In vitro culture of intramolluscan stages of the avian schistosome Trichobilharzia ocellata

Several different procedures for in vitro cultivation of intramolluscan stages of the avian schistosome Trichobilharzia ocellata were tried. A medium was found and culture conditions were established that not only supported in vitro transformation of miracidia into mother sporocysts, but also resulted in substantial subsequent growth; moreover, some degree of germinative development appeared to occur as well. Cerebral ganglia from uninfected adult snails of the intermediate host species, Lymnaea stagnalis, could produce factors promoting in vitro development of young mother sporocysts. Results are compared with data from the literature and it is concluded that greater success in in vitro culturing of young mother sporocysts of T. ocellata can be achieved than has hitherto been reported for other schistosome species. The same culture procedures were less successful when applied to other intramolluscan stages of T. ocellata, but can be used for in vitro maintenance of these stages. The procedures described here will be a useful tool in the study of schistosome-snail interactions in T. ocellata-L. stagnalis and possibly in other systems as well.     

Schistosoma mansoni: changes in the outer membrane of the tegument during development from cercaria to adult worm

Hockley D. J. and McLaren D. J. 1973. Schistosoma mansoni: changes in the outer membrane of the tegument during development from cercaria to adult worm. International Journal for Parasitology3: 13–25. The tegumental outer membrane of the cercaria is trilaminate: the adult worm, however, has a seven-layered membrane. Formation of the heptalaminate membrane commences immediately after the cercaria has penetrated the vertebrate host: multilaminate membrane-bounded vacuoles are passed from subtegumental cells into the tegument where they enlarge, join to the outer membrane and open to the exterior. The heptalaminate limiting membrane of the vacuole thus becomes the outer membrane of the tegument. At the same time the original trilaminate tegumental membrane is formed into microvilli which are cast off and thus the cercarial outer membrane is lost. Schistosomula usually have a heptalaminate outer membrane within three hours of penetration. After this time the large vacuoles are replaced by smaller membraneous bodies which presumably contribute to the outer membrane during growth of the schistosomulum. The membraneous bodies are also present in the tegument of the adult worms and there is some evidence that the outer membrane is continually renewed.     

Trichobilharzia ocellata: interference with endocrine control of female reproduction of Lymnaea stagnalis

Calfluxin (CaFl), one of the gonadotropic hormones of Lymnaea stagnalis, stimulates the influx of Ca2+ into the mitochondria of the cells of the albumen gland, one of the accessory sex organs of the snail. This effect is suppressed in glands of noninfected snails by an agent (schistosomin) present in the hemolymph of snails infected by Trichobilharzia ocellata as shown in in vitro experiments. The agent is present from 6 weeks postinfection onward. Ca2+ deposits in the mitochondria were demonstrated with the ultracytochemical antimonate precipitation technique. The percentage of Ca2+-positive mitochondria was taken as a measure for the effects of CaFl. This percentage appeared to be greatly reduced when glands were incubated in serum of infected snails (Sinf). The data showed that Ringer incubations can serve as controls for experiments with serum: no differences were found between Ringer incubations and incubations in either fresh or frozen serum of noninfected snails. Schistosomin was not affected by freezing, which enables cold storage of Sinf. The dose-response relationship of schistosomin shows that at a 1:2 dilution of Sinf with Ringer the response to CaFl was reduced more than 50%. Schistosomin is heat-stable and Pronase-labile, which indicates that it has a peptide nature. Probably schistosomin(s) is responsible for the reduction/cessation of fecundity in trematode-infected snails.     

In vitro optimization of the Gallus domesticus oviduct epithelial cells culture

The aim of this experiment was to establish an efficient method for isolation and further culture in vitro of the normal chicken oviduct epithelial cells (COEC) for cell-based research models. Different factors were tested to optimize COEC primary culture for repeatable results: the origin of isolated cells (oviduct Infundibulum or Magnum section); the oviduct tissue dissociation procedure (mechanical scrapping or mincing), tissue digestion times (15, 30 and 45 min), the culture plates coating (colagene I, polystyrene surface or 3T3 feeder layer), the growth media (classic DMEM/Ham's F12 and defined serum-free medium, Lonza Switzerland), incubation temperature (37 °C vs 41°C) and different cell seeding numbers: 0.2M, 0.5M and 1.0M cells/well. The COEC isolated by mincing the Infundibular neck and digestion of tissue for 30 min formed cell aggregates of bright colour and gave proliferating colonies of epithelial-like character which was the best result obtained from all applied procedures in our studies. The fibroblast-like cells considered as contaminants occurred only sporadically up to day 7 of culture. Seeding about 1M cells in 1 mL of serum-free medium onto 12-well dishes gave the optimal growth of colonies resulting in 5 to 7 confluent culture wells from a single oviduct sample. Feeder layer and collagen I did not improve adhesion of the COEC to the culture vessel. Adoption of 37 °C and 41 °C did not reveal apparent differences to the condition of cultured COEC. Cell differentiation and proliferation potential depends on number and replicative capacity of isolated progenitors. The progenitors are responsible for holoclones formation and good culture growth. The percentage of colonies developed from the cells isolated from Infundibulum was greater than that of other samples in our studies. We conclude that the model of COEC primary cultures from different segments of oviduct, in particular infundibulum, should be incorporated to the range of avian cells research as this work generates questions about undocumented sources of oviduct progenitor cells.     

不同培养体系对鼠-猪异种嵌合胚胎发育和嵌合能力的比较

近年来,随着干细胞分化与再生医学研究的不断深入,异种嵌合已成为当前干细胞和再生医学领域的热点问题,并有望为未来解决器官移植供体来源严重短缺等再生医学难题开辟新的方向。异种嵌合以及异种器官再造过程中面临众多科学问题和技术难题,而异种嵌合过程中嵌合胚胎时期的选择,后续培养液的选择以及这些环节所造成的供体细胞与受体胚胎之间的发育平衡成为建立异种器官再造的第一个科学问题。猪由于具有与人类器官大小相似、繁殖快等特点,成为异种嵌合最适合的潜在研究对象。为了提高鼠-猪异种嵌合胚胎中小鼠供体细胞——诱导多潜能干细胞(Induced pluripotent stem cells,i PSCs)的存活率和增殖率,我们尝试以i PSCs培养液(N2B27)以及N2B27→PZM-3梯度更换的培养液(N2B27(3.5 h))作为研究异种嵌合胚胎体外发育培养的对象,并与猪胚胎培养液(PZM-3,Porcine zygotic medium)体系下发育进行比较,从而评价了这3种培养液在8-细胞和囊胚期注射后,对嵌合胚胎后续发育的影响及嵌合情况。结果显示,8-细胞期注射后,PZM-3不仅对嵌合胚胎的后续发育较为有利,更有利于小鼠i PS嵌合到猪胚胎中;囊胚期注射后3种培养体系下GFP阳性嵌合率差异不显著,但其嵌合率显著低于8-细胞期嵌合率。结果表明,PZM-3培养体系更有利于鼠-猪异种嵌合胚胎的体外发育,对8-细胞期胚胎进行嵌合操作有益于提高鼠-猪异种嵌合后胚胎的嵌合率。     

Navigation within host tissues: Schistosoma mansoni and Trichobilharzia ocellata schistosomula respond to chemical gradients

After penetration of human or duck host's skin schistosomula of Schistosoma mansoni and Trichobilharzia ocellata migrate parallel to the surface in the epidermis, then they enter the dermis and venules prior to further migration. This study focuses on potential behavioural mechanisms and host cues which may enable this navigation within host tissues. We stimulated cercariae to penetrate into agar substrates and to transform to schistosomula, and analysed their orientation behaviour within chemical concentration gradients. Both species were chemotactically attracted by low molecular weight fractions of their host's serum (human, duck) and D-glucose and L-arginine were identified as attractive components in serum. They responded to gradients, which established after addition of very low concentrations of D-glucose (1 microM in T. ocellata and 2 microM in S. mansoni) and L-arginine (0.025 microM in T. ocellata and 1.0 microM in S. mansoni). The response to D-glucose was specific as other saccharides had no stimulatory activity. L-Arginine stimulated chemotactic orientation both when free and bound in peptides. However, the two species responded differently to the position of L-arginine within the peptide (terminal or subterminal), and only S. mansoni, not T. ocellata, responded to peptides occurring in serum and endothelial cells: fibronectin (1 microM), bradykinin (25 pM) and its fragment 1-5 (2.5 microM). Both species adjusted their body axis with the ventral side towards the higher concentrations of D-glucose and of L-arginine. We argue that the chemotactic orientation and the alignment of the body axis enable the parasites (i) to orientate towards deeper skin layers and avoid accidental perforation of the covering skin surface layers, (ii) to determine their position during their surface-parallel migration within the epidermis, (iii) to locate blood vessels.     

In vitro culture of Ginkgo

Summary Ginkgo biloba L. is an important landscape tree, is resistant to insect, fungi and other pests, and produces a number of chemicals that have pharmaceutical properties (termed ginkgolides). Studies were initiated to establish an in vitro culture protocol for Ginkgo. Explants (intact embryos, embryos with cotyledons removed, and cotyledon tissue) were removed from disinfested seeds and cultured on Murashige and Skoog minimal organics medium with various combinations of either 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthaleneacetic acid (NAA) and either kinetin or benzyladenine (BA). Cultures were incubated in the light and morphological development was recorded. Both embryo and cotyledon explants produced callus (cotyledon tissue produced the most callus). Ginkgolides A and B were detected in callus tissue extracts. Intact embryo cultures initiated on media with 2,4-D plus NAA for 5 wk produced shoots and roots when transferred to media with 4.5 μM 2,4-D alone for an additional 5 wk. Plants were transferred from the 2,4-D media to pots and maintained in the greenhouse.     

In vitro androgenesis of triticale in isolated microspore culture

Culture conditions for triticale (X Triticosecale Wittmack) androgenesis were studied using microspore culture. Sporophytic development of isolated triticale microspores in culture is described in five winter hexaploid triticale genotypes. Microspores were isolated using a microblendor, and embryogenesis was induced in modified 190-2 medium both in the presence and absence of growth regulators. The highest induction of microspore embryogenesis was obtained in a growth regulator-free medium. Adventitious embryogenesis was observed during in vitro development of triticale microspores. Albino and green plantlets were regenerated from embryo-like structures. More than 50% of regenerants were albino. In total, 126 green plantlets were produced, transplanted and established in soil. Cytological evidence revealed that 90% of the transplanted regenerants were haploid. This revised version was published online in June 2006 with corrections to the Cover Date.     

Morphological peculiarities of schistosomatid cercariae of Trichobilharzia cf. ocellata group occuring in Moscow and Saint-Petersburg populations

Morphology of schistosomatid cercariae of the genus Trichobilharzia (ocellata group) is described based on original and reference data. The material (Trichobilharzia cercariae) was collected in 1999-2000 from naturally 'infected snails Lymnaea stagnalis from Moscow and Saint-Petersburg megalopolises. The more accurate flame cell formula for these cercariae, as well as some aspects of chaetotaxy and eye morphology are given. The differences between cercariae from these regions were found. Based on a comparison of nef data with the reference data, it was found that the cercariae examined differ from those from the Central Europe.     

In vitro culture of blood cells from the colonial protochordateBotryllus schlosseri

Summary Primary cultures of circulatory blood cells from the colonial tunicateBotryllus schlosseri were cultivated in 96-well plates for up to 3 mo. in a medium based on Dulbecco’s modified Eagle’s medium, supplemented with salts to the botryllid ascidian hemolymph osmolarity, HEPES buffer,l-glutamin, fetal bovine serum, and antibiotics. Intercellular bridges between granular pigment cells were established within 24 h. The viability of these cells decreased slowly, and most died within 1 mo. without any sign of cell proliferation. Other cell types remained in an arrested state and were subjected to a weekly medium exchange. Spontaneous cell proliferation was randomly recorded in 6 to 10% of the wells from 2 wk to 1 mo. This proliferation was followed by the formation of masses of cell clumps, from which uniform hemocytes (5 μm, lymphocytelike cells) migrated peripherally. Stress conditions, which included longer intervals between medium exchanges and partial medium replacement, increased the probability of cell proliferation. From each proliferating primary culture, we successfully performed up to 10 plating cycles over a period of 15 wk, during which the cells differentiate in size but are uniformly structured. This produced the firstBotryllus lymphocytelike cell line. From this stage, cell numbers remained constant for up to 6 mo. without increase in cell number. Several mitogenic factors were employed on primary cultures.Botryllus and sea cucumber hemolymphs and mixed interleukins were found to augment significantly proliferation of at least one specific cell size, whereas cells were not markedly responsive to lectins (Concavalin A, wheat germ agglutinin,Ulex europaeus agglutinin), insulin, and retinoic acid. The results are discussed with respect to future efforts in the development of tunicate blood cell cultures.     

Trichobilharzia ocellata: Quantification of effects on haemocytes of the pond snail Lymnaea stagnalis by morphometric means

To identify functionally different subpopulations, we quantified by morphometric means the spreading activity of circulating haemocytes of the pond snail Lymnaea stagnalis, recognized by monoclonal antibodies (5 surface and 5 cytoplasmic). The influence of snail age and of the different intramolluscan stages of the compatible avian schistosome Trichobilharzia ocellata on this activity were studied. The antibody-recognized cells could be separated into two groups, differing in their spreading activities. The probes detecting cytoplasmic markers recognized the majority of cells (78-95%). These were active (well spreading), differentiated cells. The surface probes recognized a smaller part (12-38%) of the total haemocyte population. These harmocytes were less active (less spreading), and were predominantly immature cells. The relative sizes of the antibody-detected subpopulations were not affected by snail age or infection with T. ocellata. The maximal size (planimetric area) attained after attaching to glass of all cells increased between day 0 (juvenile snails) and about 6 weeks post (sham) exposure and then decreased. Infection had little effect on the spreading activity of the more differentiated cells. The less differentiated cells showed a larger spreading activity when the parasite was present as mother sporocyst and also when the digestive gland area became colonized by growing daughter sporocysts.     

Snail host finding by Fasciola hepatica and Trichobilharzia ocellata: compound analysis of "miracidia-attracting glycoproteins"

The glycoconjugates from snail-conditioned water of Lymnaea truncatula and L. stagnalis which elicit typical host finding behavior in miracidia of Fasciola hepatica and Trichobilharzia ocellata were separated by anion-exchange chromatography and a two-step size-exclusion chromatography. We obtained fractions attractive for the parasites with MW of about 10(6) Da in both snail species. These fractions still contained species-specific information since miracidia responded only to molecules from their respective host snail. Analysis of the amino acid composition from the protein backbone revealed a similar composition in the effective fractions of both snails. Amounts of serine and threonine were higher than 30 mol %, which is typical for mucin-type glycoproteins. The carbohydrate moieties consisted mainly of galactose and fucose, but nine different other monosaccharides also were identified in smaller amounts. The heterogeneity of the molecules was also confirmed by the binding of six different lectins. Because of these characteristics, the effective molecules were termed "miracidia-attracting glycoproteins" (MAGs). MAGs may play an important role for parasite transmission, as they may increase the chance of an encounter between parasite and host and enable the miracidia to discriminate between their specific intermediate host and other unsuitable snail species.     

In vitro culture of macrobrachium eggs

Causes for the death of the eggs in the prawn Macrobrachium nobilii are: i) shedding of eggs by ovigerous female, and ii) infection by epibionts: a Saprolegnial fungus, bacteria (gram negative) and protozoans (Vorticellids and Paramecium). A cause for the death of freshly hatched larvae of some decapods is the reduction in reserve yolk energy in the larvae hatched in the last few batches. To circumvent these disadvantages, an artificial incubator was designed, in which 70% of the 3-day old eggs can successfully be incubated and hatched simultaneously. The isolted eggs are irrigated with filtered and aerated water over a diaphragm in the incubator; the water flushed from below through the diaphragm in the artificial incubator, sways and keeps the eggs continuously in a suspended motion, simulating the irrigation technique of the mother.Presented in the Second International Symposium on Invertebrate Reproduction held in Davis, California during August, 1979     

Snail odour-clouds: spreading and contribution to the transmission success of Trichobilharzia ocellata (Trematoda, Digenea) miracidia

Chemical communication among freshwater organisms is an adaptation to improve their coexistence. Here,we focus on the chemical cues secreted by the freshwater gastropod Lymnaea stagnalis, which are known to stimulate behavioural responses of Trichobilharzia ocellata (Plathelminthes, Digenea, Trematoda) miracidia. Such responses are commonly claimed to influence transmission positively, but in response to chemical cues miracidia randomly change their swimming direction. This kind of response does not necessarily increase transmission, because miracidia may be trapped at the periphery of very large snail odour-clouds, which may prevent them from approaching the snail. On the other hand, the odour clouds may be too small to improve host-localisation. To shed light on these scenarios, the spreading of molecules released around L. stagnalis (active space) was visualised by recording host-finding responses of T. ocellata miracidia when they approached snails. Behavioural responses of miracidia indicated the spreading of compounds forming an attractive active space only around the host-snail L. stagnalis, but not around sympatric non-host-snail species. The active space increased approximately linearly with the time the snail rested at the same spot and within 5 min it reached a volume of more than 30 times that of the snail. We also demonstrated in a large-scale experiment, that the active space of L. stagnalis significantly increases the transmission success of T. ocellata miracidia. Additionally, the microhabitat selection of T. ocellata miracidia was studied, demonstrating that peripheral locations near the water surface were preferred, which are also preferred sites of L. stagnalis. Improved chemoperception and microhabitat selection may have been a consequence of coevolution with snails and benefited miracidia, which became efficient transmissive stages. Electronic Supplementary Material Supplementary material is available for this article at