Histochemical lipid studies on Schistosoma mansoni adults maintained in situ and in vitro

Haseeb M. A., Eveland L. K. and Fried B. 1984. Histochemical lipid studies on Schistosoma mansoni adults maintained in situ and in vitro. International Journal for Parasitology14: 83–88. Schistosoma mansoni male and female adults were incubated at 37°C for 0.5 and 1.0 h in Earle's balanced salt solution containing 0.1% glucose and 0.5% lactalbumin hydrolysate, then examined by histochemistry and scanning electron microscopy. Histochemical analysis of cryostat sections stained with Oil Red O showed that males contain neutral lipid mainly in the parenchyma and tubercles, while females contain neutral lipid in the vitellaria. Neutral lipids are released from the tubercles of both paired and unpaired males maintained in vitro. There is evidence of in situ lipid transfer from males to blood vessel walls. Neutral lipid was not seen in females from unisexual infections. Sudan Black B staining fo total lipids is positive in tubercles, parenchyma, and vitellaria. Nile Blue Sulphate stains acidic lipids in male caecal walls. Scanning electron microscopy reveals no tegumental damage.     

Teratological Development in the Cephalic Anatomy of the Nematode Romanomermis culicivorax

Adults of Romanomermis culicivorax obtained from mass cultures were examined by scanning and transmission electron microscopy to determine the organization of their anterior sense organs. The normal pattern apparently consists of two lateral amphids plus six cephalic papillae. Lateral cephalic papillae contain two sense organs, each with a cuticular pore, while subdorsal and subventral papillae have three sense organs, each with a cuticular pore. About 30% of females and 80% of males examined showed aberrant developments in these sense organs. Both cuticle and underlying cells (hypodermis and neurons) are affected; some sense organs are absent while others are incompletely formed. Few aberrant worms were found in a smaller collection of worms reared in lower population densities. Perhaps aberrant forms are examples of teratological development resulting from, or promoted by, conditions used for mass rearing of biological control agents.     

Identification of human hookworm in failed-treatment cases using Chinese hamsters (Cricetulus griseus) and scanning electron microscopy

An attempt was made to identify the human hookworm involved in failed-treatment cases using abnormal hosts and scanning electron microscopy. Thirty-seven, 2 to 6 month old Chinese hamsters (Cricetulus griseus) from a closed, outbred, conventional colony, were each given between 20 and 120 filariform larvae per os. The larvae were cultured from faeces from mebendazole (Vermox) 500 mg single-dose, failed-treatment cases living in the lowveld farming area of the Transvaal Province, South Africa. About 60 to 78 days after inoculation, the animals were killed and adult worms were removed from their small intestines. Eleven (30%) of the 37 hamsters harboured a total of 31 adult worms (19 males and 12 females), while 26 hamsters were refractory to infection. The greatest number of worms recovered from a single animal was six. A total of 27 worms (17 males and 10 females) were subjected to examination by scanning electron microscopy. Micrographs showed male and female worms to be morphologically all of the Necator americanus species, as identified by a pair of ventral and dorsal cutting plates, a dorsal tooth and the fused terminus of spicules in the male bursa. The transverse cuticular striations were distinct and smooth. Several points of interest arose from the results of this study and are discussed.     

Scanning electron microscopy of Echinostoma revolutum (Trematoda) during development in the chick embryo and the domestic chick

Fried B. and Fujino T. 1984. Scanning electron microscopy of Echinostoma revolutum (Trematoda) during development in the chick embryo and the domestic chick. International Journal for Parasitology14: 75–81. Scanning electron microscopy (SEM) was used to study the development of chemically excysted metacercariae of Echinostoma revolutum on the chick chorioallantois. SEM studies were also made on preovigerous adults of E. revolutum grown in the domestic chick. During worm development on the chorioallantois the tegument changed from smooth to granular and sensory papillae on the suckers became well-defined. As worms developed on the chorioallantois the cephalic collar spines became thicker and more curved and the tegumentary spines showed marked changes in shape, size and distribution on both ventral and dorsal aspects of the body. Changes in the surface ultrastructure of worms grown on the chorioallantois were essentially similar to those observed in preovigerous worms from chicks.     

First Record of Cosmocephalus obvelatus (Acuariidae) in Common Gulls (Larus canus) from Gangneung,Korea

A nematode species belonging to the genus Cosmocephalus was collected from the stomach of 2 common gulls, Larus canus. The common gulls were found dead on the seaside of Gangneung City, the Republic of Korea. The worms were identified and classified by light (LM) and scanning electron microscopy (SEM) on the basis of important taxonomic characters. The nematodes were characterized by a body length 9.1-9.3 mm (males) and 15.5-15.9 mm (females) and cordons recurrent in anterior direction and anastomosing laterally at about the level of anterior quarter of the buccal cavity. The salient bicuspid deirids were located on the posterior to the cordons. Lateral alae were well-developed, extending from the level just posterior of deirids to the level about middle of the body. LM and SEM observations identified the worms as C. obvelatus. This is the first reported case of C. obvelatus infection in common gulls in Korea.     

Effect of niclosamide on the tegumental surface of Haplorchis taichui using scanning electron microscopy

The effect of niclosamide on the tegument of adult Haplorchis taichui (Trematoda: Heterophyidae) exposed in vitro was observed by scanning electron microscope. Adult worms were incubated in Tyrode's solution containing 0.01, 0.1, 1.0, and 10 microg ml(-1) of niclosamide for 30 min, 1, 6, 12 and 24 h. Control groups were incubated in Tyrode's solution without niclosamide and worms remained active until 24 h. In 0.01 microg ml(-1) of niclosamide, worms showed slightly active movements up to 1 h after incubation, while in 0.1 microg ml(-1) solution a few worms showed only slightly active movements after 30 min. Tegumental changes were determined by scanning electron microscopy. Swelling and blebbing of the tegument were observed on both ventral and dorsal sides. After longer periods, extensive swelling and blebbing of the tegument became more severe and there was a loss of the apical plasma membrane in some regions. Empty spine sockets occurred, and small perforations penetrated the basal lamina, followed by some lesions. Destruction of both surfaces was more pronounced on the posterior compared with the anterior regions.     

First evidence of maternal transmission of algal endosymbionts at an oocyte stage in a triploblastic host, with observations on reproduction in Waminoa brickneri (Acoelomorpha)

Abstract. Examination of sexual reproduction in a symbiotic acoelomorph worm, Waminoa brickneri from Eilat (Red Sea), presents the first definitive evidence for maternal transmission of dinoflagellate algal symbionts in a triploblastic organism. Sexually mature worms were removed from the stony coral Plesiastrea laxa and raised in the laboratory. Eggs were detected 18 d after the collection of the worms and hatched 4 d later. Histological sections performed on sexually mature worms showed an ovary with oocytes containing two distinct types of algal endosymbionts within their ooplasm. Transmission electron microscopy corroborated the presence of algal symbionts within the developing embryos. Our findings regarding maternal transmission of symbionts shed new light on the diversity of modes of algal symbiont acquisition known in triploblastic organisms.     

Silver Sulfadiazine Nanosystems for Burn Therapy

The objective of the present study was to formulate stable silver sulfadiazine (SSD) nanosuspensions and nanogels suitable for topical delivery with a view to increase bactericidal activity in burn therapy. SSD nanosuspensions were formulated using the microprecipitation–high-pressure homogenization technique. An optimized microsuspension of 0.5% SSD formulated with 6% Cremophor EL and 4% Lauroglycol 90 was subjected to 30 cycles of 1,000-bar pressure to give a nanosuspension with an average particle size of 367.85 nm. Transmission electron microscopy studies revealed that ovoid- to rectangular-shaped SSD particles were present as clusters. It was evident through X-ray diffraction studies that SSD was present in amorphous state both in microprecipitate and in nanosuspension. SSD (0.5%) nanogels were prepared using 1% Carbopol 974 P for topical delivery of nanosized SSD. In vitro release studies demonstrated that SSD release was faster from solutions and nanosuspensions compared to gel formulation owing to the influence of the gel matrix on SSD release. The bacterial inhibitory efficiency of SSD nanosuspension was as good as that of SSD solution against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. In vivo studies revealed that a nanogel containing 0.5% SSD was more effective in wound healing compared to 0.5% and 1% marketed cream.     

TomoJ: tomography software for three-dimensional reconstruction in transmission electron microscopy

Background  

Transmission electron tomography is an increasingly common three-dimensional electron microscopy approach that can provide new insights into the structure of subcellular components. Transmission electron tomography fills the gap between high resolution structural methods (X-ray diffraction or nuclear magnetic resonance) and optical microscopy. We developed new software for transmission electron tomography, TomoJ. TomoJ is a plug-in for the now standard image analysis and processing software for optical microscopy, ImageJ.     

Effect of host sex and litter on the population dynamics of Echinococcus granulosus in dogs.

Twenty-one Beagle dogs consisting of 10 males and 11 females and belonging to 3 litters were infected with 60,000 E. granulosus protoscolices each. They were killed on day 40, the parasites from their intestines recovered, and the number of worms, average number of proglottides per worm, average length per worm, percentage of worms with a uterine cavity, and percentage of egg-bearing worms were determined for each dog and analyzed per sex and litter. On average, the dogs had 1,253 +/- 339 worms (means +/- standard error) with 2.42 +/- 0.1 proglottides, were 1.59 +/- 0.07 mm long, and 25.6 +/- 4.8% of the worms presented a uterine cavity and 1.2 +/- 0.6% bore eggs. The number of worms exhibited a bimodal distribution with 19 dogs having less than or equal to 2,565 worms and 2 greater than or equal to 5,520 worms. Average number of proglottides also showed a bimodal distribution with 7 dogs having less than or equal to 2.1 proglottides per worm and 14 dogs having greater than or equal to 2.4 proglottides per worm. The parasites were significantly more numerous in females than in the males (1,964 +/- 573 vs. 681 +/- 202), had more proglottides (2.67 +/- 0.08 vs. 2.15 +/- 0.16), and were longer (1.72 +/- 0.07 vs. 1.44 +/- 0.11 mm). The percentages of parasites with a uterine cavity (27.8 +/- 5.9 vs. 23.2 +/- 8.1) or bearing eggs (1.0 +/- 0.5 vs. 1.5 +/- 1.8) were comparable in females and males.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phenotypic plasticity in adult worms of Schistosoma mansoni (Trematoda:Schistosomatidae) evidenced by brightfield and confocal laser scanning microscopies

A comparative morphometric study was performed to identify host-induced morphological alterations in Schistosoma mansoni adult worms. A wild parasite population was obtained from a naturally infected rodent (Nectomys squamipes) and then recovered from laboratory infected C3H/He mice. Furthermore, allopatric worm populations maintained for long-term under laboratory conditions in Swiss Webster mice were passed on to N. squamipes. Suckers and genital system (testicular lobes, uterine egg, and egg spine) were analyzed by a digital system for image analysis. Confocal laser scanning microscopy (CLSM) showed details of the genital system (testicular lobes, vitelline glands, and ovary) and the tegument just below the ventral sucker. Significant morphological changes (p < 0.05) were detected in male worms in all experimental conditions, with no significant variability as assessed by CLSM. Significant changes (p < 0.05) were evident in females from the wild population related to their ovaries and vitelline glands, whereas allopatric females presented differences only in this last character. We conclude that S. mansoni worms present the phenotypic plasticity induced by modifications in the parasite's microenvironment, mainly during the first passage under laboratory conditions.     

Ketamine Causes Mitochondrial Dysfunction in Human Induced Pluripotent Stem Cell-Derived Neurons

Purpose

Ketamine toxicity has been demonstrated in nonhuman mammalian neurons. To study the toxic effect of ketamine on human neurons, an experimental model of cultured neurons from human induced pluripotent stem cells (iPSCs) was examined, and the mechanism of its toxicity was investigated.

Methods

Human iPSC-derived dopaminergic neurons were treated with 0, 20, 100 or 500 μM ketamine for 6 and 24 h. Ketamine toxicity was evaluated by quantification of caspase 3/7 activity, reactive oxygen species (ROS) production, mitochondrial membrane potential, ATP concentration, neurotransmitter reuptake activity and NADH/NAD⁺ ratio. Mitochondrial morphological change was analyzed by transmission electron microscopy and confocal microscopy.

Results

Twenty-four-hour exposure of iPSC-derived neurons to 500 μM ketamine resulted in a 40% increase in caspase 3/7 activity (P < 0.01), 14% increase in ROS production (P < 0.01), and 81% reduction in mitochondrial membrane potential (P < 0.01), compared with untreated cells. Lower concentration of ketamine (100 μM) decreased the ATP level (22%, P < 0.01) and increased the NADH/NAD⁺ ratio (46%, P < 0.05) without caspase activation. Transmission electron microscopy showed enhanced mitochondrial fission and autophagocytosis at the 100 μM ketamine concentration, which suggests that mitochondrial dysfunction preceded ROS generation and caspase activation.

Conclusions

We established an in vitro model for assessing the neurotoxicity of ketamine in iPSC-derived neurons. The present data indicate that the initial mitochondrial dysfunction and autophagy may be related to its inhibitory effect on the mitochondrial electron transport system, which underlies ketamine-induced neural toxicity. Higher ketamine concentration can induce ROS generation and apoptosis in human neurons.     

Protoplast induction in Chlorella pyrenoidosa

Chlorella pyrenoidosa (UTEX 1230) cells in late log phase of growth were induced to form viable protoplasts by enzymatic digestion only when incubated in 2-deoxy-d-glucose (2DG) for 24 h. The combination of hemicellulase (4% w/v), Cellulysin (4% w/v), and glucuronidase (5% v/v) with 0.8 M mannitol and 8 mM CaCl₂ in modified Bristol's solution, was most effective for obtaining viable protoplasts as determined by light and electron microscopy, and vital staining with primuline (0.01% w/v). Resistance of cell walls to extensive extraction (acetolysis), and infrared analysis indicated that sporopollenin is a component of the cell wall. Transmission electron miscroscopy of acetolysed cell walls also allowed visualization of the laminate nature of the wall. This is the first report of successful induction of protoplasts from algae which contain sporopollenin in their cell walls.     

Schistosoma mansoni: Adult worm chemoattraction,with and without barriers

Heterosexual and homosexual chemoattraction studies were done with 8- to 12-week-old Schistosoma mansoni adults in linear chambers containing Earle's saline, in the absence and presence of both perforated and unperforated dialysis sac chimney barriers. In the absence of barriers attraction was seen in all heterosexual and homosexual combinations. After 90 min of observation at 37 C, organisms maintained at 4 or 37 C for at least 1 hr prior to the assay showed heterosexual attraction similar to those used immediately after perfusion. There was significantly more heterosexual attraction between worms from same than different pairs. In the presence of perforated barriers the greatest attraction was heterosexual, with males moving toward females; heterosexual attraction with females moving toward males was approximately equal to homosexual female attraction, and male homosexual attraction was not seen. In the presence of unperforated barriers the greatest attraction of females toward males occurred during the first 0.5 hr; within 3 to 4 hr females were no longer significantly attracted to males. However, after 0.5 hr males were more significantly attracted to females in unperforated than perforated chimneys. These studies demonstrate that adult schistosomes attract each other in vitro, and that there is a chemical basis for this attraction.     

Cadmium Accumulation and DNA Homology with Metal Resistance Genes in Sulfate-Reducing Bacteria

Cadmium resistance (0.1 to 1.0 mM) was studied in four pure and one mixed culture of sulfate-reducing bacteria (SRB). The growth of the bacteria was monitored with respect to carbon source (lactate) oxidation and sulfate reduction in the presence of various concentrations of cadmium chloride. Two strains Desulfovibrio desulfuricans DSM 1926 and Desulfococcus multivorans DSM 2059 showed the highest resistance to cadmium (0.5 mM). Transmission electron microscopy of the two strains showed intracellular and periplasmic accumulation of cadmium. Dot blot DNA hybridization using the probes for the smtAB, cadAC, and cadD genes indicated the presence of similar genetic determinants of heavy metal resistance in the SRB tested. DNA sequencing of the amplified DNA showed strong nucleotide homology in all the SRB strains with the known smtAB genes encoding synechococcal metallothioneins. Protein homology with the known heavy metal-translocating ATPases was also detected in the cloned amplified DNA of Desulfomicrobium norvegicum I1 and Desulfovibrio desulfuricans DSM 1926, suggesting the presence of multiple genetic mechanisms of metal resistance in the two strains.     

Skeletal sensitivity to dietary calcium deficiency is increased in the female compared with the male rat

We investigated potential sex differences in bone resorption and the conservation of whole body bone mass in 24-week-old Sprague-Dawley rats maintained on a 1.0% calcium diet and then fed diets containing 0.02, 0.5, 1.0, or 1.75% calcium for 31 days. Lowering dietary calcium from 1.00% to 0.02% doubled whole skeleton bone resorption (urinary 3H-tetracycline loss). Female rats were more sensitive to calcium stress, exhibiting the maximal resorptive response when fed the 0.5% calcium diet, whereas the 0.02% calcium diet was required to elicit this response in males. Despite the evidence of increased bone resorption, whole skeleton mass was unchanged in females and was significantly increased in males, indicating that switching to even the 0.02% calcium diet did not result in an overt loss of total body bone mass. Compared with controls, the skeleton mass of females (97+/-1.4%) maintained on the 0.02% calcium diet was significantly lower than males (107+/-2.4%), again suggesting a greater impact of calcium deficiency in females. The calculation of the average percentage growth of selected individual bones in male rats indicated a proportional increase in bone mass between the axial and appendicular skeleton of approximately +4% and +18% in animals maintained on 0.02 and 1.75% diets, respectively. By comparison, female rats consuming the 0.02% calcium diet showed an average 14% loss in axial bone and 7.5% gain in appendicular bone mass. The results indicate increased sensitivity to dietary calcium deficiency in female rats which involves a significant loss in axial bone mass not observed in male rats maintained under similar dietary conditions.     

The effect of praziquantel on the pseudophyllidean cestode Bothriocephalus acheilognathi in vitro

Adult Bothriocephalus acheilognathi were incubated in solutions containing 0 (control), 0.1, 1.0, 10.0 and 100 micrograms praziquantel per ml (0, 10(2), 10(3), 10(4) and 10(5) micrograms l-1) of 0.9% saline for 5, 15 and 60 min at a temperature of 18 degrees C. The worms contracted immediately upon being placed in the drug. Scanning and transmission electron microscopy revealed considerable tegumental damage particularly in the neck region. Vacuolization and 'bubbling' of the tegument occurred in all of the drug solutions tested. Exposure to drug concentrations of more than 1.0 micrograms ml-1 (10(3) micrograms l-1) praziquantel for 15 min or greater resulted in many of the 'bubbles' bursting and releasing their contents to the exterior. Mature proglottides were distorted and had occasional large swellings resulting in the mass expulsion of eggs. Praziquantel had no ovicidal activity. Exposure to drug concentrations of 100 micrograms (10(5) micrograms l-1) praziquantel per ml saline for 24 h was not lethal to the worms.     

Scanning electron microscopy of viable and calcified Onchocerca gibsoni

Holdsworth P. A. 1987. Scanning electron microscopy of viable and calcified Onchocerca gibsoni. International Journal for Parasitology17: 957–964. Scanning electron microscopy (SEM) of adult Onchocerca gibsoni revealed that the cephalic ends of male and female specimens possessed features of little value for differentiation from other species. The marked cuticular ridges of the mid-body region of female worms were highlighted using SEM, as were the cuticular annulations and the body constriction at the anal region. Male specimens revealed the uniform annulations of the worm body along with the caudal papillae and the spicules. Microfilariae (mff) possessed cephalic papillae, lateral lines and cuticular annulations similar to adult worms. The cephalic hook was the most prominent feature at the anterior end of the mff. Calcification of adult worms was found to be mainly internal, however, one specimen was recorded with external calcification. Energy dispersive X-ray analysis revealed the composition of the calcification to be of calcium phosphate.     

A small temperature rise may contribute towards the apparent induction by microwaves of heat-shock gene expression in the nematode Caenorhabditis Elegans

We have previously reported that low intensity microwave exposure (0.75-1.0 GHz CW at 0.5 W; SAR 4-40 mW/kg) can induce an apparently non-thermal heat-shock response in Caenorhabditis elegans worms carrying hsp16-1::reporter genes. Using matched copper TEM cells for both sham and exposed groups, we can detect only modest reporter induction in the latter exposed group (15-20% after 2.5 h at 26 degrees C, rising to approximately 50% after 20 h). Traceable calibration of our copper TEM cell by the National Physical Laboratory (NPL) reveals significant power loss within the cell (8.5% at 1.0 GHz), accompanied by slight heating of exposed samples (approximately 0.3 degrees C at 1.0 W). Thus, exposed samples are in fact slightly warmer (by < or =0.2 degrees C at 0.5 W) than sham controls. Following NPL recommendations, our TEM cell design was modified with the aim of reducing both power loss and consequent heating. In the modified silver-plated cell, power loss is only 1.5% at 1.0 GHz, and sample warming is reduced to approximately 0.15 degrees C at 1.0 W (i.e., < or =0.1 degrees C at 0.5 W). Under sham:sham conditions, there is no difference in reporter expression between the modified silver-plated TEM cell and an unmodified copper cell. However, worms exposed to microwaves (1.0 GHz and 0.5 W) in the silver-plated cell also show no detectable induction of reporter expression relative to sham controls in the copper cell. Thus, the 20% "microwave induction" observed using two copper cells may be caused by a small temperature difference between sham and exposed conditions. In worms incubated for 2.5 h at 26.0, 26.2, and 27.0 degrees C with no microwave field, there is a consistent and significant increase in reporter expression between 26.0 and 26.2 degrees C (by approximately 20% in each of the six independent runs), but paradoxically expression levels at 27.0 degrees C are similar to those seen at 26.0 degrees C. This surprising result is in line with other evidence pointing towards complex regulation of hsp16-1 gene expression across the sub-heat-shock range of 25-27.5 degrees C in C. elegans. We conclude that our original interpretation of a non-thermal effect of microwaves cannot be sustained; at least part of the explanation appears to be thermal.     

Calpain inhibitor suppresses both extracellular vesicle-mediated secretion of miRNAs and egg production from paired adults of Schistosoma japonicum

Extracellular vesicles (EVs) have been reported to be secreted from Schistosoma japonicum at all developmental stages. However, the reproduction and communication mechanisms between the paired adults through the EVs in dioecious Trematoda have not been reported. In this study, EVs containing many exosome-like vesicles and microvesicles were observed in the supernatants of paired adults cultured in vitro, and abundant selected miRNAs were contained in them. In particular, the female-specific miR-bantam was present only in vesicles and was hardly secreted outside the vesicles. In this study, we found that male-female pairing induced secretion of miR-3479 and miR-bantam in EVs, but not of male-specific miR-61. Furthermore, ingestion of mouse erythrocytes also increased the production of miRNAs in paired adult and single female worms. Vesicles were found in the tegument of females treated with erythrocytes under electron microscopy. After the paired worms were treated with several inhibitors against the secretion of EVs, only calpain inhibitor (calpeptin) significantly reduced the amount of miRNA in EVs. Furthermore, the worms treated with only calpeptin inhibited egg production in vitro. Together, these results indicate that qualitative miRNA production through EVs regulated by calpain plays a role in egg production in S. japonicum.