the cytoplasmic sheath of the parent flagellum, smaller organisms produced by unequal cytokinesis remain attached. If the axoneme grows free, the smaller daughter organisms become free-swimming. Passage into the rectum of the adult bugs causes a rounding up of all parasites although the leishmaniform organisms continue to divide. It is presumed that infection of clean bugs is accomplished by the ingestion of leishmaniform organisms through a common water source. The reason for the presence of flagellates in the rectum of the adult but not in the nymphal insect and the mechanism responsible for the change from binary to unequal fission are not known.     

An Electron Microscopic Study of the Bipolar Bodies in Crithidia oncopelti

SYNOPSIS. An electron microscopic study of the structure of the flagellate Crithidia oncopelti was carried out with particular reference to the nature of the bipolar body which occurs in the organism. Apart from the bipolar body, the fine structure of C. oncopelti is similar to that of the related flagellate, C. fasciculata. The bipolar body is typically a sausage-shaped organelle limited by 2 unit membranes. Outpouchings of these membranes into the cytoplasm of C. oncopelti were found, along with a constant absence of (a) ribosomes on the outer aspect of the external of the 2 membranes, (b) structures analogous to nuclear pores and (c) an internal structure analogous to a nucleolus. It is concluded that the balance of structural and biochemical evidence favors the idea that the bipolar body is a bacterial protoplast rather than a 2nd nucleus.