Accessory Filament Research Articles

First report of Neopestalotiopsis rosae causing leaf blight on Shatangju in southern China.

Shatangju (CitrusreticulataBlancocv.Shatangju) belongs to genus Citrus and was cultivated extensively in southern China. In April 2022, a leaf blight-like symptom (firstly brown spots appeared on infecting leaves, then these brown spots extended, finally the whole leaves displayed blight-like symptom) was observed on 5%~10% of Shatangju seedlings (around five hundreds in total) in an orchard located in Wuhan city, Hubei, China. Diseased leaves from three seedlings were collected and cut into pieces (0.2 to 0.5 cm). These pieces were surface-sterilized using 75% ethanol for 3 min, rinsed with sterile distilled water for several times, then placed on potato dextrose agar (PDA) and incubated at 26°C with 12-h light/dark cycle. Over 20 pieces plated, wherein 30% were identified as Colletotrichum fructicola, 60% as Neopestalotiopsis spp., and 10% developed saprophytes. C. fructicola was a known pathogen on citrus, thus Neopestalotiopsis spp. was further investigated. Eight single-conidium colonies of the Neopestalotiopsis spp. were obtained, wherein STJ-8 was chosen as a representative for further study. The average growth rate of STJ-8 was 15.1±0.5 mm/day (n=5). Fungal colonies produced white cottony mycelium with abundant black acervuli distributed in concentric rings 6-8 days after planting, which ranged from 342.3 to 710.5 μm in diameter (n=100). Conidia were fusoid, five cells, four septa with average dimensions of 25.36×5.47 μm (n=100). Basal and apical cells were hyaline, wherein three middle cells were brown with darker septa. The apical cell was cylindrical with two to three transparent accessory filaments (13.7 to 30.5 µm in length, n=80). Basal cell was conic with an appendage (4.1 to 8.8 µm in length, n=40). Partial sequences of internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF-1α), and β-tubulin (TUB2) were amplified with reported primers (White et al. 1990; Lee et al. 2006; Maharachchikumbura et al. 2014), sequenced, and submitted to GenBank (accession nos. ITS: OP236541; TEF-1α: OP250124; TUB2:OP263094). BLASTn results showed 100% identity with the corresponding sequences of Neopestalotiopsis rosae. A multilocus phylogenetic analysis showed STJ-8 was closest to N. rosae. Thus, STJ-8 was identified as N. rosae. Pathogenicity tests were performed on one-year-old Shatangju seedlings and detached primary leaves by inoculating needle-wounded leaves with seven days old 5-mm mycelial plugs/acervuli (about 5000 spores) of STJ-8. Control seedlings/leaves were inoculated with 5-mm PDA plugs/sterile water drops. All inoculated detached leaves were cultured at same the place with STJ-8 cultured, while inoculated seedlings were put in a growth chamber at 26°C under a 16-h light/dark cycle (60% humidity). Symptoms developed on all inoculated leaves (except healthy control) 2 and 4 days post-inoculation by mycelial plugs and acervuli, respectively. N. rosae was re-isolated from the inoculated leaves, confirming Koch's postulates. N. rosae has been reported to cause diseases on various plants worldwide (Rebollar-Alviter et al. 2020; Xavier et al. 2021; Lawrence et al. 2022). In China, N. rosae has been reported to cause leaf spot/blight on pecan and strawberry (Wu et al. 2021; Gao et al. 2022), which caused great loss on these crops. To our knowledge, this is the first report of N. rosae causing leaf disease on citrus. Our study is important for developing control strategies against N. rosae in future.

First Report of Pseudopestalotiopsis theae Causing Leaf Spot on Euonymus japonicus in China.

Euonymus japonicus Thunb., an evergreen shrub, is popular for landscaping in China. In 2021, leaf spot was observed on E. japonicus (about 150 trees) leaves with 40 to 50% disease incidence in Wanzhou urban forest (30°45'N; 108°27'E) of Chongqing, the infected plants were between 5 and 6 years old. The symptoms started to occur from June to July and approximately 30 to 40% of the leaves exhibited leaf spot symptoms from August to September. Initial symptoms appeared as yellow spots of 1.2 to 4.9 mm in diameter, and then expanded to become large and irregular lesions, having white center surrounded by a brown halo. Under humid conditions, black dots appeared in the central part of the spots. In later stage, split and fall of the tissues occurred from the infected spot. To identify the causal agent, infected tissues from 20 samples (from 5 trees) were cut into small pieces (5 mm2), surface-sterilized for 30 s in 75% ethanol and 3 minutes in 3% sodium hypochlorite, rinsed three times in sterile water, placed onto potato dextrose agar (PDA) amended with streptomycin sulfate (50 μg/ml) and incubated at 25°C in dark conditions. Purified eight fungal colonies were white with undulating margins and light cream on the reverse side, measuring 85 mm diameter after 7 days, dark brown to black conidiomata were irregularly scattered and Conidia were observed in 20 days old colonies. Conidia were spindle-shaped, 4.5 to 6.8 × 15.2 to 23.5 μm (n=50), with 4 diaphragms, the three median cells were light to dark brown and the two end cells were colorless. 1 to 3 accessory filaments (5.2 to 22.5 μm long) protrude from theapical cell while a short stalk (3.5 to 5.5 μm long) was attached to the basal cell, these morphological features suggested that the isolates were most likely Pestalotiopsis. sp. Eight colonies were confirmed to be identical based on morphological characteristics. For molecular identification, DNA was extracted from representative strains (YF-5, YF-13, YF-24). The internal transcribed spacer (ITS) region, β-tubulin (TUB2), the translation elongation factor-1 alpha gene (TEF1), genes were amplified using primers ITS5/ITS4, TUB2F/TUB2R, and EF-526F/EF-1567R, respectively (White et al.1990; Glass & Donaldson 1995; O'Donnell & Cigelnik 1997; Carbone &, Kohn.1999). The sequences were deposited in NCBI GenBank YF-24, [ITS; ON204233: TUB2; ON304156: TEF1; ON400075]: YF-5, [ITS; OP379570: TUB2; OP413495: TEF1; OP413496]: YF-13, [ITS; OP379589: TUB2; OP413494: TEF1; OP413497]. Which revealed a 95% similarity to the Ps. theae NTUCC 18-067 [ITS; MT322086: TUB2; MT321888: TEF1; MT321987] ex-type sequences. Based on morphology and multilocus phylogenetic analysis, representative strains were identified as Pseudopestalotiopsis theae. For Koch's postulates, wiped the leaves of six healthy plants of E. japonicus (two-year-old) grown in pots with sterile water, 10 μL of spore suspension (106 spores/ mL) was brushed on five leaves per plant (three plants in total) with a sterile brush, and the other three plants were treated with sterile water instead of spore suspension as control, the plants were placed in a greenhouse at 28°C and 95±1% relative humidity. Seven days after inoculation, brown lesions appeared, similar to those observed in infected plants. Black dots surrounded by a brown halo reappear on the lesions after 12 days, whereas control plants remained healthy. Ps. theae culture was re-isolated from the infected leaves and identified using morphological characteristics and DNA sequence analysis. To our knowledge, Ps. theae can cause diseases on tea plants and has been found in Japan, Thailand and China, this is the first report of leaf spot infection of E. japonicus caused by Ps. theae in China. This disease is reducing the ornamental value of E. japonicus. Our results will contribute to the prevention and cure of leaf spot disease in E. japonicus.

First report of leaf spot on Elaeagnus pungens caused by Neopestalotiopsis clavispora in China.

First report of leaf spot on Elaeagnus pungens caused by Neopestalotiopsis clavispora in China.

First Report of Pestalotiopsis microspora Causing Leaf Spot on Moyeam in China.

Ampelopsis grossedentata, commonly known as moyeam, has been widely used as health care herbal tea since it contains natural plant protein cream, 17 amino acids, 14 micronutrients and lots of functional flavonoid and provides a wide range of pharmaceutical functions such as antioxidant, anti-inflammatory, antitumor (Carneiro et al. 2021; Zhang et al. 2020). Moyeam is primarily produced in Zhangjiajie, stretching over the area from between 109'40 to 110'20E to between 28'52 to 29'48N, at 1300 to1890 meter above the sea level, with subtropical humid monsoon climate. Its economic value surpasses $1.25 billion in China (Liang et al. 2020). In July 2020, leaf spots were observed on some moyeam plants in Zhangjiajie. Initial spots were pinhead-sized with a yellow halo margin. The spots developed into light brown necrotic spots 6 to 8 mm in diameter, often with a dark brown margin. After 4 days of development, the spots enlarged and coalesced into irregular shape, frequently falling out and giving the leaves a tattered appearance. The infected plants eventually died with disease incidence ranging from 18 to 23%. This disease resulted in production losses of up to $1.7 million in 2020. One fungal isolate was isolated from the symptomatic leaves based on our previously published methods (Yi et al. 2019). Colonies on potato dextrose agar (PDA) were thick and villous with white at the front of the plate and yellowish at the back. After 1 week, the fungus produced conidia, which were spindle-shaped, straight or slightly curved, with 5 cells, 4-euseptates and 2-3 apical accessory filaments. Morphologically, the fungus was similar to Pestalotiopsis spp. Aerial hyphae with vigorous growth were collected for molecular identification. ITS nucleotide sequence of the rDNA and β-tubulin gene were amplified and sequenced with universal primers ITS1/ITS4 and self-designed primers based on β-tubulin gene conserved motif. BLAST searches against GenBank indicated that the ITS nucleotide sequence shared 99% similarity with that of P. microspora (MG808374.1) and the β-tubulin gene sequence shared 99% similarity with that of P. microspora (AF115396.1). Based on morphological and molecular characteristics, the fungus was identified as P. microspora. ITS and the β-tubulin nucleotide sequences were deposited in GenBank (accession no. MW350011 and MW816914). Pathogenicity tests were carried out with the following procedure. Three healthy moyeam seedlings were sprayed with a conidial suspension of 1 x106 conidia/ml while the other three seedlings were sprayed with distilled water as the controls. Plants were maintained in a greenhouse at 28±1°C. After one day of inoculation, symptoms identical to those in the field developed in the plants inoculated with the fungus. In contrast, no symptoms developed on the control plants. P. microspora has been reported to cause diseases in many crops in China. However, this is the first report of P. microspora causing leaf spot in moyeam in China. Identifying the pathogen causing the disease is important to the development of effective disease management strategies for control of this disease.

Occurrence of Neopestalotiopsis clavispora Causing Leaf Spot on Dendrobium officinale in China.

Dendrobium officinale Kimura L., an endangered orchid plant, is a rare and precious Chinese herb and widely used to prepare Chinese traditional medicine (Zheng et al. 2005). In August 2021, significant indications of an unknown leaf spot disease were observed on greenhouse-grown D. officinale in Yueqing of Wenzhou (28.39°N, 121.04°E), Zhejiang Province, China, the main producing location of this orchid plant. Approximately twenty percent of plants surveyed showed typical infection symptoms. Initially, the symptoms appeared as small, circular black spots. As the disease developed, the center of the lesions was sunken with a black border. To determine the causal agent, 10 symptomatic plant samples were collected and all pieces from symptomatic plant leaves were used for isolating pathogen. Tissues between healthy and necrotic area were cut into pieces (5 × 5 mm, n=10), disinfected with 10% sodium hypochlorite for 1 minute, rinsed 3 times with sterile water, and dried on sterile tissue. Samples were then placed on potato dextrose agar medium (PDA) for 1 piece per plate, and incubated at 25℃ in a dark biochemical incubator. After 3 days, hyphal tips growing from the disinfected tissues were individually transferred to new PDA plates and incubated at 25℃ in the dark. Twelve same fungal isolates were obtained from all symptomatic leave fragments, then DDO11 was chosen as a representative isolate for further study. The colonies showed white aerial mycelium after 5 days culture at 25°C on PDA. Black viscous acervuli appeared and scattered on the surface of the colony after 8-12 days culture. Conidia were spindle shape, five cells, four septa, average 29.3 × 8.5 μm (n = 30; length × width). The apical and basal cells were lighter in color, and most of them were hyaline. The middle three cells were darker in color, and mostly brown. There are 2 to 4 colorless and transparent unbranched accessory filaments at the top, 32.5 µm in average length, and the basal cell has a small appendage, 9.2 µm in average length, n=30. For fungal identification to species level, Internal transcribed spacer (ITS) region, β-tubulin gene (TUB2) and translation elongation factor-1α (TEF-1α) were amplified (Qiu et al. 2020), respectively. The ITS, TUB2 and TEF-1α gene sequences of the representative isolate DDO11 were deposited in NCBI GenBank nucleotide database with accession numbers OK631881, OK655895 and OK655896, respectively. BLASTn analysis respectively showed 100%, 100% and 99.6% nucleotide sequence identity with Neopestalotiopsis clavispora strain accessions MG729690, MG740736 and MH423940, which indicated that the pathogen belonged N. clavispora. A maximum-likelihood phylogenetic analysis based on multi-locus sequence (ITS, TUB2, and TEF-1α) using MEGA X showed the similar result (Kumar et al. 2018). To verify pathogenicity, thirty 1-year-old healthy D. officinale plants of cultivar Yandang1 were used for inoculation tests. Spores of N. clavispora DDO11 were produced on PDA for 7 days at 28°C and washed with sterile distilled water, and the concentrations were adjusted to 1 × 106 spores/ml using a hemocytometer. Fifteen surface disinfected healthy plants were inoculated by spraying the suspension (2 ml, 1 × 106 spores/ml) and covered with plastic bags for 24 h, and another 15 plants treated with sterile distilled water were used as control. The plants were placed in a humidified chamber (>95% relative humidity) at 25°C for 48 h after inoculation and kept in a growth chamber (Kiangnan, China) at 25°C with 12-h day/night cycle for 8 days (Cao et al. 2019). All inoculated leaves showed symptoms identical to those observed in the field. No disease occurred on the controls. The Neopestalotiopsis isolate was reisolated from the symptomatic leaves, and species identification was confirmed by the morphological and molecular method described above. N. clavispora has been reported to cause diseases on a variety of plants all over the world, such as strawberry (Gilardi et al. 2019), blue berry (Shi et al. 2021), Syzygium cumini (Banerjee et al. 2020), Macadamia (Qiu et al. 2020), and so on. To the best of our knowledge, this is the first report of N. clavispora causing leaf spot on D. officinale in China. This report will help us to recognize the leaf spot disease of D. officinale and establish a foundation for future studies on N. clavispora to address effective management strategies.

First Report of Neopestalotiopsis clavispora Causing Calyx and Receptacle Blight on Strawberry in China.

Strawberry (Fragaria × ananassa) is widely cultivated in Liaoning Province, the most important strawberry production area in China. In November 2020, extensive brown spotting on the calyces and receptacles and dark brown necrotic lesions on the leaves of 'YanLi' strawberry plants were observed in the greenhouse, College of Horticulture, in Shenyang Agriculture University of Liaoning Province, China. The incidence ranged from 15% to 20% in the greenhouse. As the disease progressed, the spots extended to cover the entire receptacles and entire leaves. Fresh samples (5 mm2) were cut from the margin of healthy and infected tissues. The isolation and purification of methods were as described previously (Obregón et al. 2018). Fungal isolations are suborbicular with irregular edges. The colonies showed white aerial mycelium at the early stage and formed concentric circles after 5 days culture at 28°C on PDA. Black viscous acervuli appeared and scattered on the surface of the colony after 7~10 days culture. Acervulus was 162.4 µm in average diameter, n= 30. Conidia were spindle shape, five cells, four septa, 7.0 µm (average width) and 24.0 µm (average length), n=30. The apical and basal cells are lighter in color, most of them are hyaline, the middle three cells are darker in color, mostly brown. There are 2 to 4 colorless and transparent unbranched accessory filaments at the top, 30.8 µm in average length, and the basal cell has a small appendage, 9.0 µm in average length, n=30 (Fig. 1c-g). For fungal identification to species level, genomic DNA was extracted by the 2% CTAB method. Internal transcribed spacer (ITS) region, β-tubulin gene (TUB2) and translation elongation factor-1α (TEF-1α) were amplified (Qiu et al. 2020), respectively. Purified PCR products were sequenced in single direction. BLASTn searches of GenBank, using the partial ITS (MW861730), β-tubulin (MW856302) and TEF-1α (MW861729) sequences of representative isolate NP102 as the queries, revealed 97%, 100% and 100% sequence identity, respectively, to Neopestalotiopsis clavispora strain WJ31 accessions MG729690, MG740736 and MG740759. The phylogenetic tree was constructed by MEGA X (Fig. 2) (Kumar et al. 2018). Pathogenicity assay was carried out by spraying a spore suspension (1 × 106 spores/ml) of 8-day-old culture of fungus on six healthy 'YanLi' strawberry plants. An additional six control plants were only sprayed with sterile distilled water, and the inoculated plants were maintained in a light incubator at 25 ± 1°C, 90% relative humidity, with a photoperiod of 12 h. Calyx and receptacle blight and leaf spot symptoms resembled those observed in the greenhouse on all inoculated tissues after 10 days (Fig. 1h and i), whereas calyces, receptacles and leaves on control plants remained asymptomatic. The experiment was repeated twice. Neopestalotiopsis clavispora was successfully reisolated from the infected tissues, fulfilling Koch's postulates. Researchers in Spain (Chamorro et al. 2016) reported N. clavispora for the first time on strawberry. After two years, N. clavispora was found in Argentina (Obregón et al. 2018). The reports characterized it mainly as a root rot pathogen. In January 2021, Neopestalotiopsis spp. were reported that it is a pathogen of strawberry leaves and fruits in Florida strawberry fields (Juliana et al. 2021). Our studies indicated that isolate NP102 was more aggressive. In the warm and humid greenhouse, the disease struck suddenly at the early stages of strawberry fruiting. Unlike previous reports, symptoms were a calyx and receptacle blight as well as a leaf spot. This pathogen can cause serious economic losses to strawberry growers. To the best of our knowledge, this is the first report of calyx and receptacle blight by N. clavispora on strawberry in China.

Structure of the trypanosome paraflagellar rod and insights into non-planar motility of eukaryotic cells

Eukaryotic flagella (synonymous with cilia) rely on a microtubule-based axoneme, together with accessory filaments to carryout motility and signaling functions. While axoneme structures are well characterized, 3D ultrastructure of accessory filaments and their axoneme interface are mostly unknown, presenting a critical gap in understanding structural foundations of eukaryotic flagella. In the flagellum of the protozoan parasite Trypanosoma brucei (T. brucei), the axoneme is accompanied by a paraflagellar rod (PFR) that supports non-planar motility and signaling necessary for disease transmission and pathogenesis. Here, we employed cryogenic electron tomography (cryoET) with sub-tomographic averaging, to obtain structures of the PFR, PFR-axoneme connectors (PACs), and the axonemal central pair complex (CPC). The structures resolve how the 8 nm repeat of the axonemal tubulin dimer interfaces with the 54 nm repeat of the PFR, which consist of proximal, intermediate, and distal zones. In the distal zone, stacked “density scissors” connect with one another to form a “scissors stack network (SSN)” plane oriented 45° to the axoneme axis; and ~370 parallel SSN planes are connected by helix-rich wires into a paracrystalline array with ~90% empty space. Connections from these wires to the intermediate zone, then to overlapping layers of the proximal zone and to the PACs, and ultimately to the CPC, point to a contiguous pathway for signal transmission. Together, our findings provide insights into flagellum-driven, non-planar helical motility of T. brucei and have broad implications ranging from cell motility and tensegrity in biology, to engineering principles in bionics.

A new genus and species of Renaudarctidae (Heterotardigrada: Arthrotardigrada) from Ryukyu Archipelago, Japan

ABSTRACTA new armoured marine tardigrade, Nodarctus hallucis gen. et sp. nov., is described from specimens collected from sandy beaches of Haterumajima, Ishigakijima and Okinawajima Islands, Ryukyu Archipelago, Japan. It is a member of the rare family Renaudarctidae, which is characterized by a stocky body with dorsal and ventral plates and stumpy legs with digits, claws and accessory filaments. However, the collected animals have prominent characters that differ from the two known species of the family: subdivided dorsal segmental plates I–III lacking lateral processes, an acute caudal spike, indented sensory plates, short spikes and peculiar papillae on legs I–III, basal spikes of legs IV and only two digits on legs IV. These differences justify the erection of a new genus and species for the animal. Morphological affinities with other marine taxa, the function of the leg papillae and the distribution pattern of the new species are discussed.http://zoobank.org/urn:lsid:zoobank.org:pub:041878AA-B437-45BF-80E3-32591D21D965

High-resolution scanning electron microscopy of the cytoskeleton of Tritrichomonas foetus

Tritrichomonas foetus is a pathogenic protozoan that causes bovine trichomoniasis. In addition to its importance in veterinary medicine, this parasite is also a good representative of one the earliest eukaryotic cells available for study. T. foetus contains organelles that are common to all eukaryotic cells as well as uncommon cell structures such as hydrogenosomes and a complex and elaborate cytoskeleton that constitutes the mastigont system. The mastigont system is mainly formed by several proteinaceous structures that are associated with basal bodies, the pelta-axostylar complex and the costa. Although the structural organization of trichomonad cytoskeletons has been analyzed using several techniques, observation using a new generation of scanning electron microscopes with a resolution of 0.8nm has allowed more detailed visualization of the three-dimensional organization of the mastigont system. Moreover, this study revealed the presence of new structures, such as the costa accessory filament, and the presence of two groups of microtubules that form the pelta-axostylar system.

Observations on the rectal flagellates of Indian amphibians. II. Genus: MonocercomonasGrassi, 1879

Summary The paper describes two species of flagellates of the genus Monocercomonas Grassi , 1879 from anuran amphibians in India. The species described are Monocercomonas maharashtrae n. sp. from Rana cyanophlyctis and M. sultanae from Rana tigrina. M. maharashtrae is characterised by a slender axostyle without the intensely staining peri-axostylar granules and distinct endoaxostylar granules in the middle and posterior thirds of the axostyle. M. sultanae is characterised by the presence of an accessory filament and the absence of a pelta and periaxostylar chromatic granules.

Structure of Trichomonads as Revealed by Scanning Electron Microscopy*†

A scanning electron microscope (SEM) study of Hypotrichomonas acosta (Moskowitz), Trichomonas vaginalis Donné, Pentatrichomonas hominis (Davaine), and Tritrichomonas foetus (Riedmüller) provided new information about the structure of the periflagellar canal; emergence of the flagella from the cell body; structure of the undulating membrane; and position, shape, and size of the pelta. Of special interest were the spatial relationships of the attached part of the recurrent flagellum and the accessory filament in Hypotrichomonas and in the members of Trichomonadinae, i.e. Trichomonas and Pentatrichomonas.

Studies on the morphology of three new species of monocercomonad flagellates from amphibians in India

Summary The communication gives an account of three new species of the genus Monocercomonas Grassi , 1879 from the frogs and toads in India. The species described are Monocercomonas cyanophlycti n. sp. from Rana cynophlyctis, Monocercomonas melanosticti n. sp. and M. aurangabadensis n. sp. from Bujo melanostictus. M. cyanophlycti is characterized by a moderately developed axostyle with distinct periaxostylar granules and a definite pelta. M. melanosticti n. sp. is distinguished by a trailing flagellum attached to the body surface for some distance and slender axostyle without chromatic rings. M. aurangabadensis n. sp. is characterized by a distinct and moderately long accessory filament and robust axostyle with distinct chromatic granules.

Unusual tubular structures in the urinary bladder of the crayfish Astacus fluviatilis

The cells of the bladder of the crayfish Astacus fluviatilis were found to contain bundles of tubular elements, 52 nm in diameter, whose construction may be similar to that of microtubules. They are linked with one another and with accessory filaments by cross bridges. The tubules are made up of 13 protofilaments. Their walls contain a helical component which may be attributable to the arrangement of subunits in the protofilaments. The function of the tubules is wholly unknown, but some speculations on this subject are made in the text.

Structure of Pentatrichomonas hominis (Davaine) as revealed by electron microscopy.

SYNOPSIS. Fine structure of Pentatrichomonas hominis is described in the light of previous light microscopic findings. The relationships among kinetosomes #1‐#4 and R are like those previously reported orhomonas gallinae, and the same is true of the rootlet filaments associated with the several kinetosomes. The kinetosome (I) of the independent flagellum is situated just behind the reflection of the sigmoid filaments of kinetosome #2 onto the pelta and parallels these filaments for a considerable distance. The peltaraxostylar junction consists of 3 layers: the capitulum of the axostyle (outer, the pelta (intermediate, and the sigmoid rootlets of kineto some #2 (inner). The pelta overlaps the axostylar capitulum to a variable extent. The parabasal body consists of elongate and flattened cisternae of smooth endoplasmic reticulum surrounded by numerous small vesicles. There are 2 typically cross‐striated parabasal filaments, filament 2 probably contributing most, if not all, the material to the slender, periodic organelle that underlies the parabasal body and usually does not extend far beyond the posterior end of the nucleus. The periodic costa is paralleled by paracostal granules, but there are few, if any, paraxostylar granules. The ultrastructure of the costa appears to be a network of flattened hexagons, with a single fibril projecting thru each of the hexagonal areas. The major cross‐striations are made up largely of densely‐stained filaments which are occasionally cut in cross section. The undulating membrane consists of a cytoplasmic fold extending from the dorsal surface of the organism and of the attached part of the recurrent flagellum, which is closely applied to the fold. The segment of the membrane dorsal to the flagellum, presumably the “accessory filament,” contains the marginal lamella, a membrane folded upon itself and with periodicity virtually indistinguishable from that of the rootlet filament of kinetosome #1.

Development of the flagellar apparatus of Naegleria.

Flagellates of Naegleria gruberi have an interconnected flagellar apparatus consisting of nucleus, rhizoplast and accessory filaments, basal bodies, and flagella. The structures of these components have been found to be similar to those in other flagellates. The development of methods for obtaining the relatively synchronous transformation of populations of Naegleria amebae into flagellates has permitted a study of the development of the flagellar apparatus. No indications of rhizoplast, basal body, or flagellum structures could be detected in amebae. A basal body appears and assumes a position at the cell surface with its filaments perpendicular to the cell membrane. Axoneme filaments extend from the basal body filaments into a progressive evagination of the cell membrane which becomes the flagellum sheath. Continued elongation of the axoneme filaments leads to differentiation of a fully formed flagellum with a typical "9 + 2" organization, within 10 min after the appearance of basal bodies.

Hypotrichomonas acosta (Moskowitz) Gen. nov. from Reptiles. I. Structure and Division.*

SYNOPSIS.Hypotrichomonas gen nov., with a feeble undulating membrane but lacking a costa, is placed in the family Monocercomonadidae Kirby of the order Trichomonadida Kirby. H. acosta (Moskowitz) from squamate reptiles is designated as the type species. It has 3 unequal anterior flagella. The proximal part of its recurrent flagellum is attached to the surface of the body along an accessory filament. A low membrane with typically a few undulations extends between the accessory filament and the body surface. The membrane, which varies greatly in length in relation to the length of the body among strains of the species, is not supported by a costa. The recurrent flagellum continues as a free posterior flagellum beyond the end of the undulating membrane and terminates in an acroneme. The capitulum of the axostyle, spatulate or scoop‐shaped, is closely applied to the anteriorly located nucleus. The capitulum extends anteriorly into a crescent‐shaped pelta. The relatively stout trunk of the axostyle is straight or twisted. It projects for some distance from the posterior body surface, and the terminal segment of the projecting part tapers gradually to a point. The parabasal apparatus consists of a rod‐ or shaped body and of a filament or filaments.In division, the full complement of flagella in each of the mastigonts is restored relatively late during division but before cytokinesis. One of the daughter individuals retains the parental undulating membrane; the other develops a new organelle. The development of the new membrane is first marked by the appearance of an acroneme, presumably recurrent flagellum, early in division. The proximal part of this flagellum becomes progressively attached to the body surface. The process of attachment is paralleled by differentiation of the accessory filament and is followed by the appearance of the undulating membrane. The parental axostyle is resorbed during division, and each daughter individual develops this organelle de wove. In the very early stages of division the parabasal consisted of two arms and in all more advanced stages of a single rod‐shaped body applied to a single filament.The significance of Hypotrichonionas in the evolution of trichomonads is discussed. Its morphology and morphogenesis suggest that this genus may represent an important step in the evolution of Trichomonadidae from Monocercomonadidae.

The Electron Microscopy of Trichomonas muris*†

SUMMARY. Thin sections of Trichomonas muris, from the caecum of Mesocricetus auratus, were observed with the aid of the electron microscope in order to determine structural details of organelles of this species.The blepharoplast appears to be limited by a membrane. What seem to be basal granules occur in the blepharoplastic area for the axostyle and the lamellae of the undulating membrane and measure approximately 500 Å in diameter.In cross section the anterior flagella and posterior flagellum are composed of eleven fibrils, two centrally and nine peripherally located. Each fibril measures about 380 Å in diameter.The so‐called “accessory filament” of the undulating membrane is devoid of any filamentous structure and appears to be composed of two differentiated mesh‐work areas. The undulating membrane is composed of a series of lamellae ranging from 300 to 400 Å in thickness. This organelle is attached to the external body surface by fine fibers ranging from 167 to 300 Å in thickness.The costa consists of a series of disks embedded in a matrix. The costa is attached to the internal body surface by what appear to be extensions of the costal disks. These disks are approximately 370 Å in thickness and spaced 490 Å apart.The axostyle is limited by a double, corrugated membrane. The corrugations are about 150 Å in thickness and spaced 110 Å apart. The so‐called chromatic ring is composed of a series of rods approximately 640 Å in thickness.The parabasal body consists of a series of filaments approximately 190 Å in thickness.Mitochondria, spheroidal in shape, are limited by a membrane about 180 Å in thickness. The internal structure consists of a varying number of projections which make this organelle appear in section as a series of compartments.Chromatic granules (paracostal, para‐axostylar, endo‐axostylar and those scattered in the cytoplasm) are irregular in shape and limited by a membrane approximately 200 Å in thickness. These granules typically display a vacuolated internal structure.

Comparative Morphology of the Trichomonad Flagellates of Man

Summary Morphologically, the three species of Trichomonas of man are distinct, although T. vaginalis and T. tenax resemble each other more than either resembles T. hominis. T. vaginalis is much the larger, the other two being about the same length. T. vaginalis is more robust in shape and T. tenax is more slender. T. vaginalis and T. tenax normally have a single compound blepharoplast which may occasionally appear as two approximately equal elements. In T. hominis there are characteristically two blepharoplasts of unequal size, the smaller being ventral in position. The oral and vaginal species have regularly four anterior flagella. T. hominis has a variable number, usually four or five, but more characteristically five. One of these is attached to the smaller ventral blepharoplast and the other four to the larger one. The undulating membrane of T. tenax is relatively longer than that of T. vaginalis, although typically less than body length in both and without an extension of the marginal filament into a trailing flagellum. T. hominis has a full-length undulating membrane with a trailing flagellum. At the margin of the undulating membrane each of the three species has an accessory filament which is more slender than the posterior flagellum. The costa of T. vaginalis and T. tenax is very slender and of uniform diameter while that of T. hominis is coarser, and thicker in its midregion. Both T. vaginalis and T. tenax have a well-developed parabasal apparatus, but this is apparently absent in T. hominis. The axostyle of T. hominis is relatively somewhat coarser than that of the other two species. In large individuals of T. vaginalis it tends to split into a number of fibrils. The nucleus of T. vaginalis is typically much elongated while in the other two species it is more commonly rounded or oval. In T. vaginalis and T. tenax the chromatin is usually aggregated into granules which are uniformly distributed; these are larger and fewer in T. tenax. In T. hominis the chromatin is less often aggregated into granules and commonly forms a layer against the nuclear membrane. The siderophilic cytoplasmic granules of T. vaginalis are numerous and scattered through the cytoplasm but tend to collect along the axostyle and costa. In T. tenax they are fewer, forming a row along the costa but not along the axostyle. In T. hominis there is a row of paracostal granules at the base of the undulating membrane and a zone of perinuclear granules or a chromatic cloud in the vicinity of the nucleus. The cytostome is inconspicuous in T. vaginalis but more evident in the other two species, especially in T. hominis. In fresh material T. vaginalis usually shows no food inclusions except a leukocyte in an occasional individual while T. hominis usually shows a variety of food bodies. T. vaginalis forms pseudopodia more often than the other two species, is more sluggish, and shows greater tendency to adhere to solid objects and to each other at the posterior ends. Survey data show that T. vaginalis and T. tenax are much more prevalent than T. hominis even in the same group of persons, indicating that the latter species is not transferable to other parts of the body. Available literature provides abundant evidence that, in the matters of culture conditions, inoculation into various animals and unsuccessful transmission of intestinal and oral trichomonads to the human vagina, these three species are physiologically as well as morphologically distinct. Trichomonas vaginalis Donne is the type species of the genus and presents no taxonomic problem. T. tenax (O.F.M.) has priority for the oral species. The intestinal form appears to have a variable number of flagella, characteristically five. For the present, T. hominis (Davaine) is retained for it. Tritrichomonas fecalis Cleveland is shown to be distinct from T. hominis and very similar to, if not identical with, a species from the intestine of frogs and toads.

Studies on the morphology of tritrichomonas foetus (Riedmüller) (protozoa, flagellata) from American cows

AbstractTritrichomonas foetus (Riedmüller) which occurs in the genital tract of domestic cows and which appears to be associated with pathological conditions, including sterility and abortion, has been found in American cows and its morphology studied.The spindle‐shaped body is 10 to 25 μ in length and bears three anterior flagella and a posterior flagellum which constitutes the marginal filament of the nearly full‐length undulating membrane and continues posteriorly as a trailing flagellum. There is an accessory filament in the undulating membrane. The axostyle is of the thick, hyaline type and contains endoaxostylar granules in the capitulum and bears a chromatic ring at the point of emergence from the body. There is a well‐developed costa under the undulating membrane and a simple type of parabasal body.In its morphology this species more nearly resembles such intestinal forms as T. augusta and T. muris than it does Trichomonas vaginalis of man, since the latter has four anterior flagella, a short undulating membrane without an accessory filament, no trailing flagellum, a different type of axostyle, and a parabasal filament along with its larger parabasal body.