Steinernema shori n. sp., a new entomopathogenic nematode (Nematoda: Steinernematidae) from India.

In this study, morphological and molecular features were used to identify a new Steinernema sp. from Chhattisgarh, India. Morphological and molecular features provide evidence for placing the new species into the "bicornutum" clade. The new species is characterized by the following morphological features: infective juveniles with a body length of 587 (494-671) µm; a distance from the anterior end to excretory pore of 46 (43-50) µm; a distance from anterior end to nerve ring of 72 µm (61-85 µm); and E% of 88 (77-97). The first-generation males are characterised by 27 genital papillae and very short spicules, with a length of 61 µm (53-67) µm. The SW% and GS% ratio of S. shori n. sp. are 139 (107-190) and 75 (62-90), respectively. The new species is further characterized by sequences of the internal transcribed spacer and partial 28S regions of the ribosomal DNA. Phylogenetic analyses show that S. shori n. sp. is most closely related to S. abbasi, S. kandii, and S. yirgalemense.

In vitro liquid culture of the mollusc-parasitic nematode Phasmarhabditis (Rhabditida: Rhabditidae).

The success of the mollusc-parasitic nematode, Phasmarhabditis hermaphrodita (Schneider) Andrássy (Rhabditida: Rhabditidae), as a biological control agent in Europe has led to worldwide interest in phasmarhabditids as biocontrol agents. In this study, the mass culture potential of three phasmarhabditids, namely Phasmarhabditis papillosa, Phasmarhabditis kenyaensis and Phasmarhabditis bohemica, was assessed. In addition, ten bacterial candidates, consisting of seven associated with slugs and three associated with entomopathogenic nematodes, were investigated. The bacteria were tested for their ability to cause mortality to Deroceras invadens, as well as to support nematode growth. Initial mortality studies demonstrated that Kluyvera, Aeromonas and Pseudomonas spp. (AP3) caused 100% mortality when they were injected into the haemocoel of D. invadens. However, in growth studies, Pseudomonas sp. (AP4) was found to be the most successful bacterium, leading to recovery and reproduction in almost all nematode species, except for P. kenyaensis. In flask studies, P. bohemica, which showed exceptional growth with Pseudomonas sp. (AP1), was chosen for further investigation. The effect of inoculating flasks with different concentrations of Pseudomonas sp. (AP1), as well as with different concentrations of P. bohemica, was evaluated by assessing the nematode populations for 14 days. The results indicated that the lowest, 1% (v/v), bacteria inoculation led to higher total nematode and to infective juvenile (IJ) yield, with flasks with the highest IJ inoculum (3000 IJs/ml) having a positive effect on the total number of nematodes and IJs in cultures of P. bohemica. This study presents improvements for the mass-culturing of nematodes associated with molluscs.

Steinernema populi n. sp. (Panagrolaimomorpha, Steinernematidae), a new entomopathogenic nematode species from China.

Steinernema populi n. sp. was recovered by baiting from beneath poplar trees in China. Morphological and molecular features provided evidence for placing the new species into the Kushidai clade. The new species is characterized by the following morphological features: third-stage infective juveniles (IJ) with a body length of 1095 (973-1172) µm, a distance from the anterior end to excretory pore of 77 (70-86) µm and a tail length of 64 (55-72) µm. The Body length/Tail length (c) ratio and Anterior end to Excretory pore/ Tail length × 100 (E%) of S. populi n. sp. are substantially greater than those of all other 'Feltiae-Kushidai-Monticolum' group members. The first-generation males can be recognized by a spicule length of 66 (57-77) µm and a gubernaculum length of 46 (38-60) µm. The new species is further characterized by sequences of the internal transcribed spacer and partial 28S regions of the ribosomal DNA. Phylogenetic analyses show that Steinernema akhursti and Steinernema kushidai are the closest relatives to S. populi n. sp.

Ex vivo development of Phasmarhabditis spp. associated with terrestrial molluscs.

The success of Phasmarhabditis hermaphrodita (Schneider) Andrássy (Rhabditida: Rhabditidae) as a biological control agent of molluscs has led to a worldwide interest in phasmarhabditids. However, scant information is available on the lifecycle development of species within the genus. In the current study, the development of P. hermaphrodita, Phasmarhabditis papillosa, Phasmarhabditis bohemica and Phasmarhabditis kenyaensis were studied using ex vivo cultures, in order to improve our understanding of their biology. Infective juveniles (IJs) of each species were added to 1 g of defrosted homogenized slug cadavers of Deroceras invadens and the development monitored after inoculated IJ recovery, over a period of eight-ten days. The results demonstrated that P. bohemica had the shortest development cycle and that it was able to produce first-generation IJs after eight days, while P. hermaphrodita, P. papillosa and P. kenyaensis took ten days to form a new cohort of IJs. However, from the perspective of mass rearing, P. hermaphrodita has an advantage over the other species in that it is capable of forming self-fertilizing hermaphrodites, whereas both males and females are required for the reproduction of P. papillosa, P. bohemica and P. kenyaensis. The results of the study contribute to the knowledge of the biology of the genus and will help to establish the in vitro liquid cultures of different species of the genus.

Redescription and synonymization of Oscheius citri Tabassum, Shahina, Nasira and Erum, 2016 (Rhabditida, Rhabditidae) from India and its taxonomical consequences.

A population of a nematode species belonging to the genus Oscheius was isolated in western Uttar Pradesh, India. Morphological and morphometrical studies on this species showed its high similarity with six species described previously from Pakistan (Oscheius citri, O. cobbi, O. cynodonti, O. esculentus, O. punctatus and O. sacchari). The molecular analysis of the ITS1-5.8S-ITS2 rDNA sequences of the Indian population and the six species described from Pakistan showed that all the sequences are almost identical. Thus, based on morphological and molecular characteristics, all of the six above-mentioned Pakistani species and Indian strain do not differ from each other, hence can be considered synonyms. The correct name for this taxon is the first described species O. citri. Additionally, the phylogenetic analysis of the 18S rDNA and the 28S rDNA sequences showed that Oscheius citri is sister to the clade formed by O. chongmingensis and O. rugaoensis from China. The high similarity of morphological and morphometric characteristics of O. citri and other species, O. maqbooli, O. nadarajani, O. niazii, O. shamimi and O. siddiqii, suggest their conspecificity; however, lack of molecular data for these species does not allow this hypothesis to be tested.

The first report of Xenorhabdus indica from Steinernema pakistanense: co-phylogenetic study suggests co-speciation between X. indica and its steinernematid nematodes.

During a survey in agricultural fields of the sub-humid region of Meerut district, India, two strains of entomopathogenic nematodes, labelled CS31 and CS32, were isolated using the Galleria baiting technique. Based on morphological and morphometric studies, and molecular data, the nematodes were identified as Steinernema pakistanense, making this finding the first report of this species from India. For the first time, we performed a molecular and biochemical characterization of the bacterial symbiont of S. pakistanense. Furthermore, a co-phylogenetic analysis of the bacteria from the monophyletic clade containing a symbiont of S. pakistanense, together with their nematode hosts, was conducted, to test the degree of nematode-bacteria co-speciation. Both isolates were also tested in a laboratory assay for pathogenicity against two major pests, Helicoverpa armigera and Spodoptera litura. The morphology of the Indian isolates corresponds mainly to the original description, with the only difference being the absence of a mucron in first-generation females and missing epiptygmata in the second generation. The sequences of bacterial recA and gyrB genes have shown that the symbiont of S. pakistanense is closely related to Xenorhabdus indica, which is associated with some other nematodes from the 'bicornutum' group. Co-phylogenetic analysis has shown a remarkable congruence between the nematode and bacterial phylogenies, suggesting that, in some lineages within the Steinernema / Xenorhabdus complex, the nematodes and bacteria have undergone co-speciation. In the virulence assay, both strains caused a 100% mortality of both tested insects after 48 h, even at the lowest doses of 25 infective juveniles per insect, suggesting that S. pakistanense could be considered for use in the biocontrol of these organisms in India.

Steinernema pwaniensis n. sp., a new entomopathogenic nematode (Nematoda: Steinernematidae) from Tanzania.

A new species of entomopathogenic nematode, Steinernema pwaniensis n. sp., belonging to the glaseri group, is described from Tanzania. The infective juveniles of S. pwaniensis n. sp. are characterized by a body length of 978 µm (808-1131), distance from anterior end to excretory pore of 86 µm (80-95) and a tail length of 87 µm (75-95). The ratios a, c and E% of S. pwaniensis n. sp. are substantially lower than those of all other African 'glaseri' group members. The first-generation males of S. pwaniensis n. sp. can be distinguished by a large spicule length of 92 µm (80-97) and by the absence of the caudal mucron, while second-generation males possess a short spine-like mucron. First-generation females have a peg-like tail tip bearing three spine-like projections. Second-generation females can be recognized by a slightly protruding vulva and well-developed post-anal swelling. The new species is further characterized by sequences of the internal transcribed spacer (ITS) and partial 28S regions of the ribosomal DNA. Phylogenetic analyses show that S. pwaniensis n. sp. forms a strongly supported monophyletic clade with two other East African species, S. ethiopiense and S. karii.

The significance of the tail projections in female entomopathogenic nematodes within the family Steinernematidae.

Several taxonomic studies of the entomopathogenic nematode family Steinernematidae have reported on the minute tail projections on the tail tips of females of the first generation; however, the occurrence and morphology of these traits in other steinernematid species and their taxonomic significance are still unclear. In the present study, the tail projections in females of seven steinernematid species belonging to four phylogenetic clades were examined. The projections are present in all of the tested species and their number and shape is consistent within species but significantly differs among species. It thus seems that these structures may have taxonomic significance at the species level. Based on these results, we suggest that every description of a new species in the family Steinernematidae should contain a precise morphology of the female tail, focused on these projections, using both light and scanning electron microscopy.

To complete their life cycle, pathogenic nematode-bacteria complexes deter scavengers from feeding on their host cadaver.

The life cycle of commercially used molluscicidal rhabditid nematodes Phasmarhabditis hermaphrodita and entomopathogenic steinernematid nematodes is similar: infective stages carry symbiotic bacteria, which kill their host. Nematodes complete their life cycle feeding on the proliferating symbiont and the host tissue. After 1-2 weeks, new infective stages carrying the bacteria leave the host cadaver in search of new hosts. The removal of invertebrate cadavers by scavengers is extremely fast and represents a severe threat to the developing nematodes.Two-choice trials were used to assess prey choice of the generalist predator/scavenger Pterostichus melanarius (Coleoptera: Carabidae) between Deroceras reticulatum (Mollusca: Agriolimacidae) slugs or wax moth Galleria mellonella (Lepidoptera: Pyralidae) larvae killed by infection of P. hermaphrodita/Steinernema affine and control killed by freezing. We demonstrate that the presence of either of the two nematodes tested deters the beetles from consuming infected cadavers. As P. hermaprodita cannot infect an insect host, we hypothesise the deterrent effect being an evolutionary adaptation of the nematode/bacteria complex rather than the ability of the beetles to avoid potentially infective cadavers.

Radiopaque poly(2-hydroxyethyl methacrylate) particles containing silver iodide complexes tested on cell culture.

Silver iodide complexes have been used as an effective radiopacifying agent to prepare radiopaque poly(2-hydroxyethyl methacrylate) (poly(HEMA)) particles. Incorporation of silver iodide complexes inside the poly(HEMA) particles was achieved by first swelling particles in potassium iodide solution and precipitating the silver iodide complexes using a 30 wt% solution of silver nitrate. The dry particles contained 15 wt% of silver iodide complexes. Particles were easily monitored using a standard imaging technique based on X-ray absorption. Toxicity of the particles has been determined in in vitro experiments on a cell culture. As no inhibition of growth of cells surrounding the particles was observed, they can be considered non-toxic.

Hydrogels in endovascular embolization. VI. Toxicity tests of poly(2-hydroxyethyl methacrylate) particles on cell cultures.

Cytotoxicity of poly(2-hydroxyethyl methacrylate) [poly(HEMA)] hydrogel spherical particles, prepared by radical suspension polymerization and designed for endovascular occlusion, was studied in vitro on cell cultures. Testing methods included a direct contact test and extraction test. No inhibition of growth of cells surrounding the poly(HEMA) beads and a very low inhibition of cell viability, only in concentrated extracts in long-term contact, were observed. As a result, poly(HEMA) beads can be considered non-toxic.

[Preparation trauma in stomatology]. / Preparacní trauma ve stomatologii.

In this paper authors deal with the causes of preparation trauma in stomatology. They have studied effects of high temperature on human cells cultured in vitro. Based both on literature data and on their own experience they summarize basic principles of preparation which prevent preparation trauma. They summarize how to eliminate as much as possible factors that damage hard dental tissues and pulp.

[Physical, chemical and biological aspects of treatment of pathological changes in hard dental tissues]. / Fyzikální, chemická a biologická hlediska osetrení patologických zmen tvrdých zubních tkání.

The methods of preparation of dental tissues, treatment of the dentine wound and filling materials, which replace the hard dental tissues, are discussed to evaluate their features in toward the requirements for biological harmlessness. Based on the literature data, their own clinical and practical experiences, the authors have found that none of the currently used preparation methods as well as supports or filling materials are fulfilling the biological treatment requirements. Therefore, it is necessary to be aware of this fact, and adjust the medical procedures in such a way to avoid the harmful effects or to suppress then as much as possible until the ideal preparation procedures and filling materials will be developed.

[Biological aspects of materials recommended as alternatives to amalgams]. / Biologické aspekty materiálu doporucovaných jako alternativy za amalgám.

Evaluation of biological properties of filling materials used as an alternative to amalgams shows that none of the tested materials possess the required biological properties. It is therefore necessary to search for new and better filling materials.

Apoptosis and necrosis: Dynamics of structural changes in cells cultivated in vitro after treatment with xenobiotics.

Cell death has recently become a very widely discussed topic, and a great deal of information has become available regarding physiological (programmed) cell death or apoptosis. Contrary to the situation with apoptosis, few new developments have occurred in the field of xenobiotic-induced cell death. The purpose of this study was to analyse morphological changes related to in vitro induced cell death. The main emphasis was on detailed analysis of the dynamics of structural changes. The experimental models of cell death include both apoptosis (Cisplatin) and necrosis (Tween 20). Cells of stabilized cell line Hep2 (human epithelial cells) were used in this study. The dynamics of structural changes were followed using time-lapse microcinematography of living cells observed in phase contrast. The experiments followed the same population of cells before, during and after treatment with model compounds. It is possible to distinguish characteristic types of cell death in vitro (from morphological criteria), but many intermediate types of cell death can also be found. Cellular responses to the presence of one particular xenobiotic appear to be complex, dependent on the dosage: even in a single treated cell population, different morphological responses were observed.

In vitro cytotoxicity testing of metal alloys used in medicine: Comparison of different approaches.

Metal alloys are used in medicine mainly for implantation in different branches of surgery and as indispensable materials in stomatology. The toxicity testing of these materials incurs even more technical problems than the toxicity testing of new drugs. In principle there are two approaches-direct contact of a tested material with cells, or toxicity testing of an eluate prepared from tested material. We have tested more than 20 different metal alloys, based either on titanium, gold or stainless-steel. We used three different in vitro methods: the dynamic assessment of contact cytotoxicity-cell monolayer is in direct contact with tested material (Cervinka and Puza, 1990); cell proliferation assessment-effect of an eluate on cell proliferation is measured (Cervinka and Puza, 1990); agar overlayer with neutral red uptake-both effect of eluate and direct contact with tested material with agar overlayer could be measured. Based on our results we recommended to use all three approaches simultaneously in a battery of in vitro tests.

[Cytotolerance for Nimetic Grip (ESPE), a material used for fixation of dental bridge plates]. / Cytotolerance materiálu Nimetic Grip (ESPE) uzívaného k fixaci ploténkových mustku.

Providing in vitro experimentation on cells, the authors monitor general biologic effects of the fixation composite Nimetic Grip (ESPE). Cytotoxicity of three components of supplied package is tested with both dynamic identification of contact cytotoxicity and macro contact test. Those concerned are composite cements. Nimetic Grip Opak and Transparent as well as Nimetic Bond adhesive varnish. All the observed materials were shown to induce morphologic changes on HEp-2 cells.

[Reaction of HeLa cells to the presence of Evicrol varnish studied by electron microscopy]. / Reakce HeLa bunek na prítomnost laku Evicrol varnish sledovaná v elektronovém mikroskopu.

The electron-microscopical changes of cells produced by underlying Evicrol varnish are described. The latter is used in preventive stomatology to underlay fillers, those from composite materials, especially. It is designed to prevent the pulp damage through these materials. The changes of in vitro cultivated cells have in contrast been confirmed with previous experimentation showing that the varnish itself causes toxicity for living cells and therefore is not convenient to undercoat cavities in the proximity of pulps.