In vitro culture of marine trematodes from their snail first intermediate host

The ability to culture parasites outside their host (i.e. in vitro) is essential for several aspects of parasitological research. Here, a culture medium for marine trematode parthenitae was optimized using Philophthalmus sp. rediae from the intermediate snail host, Zeacumantus subcarinatus. The medium was optimized by sequentially testing the suitability of different levels of osmolality, different commercially available media, and different concentrations of supplemented chicken serum, while controlling for genetic variation among cultures. Philophthalmus sp. rediae survived up to 56 days in cultures of the best tested medium, remaining active and continuously shedding cercariae. The broader suitability of the culture medium was tested using five other trematode species from different families (using either the same or other marine snails as first intermediate hosts): Galactosomum sp., Acanthoparyphium sp., Maritrema novaezealandensis, Curtuteria australis, and an undescribed species of the family Opecoelidae. Survivorship of rediae and sporocysts from these species ranged from eight days to 42 days. The culture procedures developed here can therefore be used in the future as a system under which to culture marine trematode parthenitae for experimental studies.     

In vitro culture of rediae of Echinostoma caproni.

Rediae of Echinostoma caproni (Egyptian strain) were dissected from Biomphalaria glabrata snails at intervals from 13-34 days post-exposure and co-cultured for up to 51 days with cells of the B. glabrata embryonic (Bge) cell line. Rediae readily ingested Bge cells and survived longer when co-cultured with cells than in cell-free cultures. Rediae released mostly motile cercariae throughout the observation period when in Bge medium and cells. Rediae cultured in 199 medium with Bge cells also produced progeny throughout most of the observation period. In the latter medium, progeny were much more likely to include rediae as well as cercariae. Some cercariae produced in vitro encysted as metacercariae. Rediae consumed cercariae released into culture but were not observed to attack one another or rediae of a different echinostome species.     

Observations on the host-parasite relations between Echinostoma revolutum and lymnaeid snails.

Echinostoma revolutum from Taiwan was studied in lymneid snails at 29 +/- 0.5 degrees C. Given 3-5 miracidia, 95% of Lymnaea ollula and 40% of Lymnaea swinhoei became positive; the prepatent periods were 18 and 25 days, respectively. The following are based on the observations in Lymnaea ollula: The time required for miracidial penetration was about one hour. The sporocysts developed only in the ventricle of the snail but mother rediae developed in the heart and other organs. Mature daughter rediae were not found in the heart cavity. The sporocysts reached the ventricle within 3 days. Mother rediae were released after 6 days and daughter rediae after 8 days. Given 5 miracidia, 1-3 sporocysts reached the heart and 2-20 mother rediae were found per snail. The number of mature daughter rediae was usually 100-200 although more than a thousand may develop in a snail. The sporocysts and mother rediae attained maximal size 9 days postinfection and started degeneration 13 days postinfection. Daughter rediae were largest at the beginning of cercarial emergence and decreased in size thereafter. Simultaneous production of daughter rediae and cercariae by the mother redia was seen only once in this snail mature cercariae were obtained in 10 days postinfection. The cercariae emerged from a small area of mantle collar near the posterior corner of shell aperture. They were negatively phototactic and positively geotactic. An estimation showed that each snail shed about 350 cercariae a day. The cercariae reached the pericardial cavity of snail in one hour via the renal orifice and metacercariae were seen 4-5 hours after exposure. The infectivity of cercariae at various times after shedding, as expressed by cyst recovery rates, were: 51.6%, O-hr old; 76.1%, 2-hr; 68% 4-hr; 32%, 6-hr; 3%, 8 and 10-hr. Cyst recovery rates were not different within the dosage of 50-500 cercariae per snail. Most metacercariae recovered 1-2 days after cercarial exposure were viable; only 5 among 6,533 cysts were dead.     

Insights into the development of Notocotylus attenuatus (Digenea: Notocotylidae) in Lymnaea stagnalis: from mother sporocyst to cercariae

Notocotylus attenuatus (Digenea: Notocotylidae) is a monostome fluke parasitizing the intestinal caeca of waterfowl that uses an injection apparatus to infect its intermediate snail host. Morphology of the invading larva (a sporocyst), and the intramolluscan larval development of this fluke have not been characterized extensively. In this study, experimental infections of Lymnaea stagnalis using N. attenuatus eggs resulted in the development of sporocysts containing one germ ball or mother redia between 12 and 21 days post exposure (p.e.) within the hepatopancreas. Independent mother rediae and developing daughter rediae were present between day 25 and day 42 p.e. Cercariae, within the body of rediae, were detected 42 days p.e. The development of daughter rediae and cercariae started posteriorly in the body of parent redia and these larvae migrated anteriorly during development towards the birth pore. A cercaria was also observed emerging from the birth pore and released cercariae maturated further within the snail hepatopancreas prior to leaving the snail. The intramolluscan development was completed 45 days p.e. when the first fully formed cercariae were shed into the outer environment. These data detail the fascinating post-embryonic development of N. attenuatus and highlight the intricate nature of larval transitions within its snail host.     

β-Galactosidase-deficient human fibroblasts: uptake and processing of the exogenous precursor enzyme expressed by stable transformant COS cells

Summary COS-1 cells were transfected by electroporation with a cDNA for human acid -galactosidase cloned in our laboratory and stable transformants expressing the enzyme activity were selected. The precursor form of the enzyme was secreted in large quantities into the culture medium. The fibroblasts from patients with G_M1-gangliosidosis or Morquio B disease showed a remarkable increase of enzyme activity, up to the normal level, after culture in this medium for 2 days; the amount of uptake was essentially the same as that for the precursor form in human fibroblasts. After endocytosis, the precursor molecules were processed normally to the mature form and remained as stable as those produced by human fibroblasts. On the other hand, cells from galactosialidosis patients did not show any increase of enzyme activity in a similar experiment. It was concluded that the transformants are useful as the source of precursor proteins for the study of intracellular turnover of enzyme molecules in mutant cells.     

The influence of hypomagnesemia on erythrocyte antioxidant enzyme defence system in mice

The effect of magnesium deficiency on antioxidant defence system was studied in RBC of mice suffering from hypomagnesemia. The animals were kept for 8, 15 and 22 days on magnesium-deficient diet with consequent reduction of magnesium level in plasma by 38% at the first 8 days and by 64% after 22 days of experiment. The activities of the most important antioxidant enzymes, catalase, glutathione peroxidase, superoxide dismutase, glutathione reductase, glutahione S-transferase were assayed in hemolysates. The level of reduced glutathione in erythrocytes was measured as well. Apart from catalase, the activities of antioxidant enzymes were decreasing. The activity of superoxide dismutase decreased gradually during the experiment and on the 15th and 22nd day of experiment was significantly (P<0,05) lowered by 30 and 32% respectively. The catalase activity was increased on each point of the experiment with the peak value up to 149% on 15th day, and by 32% on 22nd day. Glutathione peroxidase activity was insignificantly reduced. The reduction of Glutatione reductase and Glutathione S-transferase activities by 24 and 21%, respectively, were observed after 8 days of the experiment with a further downward tendency. The reduced glutathione was significantly depleted after 8 days by 33% and was kept on that level in the course of the study. These findings support previous reports on the hypomagnesemia – induced alteration in endogenous enzyme antioxidant defences and glutathione redox cycle of mice.     

Himasthla quissetensis: uptake and utilization of glucose by rediae as determined by autoradiography and respirometry

Daughter rediae of Himasthla quissetensis removed from the digestive gland of Nassarius obsoletus were placed in sterilized seawater fortified with antibiotics. When [³H]-glucose was added to this medium and autoradiographs were made after 3, 9, and 24 hr of exposure, labeling was observed associated with the redial walls and developing germ balls and cercariae within the brood chambers. Respirometric determinations on starved rediae suspended in the seawater medium with and without glucose revealed the rate of oxygen utilization by rediae exposed to exogenous glucose is significantly elevated. These results are interpreted to mean that the daughter rediae of H. quissetensis can take up and utilize glucose.     

Redial generations of Fasciola gigantica in the pulmonate snail Lymnaea truncatula.

Lymnaea truncatula, 4 mm in height, were subjected to infection by a single miracidium of Fasciola gigantica, then raised at 23 degrees C until day 60 of the experiment. Histological study of these snails demonstrated a mean redial burden of 34 parasites at day 60, of which one third were degenerating forms. The mean number of living independent rediae did not exceed 5 for the first and second generations. Conversely, in subsequent generations there were as many as 18 rediae per snail at day 60. The first living redia of the first generation in particular gave rise to daughter rediae. Mature rediae appeared at day 35 and especially concerned the first and second generations at day 60. The authors conclude that development of the first and second redial generations occurs during the same period, and that the forms of the first cohort of the second generation are produced from the first redia of the first generation which originated from the sporocyst.     

甘蓝型油菜游离小孢子培养的胚胎发生

以生长在非控温控光下的４个冬性甘蓝型油菜（ＢｒａｓｓｉｃａｎａｐｕｓＬ．）品种为供体,进行游离小孢子培养。研究发现,多数品种在开花３－７天取材最为适宜。在甘蓝型油菜小孢子培养中只有单核晚期的小孢子才可能发育成胚状体,而花药培养时处于单核早期的小孢子易于发育成胚状体。在适当花期选取发育比较一致的单核晚期小孢子培养,经数小时后,部分小孢子便开始膨大,这是小孢子发育成胚的最早标志,膨大的小孢子中,有部分形成多细胞球并进一步发育成胚。用春性甘蓝型油菜为材料进行蔗糖浓度的实验结果表明：培养３天后,在１６％蔗糖培养基中存活的小孢子最多,达１６．１３％;培养３０天后,胚状体诱导频率则以１３％蔗糖浓度为最高,每花蕾可达１４４个胚状体。如果在１６％蔗糖培养基中培养３天后,添加等体积的１３％蔗糖培养基,能够大大提高胚状体的诱导频率,为仅用１３％蔗糖培养基培养的３．７倍。这一实验体系正在用于抗菌核病的诱变与筛选,并作为外源基因导入的实验体系。     

Echinostome rediae: effect of pretreatment with cold on transplantation to uninfected snails.

When donor snails, Lymnaea stagnalis, infected with Echinoparyphium aconiatum were held at 13 C for at least 24 days, transplantation of newborn rediae from them to uninfected snails was 2.5 times as successful as when donor snails were held at 23 C. The difference does not seem to be do to the fact that rediae transferred from 13 C-snails were slightly larger than those from 23 C. This effect of temperature was not observed in analogous experiments with another echinostome, Isthmiophora melis. The mechanism of this phenomenon found in the experiments with E. aconiatum is still unknown.     

Metabolic and enzymatic characteristics of adult rat liver parenchymal cells in non-proliferating primary monolayer cultures

Parenchymal cells from normal adult rat liver, prepared with high yield (30 × 10⁶ cells/g liver) and viability index (>96%) by a non-perfusion method, were maintained in non-proliferating monolayer culture. Several metabolic functions were investigated for 7 days to evaluate functional integrity of the cultured hepatocytes. Leucine was linearly incorporated into protein for 4.5 h at each day of cultivation and the incorporation rate increased up to 2-fold after 3 days. Urea production was maintained at a rate of 0.5 μmoles/mg protein × h for at least 7 days, and its amount was enhanced 2-fold within 24 h by the addition of 3 mM NH₄Cl. Glucose was formed during the first days by the hepatocytes and was then taken up with increasing amount from the surrounding medium. Lactate consumption, on the other hand, was replaced by lactate production after one day of cultivation.Variations in enzyme levels of lactate dehydrogenase, arginase, glutamine synthetase and glucose-6-phosphatase were also studied during the whole culture period. Cell leakage, which was detected only in the case of lactate dehydrogenase (LDH), occurred through the 4th day along with a concomitant loss of intracellular LDH activity. After 4 days, however, the enzyme activity returned to the initial level. Arginase was maintained throughout the cultivation period and was stimulated 2- to 3-fold within 24 h by NH₄Cl. Glutamine synthetase declined within the first 4 h of cultivation and then remained in the hepatocytes with a transitory rise after 2 days. Its activity was also found to be inversely related to the concentration of glutamine in the culture medium up to 4 mM. Glucose-6-phosphatase gradually decreased during the cultivation period, the enzyme activity, however, was stimulated by glucagon within 24 h.     

Fasciola hepatica: cercarial productivity of redial generations in long-surviving Galba truncatula

Bimiracidial infections of Galba truncatula with Fasciola hepatica were carried out to determine the effect of food quality on the frequency of 1- and 2-sporocyst infections, to analyse its impact on the developmental patterns (normal, or abnormal) of redial generations, and to verify its consequences on cercarial production. These investigations were performed in snails reared at 20 degrees C and provided with cos lettuce and commercial fish food (Tetraphyll) as a food source until their death. Double-sporocyst infections with normal development of redial generations were recorded in 43.9% of infected snails (out of 296). Single-sporocyst infections were noted in the other snails, with normal development of generations in 53.7% and abnormal development (the first mother redia early degenerated) in 2.4%. Four successive redial generations were found in long-surviving snails (more than 90 days). In both 1- and 2-sporocyst infections, showing normal development of generations, the daughter rediae, which exited from the first mother redia (R2a rediae), constituted the greater group of free rediae and produced the highest percentages of cercariae (46.2-48.2%). However, the development of these rediae inside the snail body was slower in 2-sporocyst infections than in 1-sporocyst infections. The numbers of rediae noted in subsequent generations (R2b/R3a and R3b/R4a rediae) were similar, whatever the number of full-grown sporocysts. The number of shed cercariae recorded in the 1- and 2-sporocyst infections did not significantly differ. When long-surviving snails died, 19.8-20.7% of cercariae produced by free rediae (essentially by R2b/R3a and R3b/R4a rediae) were still present in their bodies. The increased frequency of 2-sporocyst infections demonstrated that food quality had a significant effect on the redial burden of F. hepatica developing inside G. truncatula.     

The effect of incubation temperature on the maintenance of rat palatal mucosa in organ culture

The effect of incubation temperature on the behavior of neonatal rat palatal mucosa maintained in a chemically defined medium in organ culture for periods up to 7 days was investigated. Explant survival was optimal at 37 degrees C with increasing mortality at temperatures of 34 degrees C and 30 degrees C. There was a transient increase in the epithelial mitotic activity at all temperatures, but at all time intervals mitotic activity was greatest at 37 degrees C. While the mitotic activity at 37 degrees C after 5 hr in vitro was comparable with previously described in vivo values, it was subsequently increased, only returning to values approximating those at the start of the experiment at 6 days. At 30 degrees and 34 degrees C the epithelial mitotic activity increased more slowly than at 37 degrees C; then it followed a similar pattern with time and after 5 days in vitro had fallen to values approximating initial values. At the cut edges of the explants, the rate of epithelial migration and subsequent keratinization increased with increasing temperature. It is suggested that survival of neonatal rat palatal mucosa is optimal in this organ culture system when maintained at 37 degrees C.     

Calcium dynamics of hemocytes of the gastropod Biomphalaria glabrata: effects of digenetic trematodes and selected bioactive compounds

Abstract. Two fluorescent calcium indicators, Calcium Green AM (CG) and Fura Red AM (FR), were used in conjunction with confocal microscopy to monitor hemocyte calcium dynamics following exposure to digenetic trematode larvae or relevant bioactive compounds. Changes in intracellular calcium levels, as measured by fluctuations in the CG/FR ratio, were correlated with hemocyte morphological changes. Hemocytes exposed to culture medium remained spread and had few calcium transients. However, following exposure to sporocysts, sporocyst secretory-excretory products, or small rediae of Echinostoma paraensei in culture medium, significantly more hemocytes both rounded up and exhibited calcium transients, though some hemocytes showed one response or the other but not both. Hemocytes did not respond significantly to large rediae, to sporocysts of another digenean ( Schistosoma mansoni ), or to bacterial lipopolysaccharides. Exposure to either zymosan particles or mannose BSA provoked responses similar to those seen with sporocysts of E. paraensei Caffeine caused rounding but no calcium transients, and phorbol myristate acetate provoked calcium transients but no rounding. The results show that sporocysts and small rediae of E. paraensei have pronounced effects on hemocyte rounding and calcium dynamics, and that these two events can occur independently of one another. This suggests that parasites may influence hemocytes in at least two separate ways.     

Hormonal secretion and enzyme activity of cultured roe-deer (Capreolus capreolus L.) Leydig cells, as measured by radio-immunological and histochemical assays

Leydig cells from roe-deer collected according to Steinberger's (1975) technique were cultured as monolayers in Leighton tubes for 10 days. Cultures were grown in medium 199 supplemented with 10% calf serum. Androgen and oestrogen secretion by Leydig cells into the culture medium was measured using appropriate radio-immunoassays. Using histochemical tests the activity of the following oxydoreductive enzymes in cultured Leydig cells was shown: delta 5, 3 beta-hydroxysteroid dehydrogenase (delta 5, 3 beta-HSD), 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD), succinate and lactate dehydrogenases (SDH and LDH). Strong activity of the enzymes investigated during the first 4 days of culture was observed. The androgen level was high throughout the second and fourth day of culture. A decrease in hormone secretion after day 4 occurred, and this was closely correlated with enzyme activity. The oestrogen level was very low during culture. The direct effect of the luteinizing hormone (LH) added into the culture medium caused an increase in not only enzyme activity but also androgen and oestrogen levels.     

Production of quail (Coturnix japonica) germline chimeras derived from in vitro-cultured gonadal primordial germ cells

A previous report from our laboratory documented successful production of quail (Coturnix japonica) germline chimeras by transfer of gonadal primordial germ cells (gPGCs). Subsequently, this study was designed to evaluate whether gPGCs can be maintained in vitro for extended period, and furthermore, these cultured PGCs can induce germline transmission after transfer into recipient embryos. In experiment 1, gonadal cells from the two strains (wild-type plumage (WP) and black (D) quail) were cultured in vitro for 10 days. Using antibody QCR1, we detected a continuous, significant (P = 0.0002) increase in the number of WP, but not D, PGCs. QCR1-positive WP colonies began to form after 7 days in culture. On Day 10 of culture, 803 WP PGCs were present as a result of a continuous increase, whereas no D PGC colonies could be detected and the D gonadal stroma cells were rolled up. Differences in the PGCs or the gonadal stroma cells of the two different strains might account for these differences. In experiment 2, WP PGC colonies were maintained in vitro up to Day 20 of culture, and 10- or 20-day-cultured PGCs were microinjected into dorsal aortas of 181 recipient D embryos. Thirty-five (19.3%) of the transplanted embryos hatched after incubation, and 25 (71.4%) of the hatchlings reached sexual maturity. Testcrossing of the sexually mature hatchlings resulted in three (10 days, 33.3%) and eight (20 days, 50.0%) germline chimeras respectively. This report is the first to describe successful production of germline chimera by transfer of in vitro-cultured gPGCs in quail.     

Long-term culture of adult rat hepatocyte spheroids

Hepatocytes from adult rats were cultured on poly-HEMA-coated surface to form spheroids in hormonally defined media as previously shown with newborn rat hepatocytes. Spheroidal aggregates of adult rat hepatocytes were morphologically similar to those of newborn rat hepatocytes and could also form a monolayer of uniform liver parenchyma-like cells when transferred on collagen-coated surfaces even after 2 months of culture. Under these culture conditions, albumin and transferrin secreted in vitro by adult rat hepatocyte spheroids were detectable by immunoprecipitation method at least until 2 months of culture. The production of proteins by hepatocyte spheroids could be regulated in vitro by IL-6: the secretion of alpha 2-macroglobulin was increased and the secretion of albumin was decreased in the presence of this cytokine. In addition, cytochrome P450 IA1 was strongly induced by methylcholanthrene in adult rat hepatocyte spheroids, and the induction remained relatively constant up to 22 days of culture. These cells were also able to metabolize lidocaine to monoethylglycinexylidine when measured up to 14 days of culture, showing the presence of a relatively high level of P450 IIIA2. The UDP-glucuronyltransferase activity, specific for bilirubin conjugation, decreased to 18% of the initial value after 2 weeks of culture. This work showed that adult rat hepatocytes in long-term spheroid culture kept differentiated functions, providing a new model for the in vitro study of hepatocyte functions and complementing that of newborn rat hepatocytes using the same system.     

Fasciola hepatica: the growth and larval productivity of redial generations in Galba truncatula subjected to miracidia differing in their mammalian origin

Experimental infections of Galba truncatula with 4 isolates of Fasciola hepatica miracidia differing by their mammalian origin (cattle, nutrias, rabbits, or sheep) were carried out to determine if parasite origin had an effect on the number of free rediae, their growth, and their larval productivity in each redia category. The mammalian origin of miracidia had a significant influence on the numbers of free rediae (they were higher in cattle-group snails) and the lengths of rediae (they were lower in rabbit groups). The redia category had also a significant effect on body and pharyngeal measurements. In all groups, the majority of cercariae (55.8-63.2%) were produced by the daughter rediae (R2a rediae) originating from the first mother redia. Compared with the other groups, the mean number of cercariae at day 49 postexposure was twice as high in cattle groups. In contrast, the mean number of daughter rediae produced by each second-appearing mother redia or each R2a redia was higher in the nutria, rabbit, and sheep groups. The mammalian origin of F. hepatica miracidia had an effect on the number of live rediae, their length, and their redial and cercarial productivity.     

Larval development of Fasciola hepatica in experimental infections: variations with populations of Lymnaea truncatula

A retrospective study was undertaken on 70 French populations of Lymnaea truncatula experimentally infected with Fasciola hepatica to determine whether or not susceptibility of snails to infection influenced redial and cercarial production. Results were compared with those obtained from two control populations, known for prevalences higher than 60% when experimentally infected with F. hepatica. In the 70 other populations examined, the prevalences ranged from 2 to 75%. In 55 of these populations, where the prevalence was more than 20%, a high proportion (50.1-56.8%) of snails died after cercarial shedding, whereas in the other groups (non-shedding snails with the most differentiated larvae being free cercariae, rediae containing cercariae, immature rediae, or sporocysts, respectively), snail death was significantly less. In 11 populations, where the prevalence values were 5-19%, only 14% of snails died after cercarial shedding, whereas snails with free cercariae, rediae with cercariae, or immature rediae showed significant increases in snail mortality. In the remaining four snail populations, with prevalences of less than 5%, the most differentiated larval forms were only immature rediae and/or sporocysts. Overall, the number of rediae containing cercariae significantly decreased with decreasing prevalence values. The low prevalence of experimental infection in several populations of snails might be explained by the occurrence of natural infections with miracidia originating from a mammalian host other than cattle, and/or by genetic variability in the susceptibility of snails to infection.     

Superoxide dismutase activity in callus from the C3-CAM intermediate plant Mesembryanthemum crystallinum

In light-grown callus obtained from M. crystallinum hypocotyls, three classes of superoxide dismutase (SOD): Mn-, Fe- and Cu/ZnSOD were identified. Callus cultured on a medium containing 0.4 M NaCl showed an increase in FeSOD activity on day 4 of the experiment. In contrast, Cu/ZnSOD activity was higher over 16 days of the experiment. Salinity stress induces oxidative stress mainly for the cytosolic SOD form (Cu/ZnSOD). After 16 days of callus culture on salt-containing medium, diurnal malate oscillations, and an increase in NADP-malic enzyme activity were noticed. These results strongly suggest that C₃-CAM transition can also be expressed at the cellular level. Therefore, callus tissue could be a useful model, similar to a whole plant, for investigation of mechanisms of stress responses in M. crystallinum.