Axenic Cultivation of Phytomonas davidi Lafont (Trypanosomatidae), a Symbiote of Laticiferous Plants (Euphorbiaceae)*

SYNOPSIS. Phytomonas davidi (Trypanosomatidae) originally discovered by Lafont in 1909 on the island of Mauritius was rediscovered in Euphorbia cyathophora in Florida. Successful cultures were established in diphasic medium consisting of duck blood agar and modified Phillips’medium as overlay. Optimal growth was obtained when Mansour's medium was used as overlay and poorest growth when Cowperthwaite's medium buffered at pH 5.0 was utilized for this purpose. Marked changes tending toward choanomastigotes rather than the elongate twisted promastigotes were observed in cultures.     

Axenic Cultivation of Phytomonas (Trypanosomatidae) Associated with Laticiferous Plants in Suriname1

Using variously enriched media, axenic cultures were obtained of Phytomonas spp. associated with Allamanda cathartica, Mandevilla scabra, and Rhabdadenia biflora (Apocynaceae), and Blepharodon nitidus (Asdepiadaceae) and Euphorbia hirta (Euphor-biaceae). Cultures were usually subcultured once every week for many weeks in succession. Attempts to grow the flagellates from Bonafousia tetrastachya (Apocynaceae), Cecropia palmata (Cecropiaceae), and Euphorbia hyssopifolia (Euphorbiaceae) were unsuccessful. The “phytomonads” in latex of different host plants showed morphological differences, whereas the cultured flagellates were similar in morphology whatever the host plant source or culture medium.     

The cell surface of Phytomonas davidi

Carbohydrates were located on the surface of Phytomonas davidi using ultrastructural cytochemistry, and agglutination induced by lectins which bind to residues of mannose, N-acetylglucosamine, galactose, N-acetylgalactosamine, fucose and sialic acid. The surface charge of the cells was analysed by the binding of cationic particles (colloidal iron and cationized ferritin) to the cell surface and by cell electrophoretic mobility (EPM). Based on observations of binding of cationic particles to the cell surface; a decrease in the binding of these particles to the cell surface; a decrease in the mean EPM of the cells after their incubation in the presence of neuraminidase; and detection of N-acetylneuraminic acid by paper and gas-liquid chromatography, it was concluded that sialic acid residues are exposed on the surface of P. davidi. These residues may be glycolipids or are masked on the cell surface since only after brief trypsinization were the cells agglutinated by the lectin from Limulus polyphemus.     

A PCR-based survey on Phytomonas (Euglenozoa: Trypanosomatidae) in phytophagous hemipterans of the Amazon region

ABSTRACT. We have surveyed 244 hemipterans from Western Brazilian Amazônia for the presence of trypanosomatids and identification of members of the genus Phytomonas. Examination by phase microscopy of squashes of insect salivary glands (SG) and digestive tubes (DT) revealed that 44% (108/244) of insects from seven families harbored trypanosomatids. Infections were 5 times more frequent in Coreidae than in all other families together. Smears of SG and DT of the dissected insects were fixed on glass slides with methanol and stained with Giemsa for morphological analysis. DNA was recovered from these preparations and submitted to a PCR assay that permitted amplification of all trypanosomatid genera using primers of conserved sequences flanking a segment of the spliced leader (SL) gene. Upon PCR amplification of the recovered DNA, amplicons were hybridized with an oligonucletide probe (SL3′) complementary to a SL intron sequence specific for flagellates of the genus Phytomonas. Among the trypanosomatid‐positive insects, 38.8% harbored Phytomonas spp., corresponding to an overall Phytomonas prevalence of 17.1% among phytophagous bugs, their putative vectors. Since many Phytomonas are pathogenic in plants, this high prevalence in their vectors emphasizes the permanent risk of exposure to disease by native and cultured plants of the Amazon region.     

The Catabolism of Tryptophan to Indole-3-Ethanol by Crithidia fasciculata and Phytomonas davidi1

Crithidia fasciculata and Phytomonas davidi catabolize tryptophan (TRP) to indole-3-ethanol, which was identified by both thin layer and gas chromatography. The catabolic pathway involved in this metabolic conversion is suggested to be similar to that proposed for other members of the family Trypanosomatidae. Although this catabolism occurs at both 25° and 37°C, the catabolic rate is greater at 37°C, a non-permissive growth temperature. Conditions that inhibit protein synthesis would appear to favor the catabolism of tryptophan to indole-3-ethanol. The possible importance of this catabolic pathway to these organisms is discussed.     

Axenic Cultivation and Ultrastructural Study of a Phytomonas sp. Isolated from the Milkweed Plant Euphorbia hyssopifolia1

ABSTRACT. A large number of trypanosomatids was seen in the latex of the milkweed plant Euphorbia hyssopifolia found in Rio de Janeiro, Brazil. The parasites, which belong to the genus Phytomonas, were isolated and axenically cultivated in a biphasic culture medium. The form and the structural organization of the parasites as found in the intact plant, in the isolated latex, and in the culture medium was studied by scanning and transmission electron microscopy.     

An Ultrastructural Study of Basommatophoran Spermatozoa (Mollusca, Gastropoda)

Spermatozoa of the basommatophoran pulmonate families Ellobiidae ( Ophicardelus ornatus Ferussac), Amphibolidae ( Salinator fragilis Lamarck, Salinator solida von Martens), Siphonariidae ( Siphonaria funiculata Reeve) and Lymnaeidae ( Lymnaea lessoni (Deshayes)) were studied using transmission electron microscopy. Spermatozoa of all species, like most euthyneuran spermatozoa, possess ( 1 ) an acrosome composed of an apical vesicle and acrosomal pedestal (many differences between families), (2) a helically keeled, posteriorly invaginated nucleus, ( 3 ) a midpiece composed of paracrystalline and matrix materials and a variable number of incorporated glycogen-filled helices (one in Salinator and Siphonaria , two or three in Lymnaea , three in Ophicardelus ) and (4) an axoneme associated with coarse fibres (periodically banded in "neck" region) and rows of intra-axonemal granules. The wide diversity of spermatozoon structure in the species studied, in particular the midpiece structure of Siphonaria (which resembles closely that of certain opisthobranchs) indicates that the Basommatophora may not represent a valid taxonomic unit. A comparison of basommatophoran sperm with other euthyneurans and with euspermatozoa of prosobranchs is given.     

Transmission of the Trypanosomatid Flagellate Phytomonas davidi,a Symbiont of the Euphorbiaceae,by the Hemipteran Bug Pachybrachius bilobata scutellatus1

ABSTRACT. Phytomonas davidi is found in the latex of species of the plant family Euphorbiaceae, including Chamaesyce hirta and C. hyssopifolia, to latitude 32°N in the southeastern United States. The hemipteran Pachybrachius bilobata scutellatus (family Lygaeidae) serves as an agent of transmission from plant to plant. Heavy infections of flagellates in the salivary glands were observed; trypanosomatids were less than one-half the size seen in the plants and in the gut of the bug.     

An Ultrastructural Study of Lentomonas applanatum (Preisig) N. G. (Euglenida)

ABSTRACT. Lentomonas applanatum (syn. Entosiphon applanatum Preisig) is a biflagellate, phagotrophic euglenid found in intertidal salt marshes. Lentomonas applanatum bears a superficial similarity to Entosiphon sulcatum , however, an ultrastructural study of L. applanatum revealed many features that are atypical for other described species of the genus Entosiphon . These features include number and organization of pellicular strips, construction of the feeding apparatus, nature of the flagellar transition zone and flagellar apparatus, and point of flagellar emergence. These differences show that L. applanatum is related more closely to phagotrophic genera such as Ploeotia than to E. sulcatum . The construction of the feeding apparatus and pellicle suggest that L. applanatum has retained many of the pleisiomorphic characters that were present in the earliest euglenids. The presence of similar structures in other related protists may provide important clues as to the evolution of the Euglenida.     

An Ultrastructural Study of the Extruded Polar Tube of Anncaliia algerae (Microsporidia)

All microsporidia share a unique, extracellular spore stage, containing the infective sporoplasm and the apparatus for initiating infection. The polar filament/polar tube when exiting the spore transports the sporoplasm through it into a host cell. While universal, these structures and processes have been enigmatic. This study utilized several types of microscopy, describing and extending our understanding of these structures and their functions. Cryogenically preserved polar tubes vary in diameter from 155 to over 200 nm, noticeably larger than fixed‐sectioned or negatively stained samples. The polar tube surface is pleated and covered with fine fibrillar material that projects from the surface and is organized in clusters or tufts. These fibrils may be the sites of glycoproteins providing protection and aiding infectivity. The polar tube surface is ridged with 5–6 nm spacing between ridges, enabling the polar tube to rapidly increase its diameter to facilitate the passage of the various cargo including cylinders, sacs or vesicles filled with particulate material and the intact sporoplasm containing a diplokaryon. The lumen of the tube is lined with a membrane that facilitates this passage. Careful examination of the terminus of the tube indicates that it has a closed tip where the membranes for the terminal sac are located.     

傅氏凤尾蕨精子发生超微结构的研究

超微结构研究显示傅氏凤尾蕨（Pteris fauriei Hieron）精子发生过程包括生毛体、多层结构和鞭毛等运动细胞器重新发生,环状线粒体形成,核塑形等过程,最后形成一个螺旋形的游动精子,这与其他真蕨类精子发生过程相似。本研究观察到的一些新现象包括：精细胞在分化早期呈极性,细胞核位于精细胞的近极端,生毛体、线粒体和质体等细胞器主要分布远极端;在生毛体分化早期,可见大量微管从其发出,其周围线粒体丰富;基体分化经历了前中心粒、中心粒和基体3个阶段,它们的内部结构不同;研究表明生毛体内的不定形物质是微管组织者,多层结构、附属微管带及鞭毛等细胞器均由不定形物质分化形成;精细胞在分化过程中产生了丰富的膜结构,它们可能为精核塑形提供原料。本研究报道了傅氏凤尾蕨精细胞分化的一些细节,这有助于进一步揭示蕨类植物精子发生的细胞学机制。     

Cell fusion in Leishmania (Kinetoplastida, Trypanosomatidae)

Cell fusion in Leishmania infantum and L. tropica was recorded by means of a camera and a video system. Promastigotes were obtained from cultures in both cases. Fusion began with attachment of the posterior extremities of two ovoid flagellates. Complete fusion, with disappearance of adjacent cell membranes took about five minutes. In parallel, the flagella appear larger and shorter. These observations support the hypothesis of genetic exchanges suggested by analysis of DNA and enzymatic proteins.     

An Ultrastructural Study on the Sertoli Cell in the Newt (Trituroides hongkongensis)

An electron microscopical study was made on the Sertoli cell in testicular lobules carrying mature spermatozoa in the newt, Trituroides hongkongensis. The ultrastructural features suggested that the Sertoli cells in newt might be capable of performing three possible functions: one, as a support for spermatozoa; two, carrying out phagocytosis; and thirdly, facilitate the liberation of spermatozoa.     

Trypanosomatidae: Phytomonas detection in plants and phytophagous insects by PCR amplification of a genus-specific sequence of the spliced leader gene

In this paper we describe a method for the detection of Phytomonas spp. from plants and phytophagous insects using the PCR technique by targeting a genus-specific sequence of the spliced leader (SL) gene. PCR amplification of DNA from 48 plant and insect isolates previously classified as Phytomonas by morphological, biochemical, and molecular criteria resulted in all cases in a 100-bp fragment that hybridized with the Phytomonas-specific spliced leader-derived probe SL3'. Moreover, this Phytomonas-specific PCR could also detect Phytomonas spp. in crude preparations of naturally infected plants and insects. This method shows no reaction with any other trypanosomatid genera or with plant and insect host DNA, revealing it to be able to detect Phytomonas spp. from fruit, latex, or phloem of various host plants as well as from salivary glands and digestive tubes of several species of insect hosts. Results demonstrated that SLPCR is a simple, fast, specific, and sensitive method that can be applied to the diagnosis of Phytomonas among cultured trypanosomatids and directly in plants and putative vector insects. Therefore, the method was shown to be a very specific and sensitive tool for diagnosis of Phytomonas without the need for isolation, culture, and DNA extraction of flagellates, a feature that is very convenient for practical and epidemiological purposes.     

Ultrastructural Studies on the Trypanosomatid Phytomonas serpens in the Salivary Glands of a Phytophagous Hemipteran

Electron microscopy of salivary glands of the phytophagous hemipteran Phihia picta infected with Phytomonas serpens revealed the presence of flagellates in the gland periphery beneath the gland envelope, in the gland central lumen, between gland cells in the intercellular space and inside the gland cells. In the latter case, flagellates were found in the cytoplasm whether or not it was surrounded by a vacuolar membrane. Flagellates were always of the promastigote type, sometimes displaying a large twisted body. Morphological peculiarities of flagellates in different gland locations are recorded.     

Penile Lentiginosis. An Ultrastructural Study

This study on five patients has revealed more extensive alterations to melanocytes than previously reported, and emphasizes the fact that depigmentation is an essential element of the condition. In hyperpigmented areas, melanocytes were increased in number along the basal layer of the epithelium, were hyperactive, and in some cases contained bizarre melanosomes. In two cases there was suggestion of a defect in melanosome transfer to keratinocytes. Lymphocytes were closely apposed to melanocytes, and, in hypopigmented areas, were clearly involved in their disintegration. In depigmented areas, there was complete absence of melanocytes and of melanosomes in keratinocytes, and lymphocytes were present in the basal layer. In general, the appearances did not resemble melanoma in situ with spontaneous regression, although a second biopsy of one patient after one year did reveal features of melanocytes suggestive of an early stage of this condition. The study has provided no clear information as to the initial cause of the condition, but the manner of destruction of melanocytes suggests an immune reaction. Neither has it been of assistance in suggesting a more precise name for it.     

Host-Seeking Behavior of Selected Chrysops Species (Diptera: Tabanidae) Harboring Trypanosomatidae (Kinetoplastida)

The host-seeking behavior of hematophagous arthropods can be altered by symbiotes, thereby biasing sampling techniques and inaccurately reflecting symbiote or vector prevalence. Knowledge of any altered vector behavior is essential in vector control and monitoring. Species of Chrysops are vectors of human and animal pathogens. Six species of Chrysops were collected at two locations in South Carolina to determine if diurnal host-seeking behavior was influenced by trypanosomatid infection. Fifty-five percent of the host-seeking Chrysops were infected with trypanosomatid parasites. Prevalence of infection in host-seeking Chrysops were statistically indistinguishable during both the morning and evening at both sites. The results indicate that the prevalence of parasites among wild host-seeking Chrysops might not be influenced by infection status.     

An Ultrastructural Study of Pollen Grains Consumed by Larvae of Osmia Bees (Hymenoptera,Megachilidae)

Pollen samples from provisions and faeces found in the nests of four Osmia species were analyzed by SEM and TEM. The pollen grains studied were Cistus, Sonchus type oleraceus. Primus type dulcis, and Quercus type ilex. The apertures of Cistus and Sonchus pollen stored in the provisions were slightly expanded, and the cytoplasm protruded through them. Conversely, Prunus and Quercus pollen grains showed hardly any signs of such apertural protrusions. Further, the cytoplasm of Cistus and Sonchus pollen (which have thin intines) was almost entirely lacking in the pollen grains recovered from faeces, while in the faecal pollen grains of Prunus and Quercus (with thick intines) the cytoplasm was much less modified. These preliminary results indicate that both the protrusion of the cytoplasm in the provisions and the thickness of the intine may play an important role in the digestion of pollen grains by Osmia bee larvae.     

An Ultrastructural Study of Mitosis in a Marine Dinoflagellate: Prorocentrum minimum1

An ultrastructural study on mitosis in the marine dinoflagellate Prorocentrum minimum is described. Early in mitosis the nuclear membrane invaginates in the area of the Golgi apparatus. Additional membrane-lined channels form within the nucleus as the Golgi apparatus separates and moves toward opposite spindle poles. Microtubules appear within the channels and make contact with distinct kinetochore-like structures on the cytoplasmic side of the channels adjacent to the site of chromosome attachment. By mid-mitosis two or three parallel channels dissect the nucleus perpendicular to the suture plane of the cell. Chromosome separation thus occurs perpendicular to the suture plane. An additional group of microtubules extends posteriorly from the flagellar apparatus towards the nucleus but has no apparent role in mitosis. Mitosis in P. minimum is compared to that of P. micans and to other dinoflagellates.