Worm Culture Research Articles

A Model of Elegance

GenBank, http://www.ncbi.nlm.nih.gov/Web/GenbankGenome Sequencing Center, University of Washington School of Medicine, http://genome.wustl.edu/gsc/gschmpg.htmlThe Sanger Center, http://www.sanger.ac.ukXREFdb, http://www.ncbi.nlm.nih.gov/Bassett/modelorgs

Heterogeneous population growth, parental effects and genotype-environment interactions of a marine oligochaete

Cultures of asexually reproducing populations of the oligochaete Paranaislitoralis (Muller) collected from six different patches (3 to 50 m apart) on an intertidal mud flat in Flax Pond, New York, on two occasions, June and October 1993, showed significant differences among lines in life span, number of offspring produced, and in finite rate of increase (λ). Although growth rates were significantly lower in October than in June, they were always positive (λ > 1) in the laboratory cultures reared in field-collected sediment, while field data show that the densities of P. litoralis decreased sharply in summer and autumn from a seasonal high in early June. Cultures of worms reared at high densities without renewal of sediment crashed, and effects on individuals were irreversible: worms from late (declining) stages of population growth had a significantly higher mortality and lower reproduction than worms from earlier stages, also when transferred to high-quality food. Genetical analysis using RAPDs (random amplified polymorphic DNA) confirmed the existence of several clones of P. litoralis in our cultures. Experiments where parent and offspring were cultured in sediments of different qualities showed clone–environment interactions in the number of asexual offspring produced, but not in age at first reproduction. Clones also differed in that some showed significant parental effects of sediment quality on life-history characteristics while other clones did not. Our results indicate that P.litoralis populations in Flax Pond are not an example of a population subdivided into a set of permanent source and sink subpopulations, but rather an example of a continuously shifting mosaic of local growth conditions.

Enzymatic basis for the lack of oxamniquine activity in Schistosoma haematobium infections.

The notion that oxamniquine is active against Schistosoma mansoni but inactive against S. haematobium was confirmed using in vitro cultures of adult worms. Since oxamniquine and hycanthone have been shown to become effective upon activation by a schistosome enzyme, enzymatic tests were carried out to detect possible differences between the enzyme of S. mansoni and that of S. haematobium. It was found that the S. mansoni enzyme could activate hycanthone and, to a lesser extent, oxamniquine. The S. haematobium enzyme, on the other hand, was capable of activating hycanthone but virtually incapable of activating oxamniquine. It is concluded that the different activity of oxamniquine in the two species is due to differences in the drug-activating enzyme.

Oviposition by Schistosoma mansoni during in vitro cultivation.

Observation of Schistosoma mansoni oviposition during in vitro culture of adult worms for a maximum period of 10 days showed three well distinct phases in the kinetics of oviposition: an initial phase with low egg production, a period of maximum oviposition and finally a progressive reduction in the number of eggs during the late phases of culture. The kinetics of oviposition and the number of eggs laid by the parasites are influenced by the number of worm pairs per amount of RPMI 1640 medium, time of parasite development in the vertebrate host and type of serum utilized in the culture medium.

Effect of Brugia malayi on the Growth and Proliferation of Endothelial Cells In vitro

Athymic mice (C3H/HeN) parasitized by Brugia malayi develop massively dilated lymphatics. The lymphatic endothelial lining is perturbed, and numerous mononuclear and giant cells are closely apposed to the endothelium. The hyperplastic endothelial cells and low opening pressure of the lymphatics suggest abnormal multiplication of these cells may be important in the dilation. We studied the in vitro growth rate of human umbilical vein endothelial cells cultured with adult worms and microfilariae of B. malayi. The tetrazolium salt reduction assays were used to quantify possible direct mitogenic or inhibitory effects. The growth factor-induced proliferation of endothelial cells was significantly suppressed by 44-51% on day 1, 46-81% on day 3, and 45-79% on day 5 in cultures containing adult female worms, which had greater suppressor activity on endothelial cell proliferation than male worms, microfilariae, or soluble adult worm extract. Culture supernatant containing female worm excretory-secretory products significantly inhibited the growth and multiplication of cells, suggesting that adult female worms release antigens or proteins that have inhibitory activity on growth factors necessary for endothelial cell proliferation in vitro. Excess human recombinant epidermal growth factor and bovine brain extract partly reversed the inhibitory activity of worms in culture and restored the endothelial cell proliferation when incubated with worm culture supernatant. Indomethacin and BW 775Hcl failed to restore normal endothelial proliferation in the presence of female worms, suggesting that parasite-derived prostanoids and cyclooxygenase products did not cause the inhibition. Lymph from dilated lymphatics, but not serum from infected mice, increased the proliferation of cells in vitro. Together, these data demonstrate that excretory-secretory products of B. malayi parasites suppress vascular endothelial proliferation in vitro. Furthermore, increases in the number of these cells in vitro in the presence of lymph suggest that parasite-induced host factors may be important in modulating the degree of proliferation.

Serum-Free Culturing of Adult Schistosoma mansoni in Dialysis Bags for the Production of Excretory/Secretory Antigens

Antigenic excretory/secretory (E/S) products from Schistosoma mansoni are potentially important in the development of diagnostic assays used to detect circulating antigens in schistosomiasis. The E/S products to be used as antigen(s) for this development must, by necessity, be free of exogenous proteins. The ability to extend serum-free in vitro culture of adult worms is, therefore, essential. Adult worms were perfused from mice, washed in serum-free RPMI-1640 with antibiotics, and placed in sterile dialysis bags, molecular weight cut-off 10 kDa, at a concentration of 100 worms in 1 ml of serum-free, supplemented RPMI-1640. Each bag was then placed in a flask of supplemented RPMI-1640 with 10% fetal calf serum in a humidified incubator at 37 C, 7% CO2. At days 1, 3, 5, 8, and 12, worms were collected; E/S culture medium was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Worm survival rates were 85% after 1 day in culture, dropping gradually to 65% on day 8, and then to 38% on day 12. Silver stain for total protein and immunoblot exposed to positive human infection serum showed E/S culture media from days 3 and 5 having the least complex banding pattern. The quantitative specific activity of E/S, as measured by antigen-limiting Falcon assay screening test system-enzyme-linked immunosorbent assays against human infection serum, indicates E/S antigenicity closely follows the attrition of worms and, therefore, may be directed against the release of somatic antigens by dead worms. Culturing S. mansoni in dialysis tubing is useful in deriving E/S products.

Uptake, Intracellular Distribution, and Stability of Oligodeoxynucleotide Phosphorothioate bySchistosoma mansoni

The in vitro uptake, cellular distribution, efflux, stability, and toxicity levels of an oligodeoxynucleotide phosphorothioate (PS-oligonucleotide) have been studied in mature Schistosoma mansoni worms. The intracellular accumulation of 35S-labeled PS-oligonucleotide occurred roughly in proportion to the worm body mass over a wide concentration range, whether the worms were exposed singly or in mating pairs. Cellular uptake was dependent on the extracellular concentration. A minor fraction (13%) of the PS-oligonucleotide taken up by the worm accumulated in the surface tegumental coat. Most of the PS-oligonucleotide taken up localized in the cytosol (54%) and the nuclei-enriched (33%) fractions. In a time course study on adult worms in culture, oligonucleotide uptake was observed within the first 2 h and peaked at about 36 h. A decrease in the intracellular concentration of the PS-oligonucleotide was observed by 42 h. Analysis of the extracted oligonucleotides showed that PS-oligonucleotide was digested slowly. Efflux of the oligonucleotide was time and temperature dependent. Significant toxicity to the cultured worms did not occur until the PS-oligonucleotide concentration was over 8 mg/ml (1 mM).

Necator americanus secretory acetylcholinesterase and its purification from excretory‐secretory products by affinity chromatography

Acetylcholinesterase (AChE) secretion by adult N. americanus was enhanced in vitro by incorporating insoluble collagen rafts into culture dishes. Enzyme produced in this way had preferential substrate specificity for acetylthiocholine iodide (ATC), and its activity was inhibited by eserine (1.1 x 10(-8) M). Ancylostoma ceylanicum, another hookworm species, failed to produce comparable amounts of AChE in culture. AChE was efficiently purified from culture medium by affinity chromatography on edrophonium sepharose; 81% of the AChE activity was retained by the affinity matrix, although this fraction contained only 4.3% of the protein loaded. Antisera raised against purified AChE in rabbits immunohistochemically stained the oesophageal glands of the parasite, and reacted with molecules of 32, 60, 80, 140 and 220 kDa in reduced adult ES products on Western blotting, although differential activity was observed against worm homogenates and earlier developmental stages. On IEF, purified AChE resolved predominantly with a pl of 3.55; proteins with a similar pl were recognized by rabbit anti-AChE. IgG preparations of this antiserum inhibited AChE activity in ES products, and inhibited AChE secretion by adult worms in culture. The availability of this immunological probe will allow definitive experiments to be conducted on the role of this enigmatic enzyme in the host-parasite relationship.

IgE synthesis in vitro during infection of mice with the nematodeNippostrongylus brasiliensis: Effects of mitogens and antigens

In vitro IgE synthesis by lymphoid cells was studied during the course of infection of mice with Nippostrongylus brasiliensis. The studies involved inbred strains of mice which had been shown to be high IgE responders (A.CA, B10.M), or non-responders (Balb/c, B10.D2) to parasite antigen. In addition, F1 hybrids of low and high responders and irradiated non-responders were studied. Infection with N. brasiliensis led to an increase in IgE synthesis in vitro which was most pronounced during reinfection of mice. Addition of mitogens e.g. pokeweed mitogen (PWM), lipopolysaccharide (LPS), concanavalin A (ConA) to the cultures induced enhancement, suppression or had no effect on IgE synthesis. Addition of N. brasiliensis homogenate or worm culture supernatant led to a fluctuating pattern of IgE synthesis. No correlation was found between lymphocyte proliferative response to mitogen and worm antigens and IgE synthesis. Our data suggest, that PWM is more likely to enhance IgE synthesis in vitro than LPS or ConA. An enhancement is more easily observed with the cells of non-infected animals or during the early phase of infection or reinfection. The mitogen-induced increase in IgE synthesis did not exceed the values obtained during infection or reinfection.

A major change in surface antigens during the maturation of newborn larvae of Trichinella spiralis

Newborn larvae of Trichinella spiralis were collected for 30 min from female worms in culture, incubated in vitro for various times up to 18 h, and surface-labelled with iodine. The detergent-solubilised products were examined by SDS-polyacrylamide gel electrophoresis. At time periods up to 6 h these larvae expressed only one M r 64 000 iodine-labelled surface protein. Some time between 6 h and 18 h a further three components (apparent M r 58 000, 34 000 and 32 000) became accessible to surface labelling. All four of these components are antigenic in that they can be immunoprecipitated with T. spiralis immune sera. Tryptic peptide analysis revealed that the 32 and 34 kDa antigens were structurally very similar, but the 58 and 64 kDa proteins differed from each other and the 32–34 kDa pair. Thus T. spiralis not only undergoes a total change in surface antigens between moults, but also major changes in surface antigen expression within one stage.

A simple method for maintaining large, aging populations of Caenorhabditis elegans

This paper describes a technique capable of establishing and maintaining large, age-synchronous populations of the nematode Caenorhabditis elegans. The technique has three essential components: a rich chemical medium; a method for producing and harvesting mass quantities of eggs; and 5-fluorodeoxyuridine (FUdR), an inhibitor of DNA synthesis. A culture of worms is filtered through glass wool or a wire screen to isolate young larvae. Eggs laid by these worms after they mature are collected over a period of 4–6 hours and allowed to hatch. A low level of FUdR (25 μM) is added just before the larvae reach maturity. This timing is important to avoid developmental abnormalities. The adults lay eggs in the presence of FUdR but the eggs do not hatch, which maintains the synchrony of the culture. Many aging characteristics appear to be similar in treated and untreated worms, such as the time of cessation of egg production, the appearance of visible and behavioral age-related changes, and the mean lifespan. This system thus seems suitable for large-scale biochemical analysis of certain aspects of aging in C. elegans.

Schistosoma mansoni: Adsorption of Human Blood Group A and B Antigens by Schistosomula

Immediately following skin penetration, schistosomula of Schistosoma mansoni were found capable of passively adsorbing human A and B blood group antigens onto their surfaces. The antigens were adsorbed from intact erythrocytes, boiled human saliva, or alcohol extracts of erythrocyte membranes. Evidence is presented suggesting that a glycosphingolipid structure may be important in the binding of erythrocyte antigens to schistosomes. No uptake of various other blood group antigens could be demonstrated. Mouse erythrocyte antigens have been demonstrated on the surface of adult Schistosoma mansoni reared in mice (Smithers, Terry, and Hockley, 1969; Clegg, Smithers, and Terry, 1971a), and it has been shown that at least one such antigen is passively adsorbed by schistosomula (Dean, 1971; Sell and Dean, 1972; Dean and Sell, 1972). Clegg, Smithers, and Terry (1971b) reported that young schistosomes reared in a culture medium containing human erythrocytes were destroyed following surgical transfer into monkeys which had been immunized against human erythrocytes, indicating that human antigens are also acquired by schistosomes. In one experiment, the erythrocytes used to immunize recipient monkeys differed from the erythrocytes in the worm cultures with respect to five major blood group antigens. Fewer worms were killed upon transfer to monkeys so immunized than was the case with homologous transfers, and this led the authors to conclude that blood group antigens might be acquired by schistosomula. The present study was carried out to deReceived for publication 13 September 1973. * Supported by the Bureau of Medicine and Surgery Work Unit No. MR041.05.01.0023A6GI. The opinions or assertions contained herein are the private ones of the author and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large. The experiments reported herein were conducted according to the principles outlined in the Animal Welfare Act (PL 89-544 as amended) and followed the guidelines prescribed in DHEW Publication No. (NIH) 72-23, formerly PHS Publication No. 1024, Guide for Laboratory Animal Facilities and Care. termine which, if any, human blood group antigens are adsorbed onto the surfaces of schistosomula in vitro. MATERIALS AND METHODS Schistosomula were prepared from Puerto Ricanstrain cercariae of S. mansoni by allowing them to penetrate excised rat skins into Hanks' Balanced Salt Solution (HBSS), by methods described previously (Stirewalt and Uy, 1969; Clegg and Smithers, 1972). Alcohol extracts of erythrocyte membranes were prepared by the method of Koscielak (1963). Wet membranes prepared by hypotonic lysis (Dodge, Mitchell, and Hanahan, 1963) were mixed with ethanol to a final alcohol concentration of 85%, mixed on a shaker at room temperature for 3 days, centrifuged, and the superatant fluid stored at -15 C for 3 days. The resulting precipitate was collected by centrifugation and stored at -70 C. Koscielak showed that the blood group active molecules in these preparations are glycosphingolipids. Saliva of a known secretor of soluble blood group A substance was boiled and centrifuged, and the supernatant fluid employed in appropriate experiments. Neutr AB is a commercial preparation (Dade Reagents, Miami, Florida) containing both A and B blood group activities. The source of this preparation (equine gastric mucin) and the method of preparation (Dade Reagents, pers. comm.) indicate that the active molecules are probably glycoproteins rather than glycosphingolipids (Baer, Kabat, and Knaub, 1950; Kabat, 1956). Specific activities of alcohol-extracted antigens and Neutr AB were measured by means of the hemagglutination inhibition (HI) assay. Serial double dilutions of antigen were prepared in Vbottom wells of Microtiter plates (Cooke Engineering Company, Alexandria, Virginia). Dilutions were made in pH 7.4 phosphate-buffered saline (PBS) at a final volume of 0.05 ml per well. An equal volume of blood group typing antiserum

In vitro detection of cytotoxic antibodies to Schistosoma mansoni schistosomules.

A simplified in vitro technique was utilized to detect cytotoxic antibodies in serum from hosts exposed to cercariae of Schistosoma mansoni. Significant destruction of cultured schistosomules occurred in the presence of immune rat, rabbit, mouse, and monkey serum. Immune sera induced heavy precipitate formation around the schistosomules. The cytotoxic activity resided in the gamma globulin fraction of immune serum and was shown to be almost wholly complement dependent. Immune serum adversely affected the in vivo viability of schistosomules before detection by the dye exclusion technique was possible. Rabbit gamma globulin that exhibited strong in vitro cytotoxicity failed to protect mice against a challenge infection. Serum from rabbits immunized with adult worm culture antigen did not possess significant in vitro cytotoxicity, although this same antigen effectively stimulated resistance in mice to cercarial challenge. These results, coupled with the findings of other investigators, make it difficult to equate the cytotoxic titer of a host's serum with its immune status.

Ascaris lumbricoides: Incorporation of linoleic acid-1- 14C into fatty acids

Male Ascaris lumbricoides were administered linoleic acid-1- 14C by injection into the pseudocoelom to determine distribution of label into other compounds. Analysis was done by thin-layer, gas—liquid, and column chromatography, and by liquid scintillation spectrometry. Water-soluble fatty acids averaged 8.2% of the administered radioactivity while 67.7% was found in the remaining fat. Volatile fatty acids with the greatest amount of 14C activity were acetic, propionic, and α-methylvaleric. No radioactivity was found in the carbon dioxide and little, if any, in the glycogen or worm culture medium. Of the radioactivity in the long-chain fatty acids, 89.5% remained in the 18:2 acid. Activity was also found in the 18:0, 18:1, and 20:2 and possibly in the 18:3 and 20:4 acids. Results indicated that the carboxyl group of linoleic acid-1- 14C was incorporated into glycogen, volatile fatty acids, and long-chain fatty acids with 18 or more carbons.

The effect of temperature on the life cycle, growth and fecundity of Branchiura sowerbyi (Oligochaeta: Tubificidae)

Branchiura sowerbyi Beddard was probably introduced to Britain with exotic plants consigned to botanic gardens. The British populations occur mainly in artificially warmed habitats such as lily ponds in botanic gardens and power station effluents but a few are found at natural temperatures. Branchiura completed its life cycle in about a year both in the river Avon, Wiltshire, at natural temperatures and in the warm effluent from a power station discharging into the river Thames. In a worm culture maintained at natural temperatures the rate of cocoon production reached a peak in summer and was closely related to temperature.In the laboratory it was found that the optimum temperature for cocoon laying in mature worms, also growth in sexually immature worms, was near 25°C. The optimum temperature for growth in sexually mature worms, however, was lower (10°C in the Avon population and 15°C in the Thames population). It followed that at 20°–25°C the growth of sexually mature worms was probably depressed by the high rate of cocoon production.

Greenhouse Horticulture in the Western Area of the Kofu Basin

Garden farming in Japan is, in some areas of the country, concentrating on the greenhouse development of agriculture and is being received favorably. One reason why this type of farming is making such great strides is because it is bringing farmers under mixed management with other agricultural branches: before greenhouse culture was introduced there were already regional difficulties.The author considers in this article some of the main garden farming regions in Japan, and then refers to the process of regional formation and facts concerning mixed management. Greenhouse horticulture in the Kofu Basin is located 300m above sea level. This area is greatly subject to the influence of cold in the winter season as compared to regions normally located along many coastal warm districts. Melons have been widely cultivated without heating three times a year in that area since 1925. It stands out in sharp contrast to melon cultivation with heating apparatuses in Shizuoka Prefecture. Then, on ground of alluvial fan land, geographically there is not an advantage over the location of garden farming with the osmotic action (permeability) of water.This report deals with the following three questions: at first the author involved consideration of “how to accommodate to unfavorable physical conditions” from various angles. Secondly, it accounts for silk worm culture concerning greenhouse horticulture and sericulture; the actual states of mixed management, and conditions of the development in greenhouse culture are restricted by the silk-raising industry. By introducing greenhouse culture in the traditional silk-worm culture, needless to say, the inclination of mixed managements, with their farmings marks the character of this area. Thirdly, though the area of greenhouse horticulture in Japan is getting more and more prosperous, a marked tendency to decrease its management size is seen lately with a few notable exceptions in this area. So we investigated their main causes thoroughly.The Kofu Basin is cold in the winter season; it is absolutely necessary for those who would carry on greenhouse culture to enrich cheap and plentiful labour with the work of keeping warm. The author believes that their production on an enlarged scale by greenhouse culture has been less prosperous. Another condition is prevented by mixed management with the silkworm culture. According to the labour productivity and the rationalization of management, greenhouse culture in other places outside the area of the Kofu Basin is getting more and more prosperous. In this area, on the contrary, far from trying to expand production, the farm-houses occupied mainly with greenhouse culture have a tendency to decrease with the diversified classes.