Chapter 1: Introduction
Members of the phylum Nematoda are able to adapt to a free-living existence in most terrestrial and marine environments. Nematodes also parasitize a wide number of plant and animal hosts. Agricultural losses from plant parasitic nematodes worldwide were estimated at greater than US $100 billion (Perry, 1996). Over one quarter of the world's human population suffers from nematode parasitic infections such as hookworm or pinworm.
The identification of substances active against nematodes may help to control both plant and animal parasites. The study of nematode reproductive inhibition by plant isolates provides insight toward the understanding of nematode nutrition, comparative biochemistry and physiology (Rothstein, 1965). Ultimately this fundamental knowledge may contribute to a better understanding of parasitism (Vanfleteren, 1978; Sayre, Hansen and Yarwood, 1963). The purpose of this study is to develop and test an assay system for the selection of peptides or protein extracts which inhibit nematode reproduction.
Plant parasitic nematodes are important antagonists in agricultural ecosystems. Currently, both naturally occurring and applied biological controls are being utilized to help control plant parasitic nematodes. Plant parasitic nematode populations may be reduced by the addition of chemicals, biological predators or the use of certain cultural practices. The most effective method to control nematodes is soil fumigation with methyl bromide. The application of methyl bromide provides a 90% reduction in nematode populations (Vanfleteren, 1978). The non-fumigant nematicides, applied at standard rates, only inhibit mobility and reduce the number of parasites which allows plants to become established reducing the effect of parasitism. The use of nematicides is expensive and not always effective at reducing the damage caused by plant parasitic nematodes. In less developed countries, the application of nematicides may be cost prohibitive. The other restrictive factor is the inevitable loss of registration of many nematicides due to their broad range toxicity, possible danger, perceived danger and potential danger to environmental safety. The use of biological controls, on the other hand, may be safer.
Biological controls include a variety of parasites, bacteria, fungi and predators. These are not always commercially available, may be slow in producing results and often have limited ability to reduce parasite populations. To be successful a biological control must attack the weakest link of the parasite life cycle. Factors limiting the use of biological controls include the variability of success, the large quantities needed, the high costs of formulation and production, the poor long-term establishment of antagonists and the lack of a consistent increase in yield (Sikora, 1992).
Use of physiological manipulation or biological therapy as a control method include chemical or biological additions to the rhizosphere or phyllosphere. These therapies may help elicit a plant's normal defenses and improve its health and resistance to parasites (Sikora, 1992). Foliar treatments with agrochemicals such as antibiotics, urea, pesticides and phytonutrients can stimulate the plant, increasing its own natural defense mechanisms (Sikora, 1992). Agrochemicals may also stimulate natural biocontrol agents which exist in the environment. For example, the use of urea as a leaf treatment on wheat helps stimulate rhizobacterial populations. Some of these growth promoting rhizobacteria may antagonize plant parasitic nematodes.
Other cultural practices, such as solarization, fallowing and crop rotation help to reduce the population of parasitic nematodes by removing their host or modifying the environment, making it unsuitable for nematode reproduction. Solarization increases the temperature and dehydrates the soil reducing nematode populations. Crop rotation or fallowing remove the host and decrease nematode production (Sikora, 1992).
Non-host plants are either not capable of supporting the nutrient requirement of the nematode or produce substances toxic to the nematode. Rotation or intercropping with non-host plants successfully control parasites. Some plants or associated bacteria control nematodes by producing toxic metabolites which either inhibit, interrupt or disrupt the nematode's life-cycle.
As the understanding of nematode parasites increases, the benefits to man become more apparent. Major advances in the control of nematodes have begun to affect world health. According to the World Health Organization (WHO), intestinal worm diseases still kill at least 135,000 people every year. However, dracunculiasis (guinea-worm disease) is on the verge of eradication and could be completely eradicated within the next few years. In 1986, 3.5 million cases of dracunculiasis were reported while only 120,000 cases were reported in 1995. Only ten of these 120,000 cases required treatment in 1996. The discovery of new therapeutic compounds is dependent on the ability to detect and develop these compounds. This study is the first step in the discovery of bioactive compounds with nematicidal or antihelminthic activity.
Nematode Bioassays and Caenorhabditis elegans
Nematode bioassays are based on the nutritional assay systems developed in the early search for a defined media. Axenic cultures were used to define the minimal nutritional requirements of nematodes (Dougherty, 1959). Nutritional studies focused on the required nutrients necessary for growth, development and production of viable progeny. These studies usually followed a single nematode, preferably freshly hatched from an egg, through growth and development. Changes in width and length were used as growth parameters. Nutritional assays for biologically active growth factors began with these early studies.
C. elegans was chosen as a model organism due to the large amount of anatomical, genetic and biochemical data presently available. These data provide a launching point for further study after preliminary investigations have been accomplished. The site of activity, of a reproductive inhibitor causing any anatomical abnormality, may be localized due to the detailed information about development and embryogenesis. The simplicity and convenience of manipulation of C. elegans make it a useful experimental organism.
C. elegans has been exploited as a model system for over 27 years. The majority of early work was focused on cellular function (Riddle and Georgi, 1990). When Sydney Brenner adopted C. elegans as a laboratory model for studies in neurobiology, he initiated a worldwide interest in nematodes. Although C. elegans is not a parasite, C. elegans shares the basic mechanisms of development and behavior with parasitic nematodes. Newly hatched larvae of all nematodes are similar both in anatomy and development. The morphological and anatomical structure of parasitic and non-parasitic nematodes are very similar. Nematodes are pseudo-coelomate with a body consisting of two concentric tubes separated by the pseudocoelom. The outer tube consists of the cuticle, hypodermis, musculature and nerve cells. The inner tube is the intestine.
All nematodes develop through four larval stages defined by the molting of the cuticle (Figure 1). Although moulting is not necessary for growth, it does define developmental stages. The cuticle is made up of layers composed of crosslinked collagen and other modified proteins and is secreted by the hypodermis (Riddle and Georgi, 1990).
The nematode cuticle is a complex structure of plates, fibers, and several layers of protein. The cuticle contains pores, ducts, glands, and dendrites. The specific structure varies between different species and different growth stages. The replacement of the cuticle allows for growth and may serve in shedding antibody complexes formed by the host immune response. The cuticle is a physiologically active component and is probably maintained by the underlying hypodermis (Bird and Bird, 1991).
Figure 1. Life Cycle of C. elegans.
William B. Wood (1988) provided the following description of C. elegans. C. elegans is a small, free-living, bacterial-feeding terrestrial nematode (adults about 1 mm in length) found throughout the world. C. elegans has only two sexual forms, hermaphrodites and males. Hermaphrodites can be self-fertilized or they may be fertilized by male sperm. Hermaphrodites lay about 9 eggs per hour during peak egg production (Spence et al., 1982) and may produce a total of 250 to 350 eggs during the reproductive life cycle (Hodgkin and Barnes, 1991). C. elegans reaches sexual maturity after it emerges from its fourth molt and is fertile for about four days. Hermaphrodites normally live an additional 10 to 15 days after cessation of egg production is complete (Klass, 1977; Cassada and Russell, 1976). Males have a somewhat shorter lifespan (Wood, 1988).
The volume of knowledge currently available on C. elegans provides excellent resources for investigative studies. C. elegans possess biochemical and developmental mechanisms conserved throughout the animal kingdom. C. elegans has a fixed cell number of which the complete cell lineage is known (Sulston et al., 1983). Basic physiological, structural or genetic changes of C. elegans, may be followed and identified by current knowledge of anatomy, neurobiology (Lewis, 1980), biochemistry, physiology (Lee and Atkinson, 1977), developmental biology and genetics. The identification of the genetics controlling the neural development of C. elegans was simplified by the fixed cell number and predictable cell differentiation in C. elegans. A detailed genetic map of C. elegans has been constructed. This map includes a variety of biochemical, behavioral, morphological and developmental mutations.
Cell constancy makes C. elegans a good organism for the study of aging and development. C. elegans is often used in biochemical aging research (Klass, 1977; Klass and Hirst, 1976; Klass, Wolf and Hirsh, 1976; Klass and Johnson, 1985). Once the cells of C. elegans are developed, they are maintained throughout the life cycle. Studies have looked into several aging phenomenon focusing on the catalytic quality of an enzyme as it ages, age dependent alterations of enzymes and their ability of the enzyme to function properly (Rothstein, 1975).
The nematode has a short generation time and is easily cultured in large quantities on a defined media. In contrast, most parasites are obligate parasites and have not been cultured outside of their host. As early as 1950, C. elegans became a model system for the study of genetics. Genetic analysis is facilitated by the ability to self fertilize and produce offspring with minimal genetic variation and a small genome size. C. elegans is a self-fertilizing hermaphrodite reducing the problems associated with mating and sex ratios. The ability to transfer genetic information by conventional breeding and genetic transformations has facilitated the use of C. elegans ( Byerly, Scherer and Russell, 1976; Wood, 1988).
Culture Requirements of C. elegans
C. elegans can be cultured on a defined or complex liquid nutrient medium and is relatively tolerant to a wide variety of tonicity and pH. The worms do not succumb to their own waste products easily, allowing stock cultures to be kept viable for three to five months (Nicholas, Dougherty and Hansen, 1959).
The culture media used by this study was a modified Caenorhabditis briggsae Maintenance Media (CbMM) developed in the early 1950's by Dougherty (1953). The historical development of CbMM was reviewed in great detail by Vanfleteren (1978). The detailed composition of the media used herein was developed by Professor N. C. Lu of San Jose State University, San Jose, California (Lu, 1995; Lu and Goetsch, 1993). Dr. Lu's media was the first chemically-defined medium to provide necessary nutrients for the growth and reproduction of C. elegans. The development of a media providing the minimal requirements for growth was based on qualitative growth analysis by Dougherty and Hansen in the late 1940's. The improvements of the media required the use of quantitative studies of larval development and reproduction (Dougherty, 1951; Dougherty and Hanson 1956).
Major factors limiting nematode culture are: nutrient supply, accumulation of waste products and the availability of oxygen. Under standard conditions, the availability of oxygen becomes the major factor limiting growth and reproduction (Nicholas and Jantunen, 1966). Adequate gas exchange in a culture reduces the buildup of waste products such as ammonia which will cause a rise in the pH of the media increasing nematode mortality (Vanfleteren, 1978). Limitations of oxygen diffusion can be overcome by bubbling oxygen into the media (Buecher and Hansen, 1971) or by shaking the culture. Rotation of tube cultures also allowed increased gas exchange and can provide higher peak populations above those reported by Tomlinson and Rothstein (1962). However, shaken cultures may give varied results.
Thin layers of culture media allow enough gas exchange for the growth of the nematode to progress at a more optimal rate than shaken cultures. The use of thin film continuous culture over a glass wool in a packed separation column provides adequate aeration and high nematode production from the same culture for several months (Hansen and Cryan, 1966). A simple routine culture of C. elegans is best accomplished with a thin layer of medium (5 ml in a 50-ml flask).
Success of nematode culture is dependent on the media and the nematode used. Until the development of the current chemically-defined media, the results and culture methods have varied. Murfitt, Vogel and Sanadi (1976) cultured C. elegans in 2.5 cm deep solutions in shaken flasks with good results. However, C. briggsae did not do well for Tomlinson and Rothstein (1962). Yet C. briggsae has been cultured in a semi-defined medium by Rothstein and Coppens (as reported by Vanfleteren, 1978). Ohba and Ishibash (1982) also report the successful use of a shaken culture with C. briggsae.
Most methods and culture systems generally follow the methods of Tomlinson and Rothstein (1962) and Rothstein and Tomlinson (1962). Tomlinson and Rothstein (1962) used 5 ml of media in 50-ml flasks to grow nematodes for biochemical studies (Rothstein and Tomlinson, 1962; Rothstein, 1963; Rothstein, 1974). The media was composed of a previously defined media plus the addition of a 10% liver extract developed and discussed by Dougherty et al. (1959) in a series of papers. This small volume culture allowed ample gas exchange with the media. Increased aeration was accomplished on a reciprocal shaker operating at 80 shakes per minute. The cultures produced 5,000 to 15,000 worms per milliliter of solution based on estimates using an nephlometer.
The liver extract usually precipitates at room temperature and makes automated measurements of population difficult. The addition of trypsin, 18 hours prior to harvesting, helped clear precipitated liver extract from solution. No direct change in the growth or reproduction was caused by the addition of trypsin. This method was used by Pinnock, Shane and Stokstad (1975) to show how population trends increase in cultures with the addition of peptide supplements.
Researchers have modified sizes of the culture flasks, media and aeration. While testing the effects of antihelminthic drugs on population growth, Vanfleteren and Roets (1972) used 2.5 ml cultures for testing purposes while larger cultures were used to keep the stock healthy. More recently, a project was undertaken to isolate membrane channel proteins. This required the use of the use of 500 gram samples of nematodes. A 150-liter bioreactor was used to successfully produce this amount of nematodes. The project was successful in the isolation of a chloride channel sensitive to ivermectin and glutamate (Gbewonyo et al., 1994).
Methods of Measuring Culture Growth of C. elegans
as Individuals and in Populations
Growth is measured by two main methods, the maturation rate of the nematode and the rate of change in the population of a culture. Assessments of growth or maturation are made by observing the change in the length of the nematode's body. Population change is expressed as the rate of change in population over time. Larval length is directly related to larval maturity. The F1 generation time has historically been used to determine the nutritional quality of media (Lower, Hansen and Yarwood, 1966). The F1 generation time is the time needed for progeny of the parental inoculum to increase in size and produce offspring (Dougherty et al., 1959).
Much of the early work to define the nutritional requirements for nematode growth and reproduction followed the F1 generation time. Vanfleteren (1973) used F1 generation time to determine the amino acid requirements for growth and maturation of C. briggsae and Hieb, Stokstad and Rothstein (1970) used it to determine the heme requirement for nematode growth and reproduction. The F1 generation time can also provide information about the effectiveness of growth inhibitors. Spence et al. (1982) used it to show the effect of mebendazole, an antihelminthic drug, on growth.
Growth of nematode cultures are expressed in terms of population size at a standard time during the log phase of growth (Pinnock, Hieb and Stokstad, 1975). Nematode cultures may exhibit a lag phase directly after inoculation (Wilson, 1976). If a culture in the stationary growth phase is used as inoculum, an initial decline in population may be seen in the first few days after inoculation (Vanfleteren and Roets, 1972). The specific time to measure growth of a culture varies widely. But, any measurement during the logarithmic growth phase is generally acceptable. Buchner, Hansen and Yarwood (1970) reported population and maturation times at 21 days after inoculation for small cultures where Pinnock, Hieb and Stokstad (1975) reported populations between 8 and 15 days for large cultures. Lu, Newton and Stokstad (1977) used 21 to 28-day old culture populations to determine growth rate but some researchers prefer to report population growth every two days (Vanfleteren and Roets, 1972).
Population measurements can be made by serial dilution, direct counts or indirect counting instruments such as nephlometers. Indirect counting by a nephlometer can reduce time required for counting cultures. Nephlometers are photometric devices used to measure absorbence or reflectance which is related to the nematode population of a culture. A nephlometer developed by Watson et al. (1974) used multiple photocells to monitor voltage changes in proportion to population changes. This device was accurate for cultures with populations between 1,000 to 100,000 nematodes per milliliter. Patel and McFadden (1976) devised a nephlometric method using a standard spectrophotometer for absorbance and reflectance measurements. Their method was accurate for cultures with populations between 10,000 to 280,000 nematodes per milliliter.
Population counting by direct microscopic examination can become very time consuming and difficult. When nematode populations become very large, counting accuracy can decrease. The optical qualities of the liquid media may change over time and the movement of the nematodes may make observation and counting difficult. Usually serial dilution or sub-sampling is used in combination with direct examination to reduce the time necessary for population determinations. This may not be practical when the number of test vials is small, e.g. five to eight, and test materials are expensive or not easily prepared in large sterile quantities. If large cultures are not used, sub-sampling may not be practical.
Sub-sampling the assay tubes can save time counting but may increase accidental contamination of the assay. A combination of sub-sampling and serial dilution was used to measure populations densities in large cultures by Patel and McFadden (1978). A sub-sample from a 10-fold serial dilution was used to estimate nematode population. A thin line of media was drawn onto a glass slide and counted after proper dilution (Patel and McFadden, 1978). This provided a reduction in nematode population to an easily counted fraction of the original culture. Nematodes do not always distribute themselves evenly in a solution reducing accuracy (personal observation). However, the dilution of large cultures is the only practical method for determining the population of cultures over 2.5 ml in volume. Obviously, the practicality of counting is relative to the one counting.
To aid in the identification of nematodes, the application of dye may be used. A neutral red dye solution of approximately 25 % saturation is mixed into a well mixed tube of nematodes. The tube is incubated in a 70 C water bath. The heat killed nematodes are stained making them easier to count. The nematode solution may then be diluted until there are 250 to 1000 nematodes for every 1 ml of solution and counted in 4 aliquots of 0.1 ml. This method provides 95 % confidence (Vanfleteren and Roets, 1972). Neutral red may be used as a vital stain at a concentration of 0.01 % (w/v) (Rothstein and Cook, 1966). This concentration allows differentiation of living and dead nematodes. Live nematodes will not be stained but dead nematodes will stain a pink color.
Use of Population Growth and Development of C. elegans as a Biological Assay
Assay methods generally use survivability, reproduction, maturation, feeding rate and egg hatching as variables. Assay methods that focus on maturation require synchronous cultures, i.e. cultures of nematodes all the same age. Synchronous cultures are obtained by specialized initial culture protocols that will not be discussed in this paper. The use of synchronous cultures allows fewer cultures to provide more samples for testing. The use of synchronous cultures provides better statistical treatments of data, but increases the amount of preparation time needed to complete an assay. The importance of such cultures is discussed by Patel and McFadden (1978).
The Larval Assay Method: The best measurement to evaluate the health of a culture is the F1 generation time (Dougherty et al., 1959; Sayre, Hansen and Yarwood, 1963). A three larvae inoculum is observed for length changes and F1 generation time. The larval assay method is not always practical due to the large amount of manipulations and necessity for sterile conditions. But, in a review of axenic culture methods and media, the determination of F1 time was considered the best factor for measurement of development, growth and reproduction (Vanfleteren, 1978).
Rapid Screening Test or Fasting Method: Larvae are inoculated into a maintenance medium supporting maturation but not reproduction. This test considers only parental maturation of growth and molting. The test substance is introduced into the media after the nematodes have been allowed to grow for a few days in a maintenance media. Maturation and F1 generation time are measured and plotted as a dose-response curve against the tested substance concentration (Sayre, Hansen and Yarwood, 1963 and Rothstein and Cook, 1966).
Mass Culture Method: The mass culture system is much more convenient than the common larval bioassay of Lower, Hansen and Yarwood (1966). A large nematode culture, greater than 2.5 ml volume, is grown and counted by sub-sampling at selected time intervals. Manual counting of sub-samples may be tedious, but it requires less sophisticated equipment and works with a larger range of solutions than some of the automated counters. Spectrophotometric counting methods do not work well with particulate or opaque solutions. Particulate matter in a solution can bias measurements due to the improper identification of particles by the sensor as nematodes (Vanfleteren, 1978).
Mass cultures are convenient. Growth rate may be used instead of population for dose response comparisons (Pinnock, Hieb and Stokstad, 1975). Growth rate can be estimated by the log of the nematode population at a standard time interval during the logarithmic growth phase. A variability of 10 % to 15 % is suggested between subsamples for this method. The 10 % to 15 % variability is comparable to standard deviations seen in single larva reproductive assays (Hodgkin and Barnes, 1991).
Short-term Toxicity Studies: Short-term toxicity studies follow survivability or motility. Nematodes are placed in an osmotic buffer or nutrient solution with a test substance for a 24 to 48 hours. These studies test for direct toxicity and do not require the ingestion of the test substance. The effectiveness of ivermectin was measured against C. elegans (Novak and Vanek, 1992) using motility and survivability as factors. This method was also used to test B. thuringiensis toxin on C. elegans (Devidas and Rehberger, 1992). These short-term studies need not be conducted in nutrient media and are easier and cheaper to use.
Measurement of survivability in a nematode solution may also provide useful information. These measurements are used to measure the activity of a fast acting toxic compound or behavioral modifiers. One toxin from Bacillus thuringiensis has been shown to be active against Meloidogyne incognita as well as C. elegans. Devidas and Rehberger (1992) demonstrated a toxicity to C. elegans and death within 24 hours, when the toxin was present in a concentration as low as 7.8 mg / kg. C. elegans showed a greater sensitivity to the toxin than M. incognita. This result is attributed to the possible absorption of toxin in the intestine of C. elegans whereas M. incognita does not take in nutrients in its free-living form.
Feeding Rate Studies: Pharyngeal pumping rate measurements may be used to show the activity of feeding and health. The feeding initiation after inoculation can be associated with the health of the inoculum and the larval stage of the inoculum. However, feeding rate can also be an indicator of starvation in C. elegans (Avery and Horvitz, 1990). It should be noted that feeding does not always start immediately after inoculation into a new media.
Egg Hatching Studies: Egg hatching studies are similar to short-toxicity studies. Eggs are incubated in a solution, nutrient or non-nutrient, containing a test substance. The successful hatching of nematodes is compared to the concentration of the test substance in a dose-response comparison. These studies are hampered by the structure of the nematode egg which is impermeable to most toxic substances.
The embryos of C. elegans are surrounded by a chitinous shell and a vitelline membrane which render it impermeable to most solutes. The membrane serves as a control element in embryo development (Schierenberg, 1984; Junkersdorf and Schierenberg, 1992 and Schierenberg and Junkersdorf, 1992). The vitelline membrane, surrounding the embryo, is impermeable to water and dissolved electrolytes. It serves as a barrier to the passage of charged substances into the embryo. It is, however, permeable to gases (Arthur and Sandborn, 1969).
Because of this virtually impenetrable protective egg covering found in most nematodes, the direct application of nematicidal compounds is not always affective. C. elegans eggs, can survive being immersed in mild solutions of 10 % hydrogen peroxide (Nicholas and McEntegart, 1957), 1 % SDS (sodium dodecyl sulfate) (Cassada and Russel, 1975) and 25 % glutaraldehyde (Murfitt, Vogel and Sanadi, 1976). For the selection of protein or peptide inhibitors the use of egg hatching studies are of limited value.
Methodological Considerations in Developing C. elegans Culture System
The major goal of this project is to develop a protocol and methods for the identification of biologically-active substances, either of protein or peptide nature, that will reduce the reproductive potential of nematodes. Because all nematode physiology is similar, it is reasonable to test for inhibitors of C. elegans in the hopes of using them against endoparasitc plant parasites. The discovery of inhibitors of plant parasites is the ultimate goal of this research.
As in any experimental procedure, it is important to control as many influences on the subject as possible. The use of axenic culture and a defined media reduced any outside biological influences on the nematode. Potential inhibitors were delivered naturally through the intestine and their effects were not dependent only on external toxicity.
C. elegans has a simple direct feeding method. Peptides or proteins in the nutrient media may affect the nematode both externally and internally. When C. elegans feeds, material is pumped through a bi-lobed pharynx which pumps food into the intestine. The intestine is a simple one-directional tube, where digestion and absorption of nutrients can take place (Wharton 1986). Because C. elegans assists the movement of the media including the test substances into its body, the use of transport compounds or forced feeding is not required. If the nematode did not feed on the test extract, then the assisted transport of the test substance into the nematode might become necessary. Transport molecules are substances that will move items through barriers. Dimethylsulfoxide (DMSO) has been used as a transport compound to move substances through non-polar barriers. Used topically, DMSO has successfully been used to transport chemicals into nematodes at concentrations not exceeding 1 % (v / v) (Vanfleteren and Roets, 1972).
B. thuringiensis toxin, a protein endotoxin which has been used extensively to control selected insect pests, has shown activity against M. incognita and C. elegans (Devidas and Rehberger, 1992). The toxin was less effective against the non-feeding M. incognita when compared to the feeding C. elegans. The results are due to the increased uptake of the toxin throughout the gut during feeding in C. elegans; whereas, M. incognita does not feed in its free-living state and therefore does not take up the toxin as easily. The cuticle, which is the external body covering, acts as a selective barrier for the nematode. The cuticle structure between C. elegans and M. incognita varies as it does with all nematodes. This difference which is related to its normal resistance to environmental hazards (Bird and Bird, 1991) may also have been a factor.
Most plant parasites do not feed in their free-living form. Meloidogyne, the root knot nematode, does not feed in its free-living form. Only after the internal feeding site has been established, does the nematode begin to feed. The nematode initiates cellular changes in the root tissue changing the morphology of the plant cell into enlarged nurse cells (Doncaster, 1971). These changes increase nutrient flow into modified plant cells. The nematode then feeds repeatedly from a small group of enlarged nurse cells. By the time feeding starts, the nematode has become immobile due to morphological changes in the plant as well as its own body. At this point the endoparasitic plant parasite is most vulnerable. If the feeding site can be altered or modified to no longer support feeding by the nematode, it will die of starvation trapped in the plant without producing any young.
C. elegans can be fed on bacteria which have been genetically manipulated to express specific proteinaceous inhibitors or effectors. Bacterial transformation is one of the steps needed to clone and move the genetic material into plants. It is simple to feed the bacterium producing the protein to the nematode C. elegans and test for toxicity or reproductive inhibition. The inhibitor gene could then be introduced into the plant to protect against plant parasitic nematodes. Once the plant is genetically transformed, then a callus feeding nematode like Pratylenchus penetrans would be a prime test organism. The use of a bacterial-feeding nematode allows testing of substances in an axenic, one organism, chemically-defined culture without the interference of other organisms.
The study for nematicidal activity is less complicated when the organism can be manipulated and maintained in a pure culture where other organisms are unable to aide or interfere with its normal biological function. The growth and reproduction of the organism must also be suitable to provide the biochemist with suitable quantities of the organism for biochemical studies. A chemically-defined media offers an opportunity to study the biochemical relationships of the nematode in a highly controlled environment. Using a chemically-defined media provides a more restrictive growing climate for the organism and helps assure the limitation of unwanted materials in the assay. The defined media offers a controlled environment for comparisons of growth and reproduction. With a defined media, several premises can be applied to interpret the results. The additions of either undefined or defined compounds may affect the test organisms or may have no measurable effect (Nicholas, Dougherty and Hansen, 1959). Test substances may supply one or more nutrients not in basal complex media; or, it may imbalance the nutrient in the media. Test substances may also convert waste material produced by the worm into a toxic compound or block the assimilation or utilization of one or more nutrients. The use of axenic systems has allowed much of the biochemistry of C. elegans to be investigated.
The expense of this culture system will be reviewed as part of the data presentation in Appendix A which will provide information concerning the chemical cost of the media and labor cost to perform the described preliminary assay. The choice of culture vessels was dependent on the population counting method and the size of the culture. Small flasks, screw top bottles, or specialized tissue culture containers were acceptable. Sterile technique was the most important factor. Early workers used a sterile glove box with ultraviolet sterilizing light; but, the use of a Laminar flow hood is sufficient for this experimental setup. Cleansing all of the surfaces with a good disinfectant was helpful. The use of Lysol-IC has been very effective as a sterilant.
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