Development of a novel in vitro method for drug development for fish; application to test efficacy of antimicrosporidian compounds.

Few drugs are approved for treating diseases caused by parasites in minor species such as fish. This is due, in part, to the expense of drug development and to the comparatively small market. In vivo effectiveness trials for antiparasitic drugs are costly, time consuming and require ethics approval, therefore an in vitro screening approach is a cost-effective alternative to finding promising drug candidates. We developed an in vitro testing system to test antimicrosporidial compounds against a microsporidian pathogen Heterosporis saurida. Five antiparasitic compounds, albendazole, fumagillin, TNP-70, nitazoxanide and lufenuron, were assayed for antimicrosporidial activity. All compounds reduced the number of H saurida spores in infected cells when applied at a concentration that did not appear to be toxic to the host cells. Albendazole inhibited replication of H saurida by >60 per cent, fumagillin and its analogue TNP-470 inhibited H saurida >80 per cent, nitazoxanide and lufenuron inhibited growth >70 per cent. The data suggest that both fumagillin and its analogous TNP-70 hold the best promise as therapeutic agents against H saurida. The ability to use fish cell cultures to assess drugs against H saurida demonstrates an approach that may be helpful to evaluate other drugs on different microsporidia and host cells.

In vitro cultivation model for Heterosporis saurida (Microsporidia) isolated from lizardfish, Saurida undosquamis (Richardson).

Heterosporis saurida is a microsporidian that infects lizardfish, Saurida undosquamis (Richardson, 1848), in the Arabian Sea. Spores were isolated from infected lizardfish and used to infect derived fish cell lines: common carp brain (CCB), epithelioma papulosum cyprinid (EPC), fathead minnow epithelial (FHM), rainbow trout gonad (RTG), bluegill fry (BF-2) and chinook salmon embryo (CHSE). Non-fish cell lines were also tested that include: insect (SF-9), rabbit (RK-13) and African green monkey (Vero E6). No growth of H. saurida was observed in any fish cell line, SF-9 or Vero E6 cell lines. H. saurida spores grew only in RK-13 cell line and were detected by immunofluorescence. Developmental stages of H. saurida were seen in RK-13 cells by light and transmission electron microscopy, and species identification was confirmed by sequencing. This study demonstrated that H. saurida was able to proliferate in the mammalian RK-13 cell line, which thus represents an in vitro model for conducting molecular genetics and cell-pathogen interaction studies of Heterosporis.

Runoff loss of pesticides and soil: a comparison between vegetative mulch and plastic mulch in vegetable production systems.

Current vegetable production systems use polyethylene (plastic) mulch and require multiple applications of agrochemicals. During rain events, runoff from vegetable production is enhanced because 50 to 75% of the field is covered with an impervious surface. This study was conducted to quantify off-site movement of soil and pesticides with runoff from tomato (Lycopersicon esculentum Mill.) plots containing polyethylene mulch and a vegetative mulch, hairy vetch (Vicia villosa Roth). Side-by-side field plots were instrumented with automated flow meters and samplers to measure and collect runoff, which was filtered, extracted, and analyzed to determine soil and pesticide loss. Seasonal losses of two to four times more water and at least three times as much sediment were observed from plots with polyethvlene mulch (55.4 to 146 L m(-2) and 247 to 535 g m(-2), respectively) versus plots with hairy vetch residue (13.7 to 75.7 L m(-2) and 32.8 to 118 g m(-2), respectively). Geometric means (+/-standard deviation) of total pesticide loads for chlorothalonil (tetrachloroisophthalonitrile) and alpha-and beta-endosulfan (6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexahydro6,9-methano-2,4,3-benzodioxathiepin 3-oxide) for a runoff event were 19, 6, and 9 times greater from polyethylene (800+/-4.6, 17.6+/-3.9, and 39.1+/-4.9 microg m(-2), respectively) than from hairy vetch mulch plots (42+/-6.0, 2.8+/-5.0, and 4.3+/-4.6 microg m(-2), respectively) due to greater concentrations and larger runoff volumes. The increased runoff volume, soil loss, and off-site loading of pesticides measured in runoff from the polyethylene mulch suggests that this management practice is less sustainable and may have a harmful effect on the environment.

An In Vitro Test for Temperature Sensitivity and Resistance to Meloidogyne incognita in Tomato.

An in vitro root explant tissue culture technique is described for determining susceptibility of tomato (Lycopersicon esculentum Mill.) breeding lines and cultivars to the root-knot nematode Meloidogyne incognita. Root explants were taken from 2-day-old seedlings cultured for 30 days at 28 C on Gamborg's B-5 medium with or without nematode inoculum. The remaining portion of the root and stem from the excised root explants was transferred to soil in pots and grown to maturity in the greenhouse. In vitro root explants were evaluated for growth and occurrence of juveniles, adults, and egg masses. The regenerated plants were used to produce more seed, The proposed technique is simple, reliable, and adapted to routine screening of large numbers of F and F samples, and it utilizes less space than tests performed on intact plants in the greenhouse or growth chamber. Evidence is presented also on the breakdown of resistance to M. incognita under high temperature stress using this in vitro root explant technique.

Pitfalls in using sodium hypochlorite as a seed disinfectant in C incorporation studies.

Seeds sterilized with sodium hypochlorite (NaOCl) retained sufficient amounts to interfere with studies of amino acid metabolism of the sterilized seeds during germination. Repeated washing in water did not remove NaOCl completely. However, soaking the seeds for 10 min in 0.01 n HCl removed NaOCl completely, without reducing germinability.Residual NaOCl reacted with the amino acids and reduced their concentrations in the incubation media. This reaction resulted in high production of CO(2) and low uptake of amino acids by the seeds. Decarboxylation of the amino acids occurred in the incubation medium outside the seed, was independent of the presence of seeds in the reaction, and therefore was not related to amino acid metabolism by the seeds. Effects of NaOCl on uptake, incorporation, and CO(2) production from indoleacetic acid were similar to those of the amino acids studied.

Hypochlorite and tissue sterilization.

Trace amounts of sodium hypochlorite that remain on the surface of seeds (Lycopersicon esculentum Mill.) after sterilization interfere with subsequent uptake and incorporation of leucine into protein when the seeds are used in metabolic studies. The hypochlorite can be washed away with 0.01 N HCl but not by washing several times with water.

Glucose metabolism of embryos and endosperms from deteriorating barley and wheat seeds.

Changes in glucose utilization into CO(2) and ethanol-insoluble material were followed in whole seeds, embryos, and endosperms of wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) which had reached different levels of deterioration through accelerated aging treatments. Excised embryos from deteriorated wheat seeds had reduced respiration and glucose utilization into ethanol-insoluble material but not into CO(2). These treatments had no effect on respiration of excised endosperms, although they reduced utilization of glucose into ethanol-insoluble material and CO(2). Changes in metabolic activity of whole seeds in response to deterioration treatments are difficult to interpret because they represent the sum of the changes that take place in the embryos and endosperms. Changes in respiration and glucose utilization in these two tissues neither proceed at the same rate nor go in the same direction during deterioration.Incubation of excised embryos and endosperms in glucose-(14)C under N(2), as compared to air, affects the utilization but not the uptake of glucose. Embryos produce more (14)CO(2) and less labeled ethanol-insoluble material under N(2) as compared to air. The responses of endosperms to N(2) are of a much lower magnitude than those of embryos.

Regulation by auxin of carbohydrate metabolism involved in cell wall synthesis by pea stem tissue.

Promotion of cell wall synthesis (from glucose) in pea (Pisum sativum) stem segments by indoleacetic acid (IAA) develops over a period of 1 to 2 hours and is comprised of a promotion of glucose uptake plus a promotion of the utilization of absorbed glucose. The effect of IAA resembles, in these and other respects, its effect on cell wall synthesis in oat coleoptile segments, but the pea system differs in not being inhibited by galactose or mannose, in involving considerably more isotope dilution by endogenous substrates, and in certain other respects.EFFECTOR INFLUENCES UPON AND TOTAL ACTIVITIES OF THE FOLLOWING ENZYMES OBTAINED FROM ETIOLATED PEA STEM SEGMENTS PRETREATED WITH OR WITHOUT IAA WERE EXAMINED: phosphoglucomutase, uridine diphosphate glucose (UDP-glucose) pyrophosphorylase, nucleoside diphosphokinase, UDP-glucose dehydrogenase, inorganic pyrophosphatase, hexokinase (particulate and soluble), and UDP-glucose-beta-1,4-glucan-glucosyl transferase (beta-glucan synthetase). The first three enzymes mentioned exhibit high activity relative to the flux in vivo, do not appear to show physiologically significant effector responses, and are concluded not to be control points. UDP-glucose dehydrogenase activity is regulated by UDP-xylose. Hexokinase is a potential control point but does not exhibit regulatory effects related to the IAA response. beta-Glucan synthetase is the only one of these enzymes with activity which is increased by treatment of tissue with IAA, and this may be responsible for the effect of IAA on wall synthesis.Assays of metabolite pools support the conclusion that stimulation of polysaccharide synthesis by IAA is due partly to changes in hexokinase reaction rate resulting from an increase in metabolic glucose pool size caused by increased glucose uptake, and partly to increased activity at the polysaccharide synthetase level.

Metabolism of barley seed during early hours of germination.

The growth process in germinating barley seeds and its inhibition by actinomycin D and puromycin were investigated. Soon after seeds are imbided, their respiratory activity increases several fold, and the protein- and carbohydrate-synthesizing systems become active. The immediate activation of protein synthesis and its inhibition by actinomycin D and puromycin suggest that the dry seed has all the components necessary for protein synthesis.Although a good correlation exists between the rate of oxygen uptake and that of protein synthesis during the first 12 hr of germination, respiration appears to be independent of protein synthesis during the first 8 hr, as reflected by the insensitivity of the respiratory process to actinomycin D and puromycin. However, after 8 hr both antibiotics reduce oxygen uptake as well as subsequent seedling growth.The distribution of (14)C, derived from labeled glucose during the early hours of barley germination, among various fractions of metabolites, indicated that 50 to 70% of the utilized glucose appeared in (14)CO(2). The rest of the incorporated label appeared in hemicelluloses and starch, water-soluble ethanol-insoluble carbohydrates, and to a lesser extent in proteins and cellulose.