Chapter 4: Discussion

Axenic cultures of C. elegans have been used in studies to determine essential nutrient for nematode growth and reproduction. The interpretation of these studies is complicated by the ability of these nematodes to grow and mature, albeit slowly, even in nutrient deficient minimal media. If given enough time and large quantities of the nutrients are not required, nematodes can synthesize some of the required nutrients; however, if placed in an environment promoting fast or accelerated metabolism, the biochemical synthesis of needed compounds may be too slow to allow proper growth and maturation of the nematode (Dougherty, Raphael and Alton, 1950). In addition, the possibility always exists that small amounts of essential nutrients will be transferred with the nematode inoculum to new cultures. These minor nutrients may be present in high enough concentration to provide for proper growth and development for several generations of nematodes. In an investigation to determine the nutrient requirement for zinc in C. elegans, Lu, Cheng and Briggs (1983) encountered this problem and were unable to demonstrate a direct requirement for zinc.

Environmental and other factors associated with axenic cultures may also complicate measurements of growth and reproduction and the interpretation of experimental results. Environmental factors such as changes in pH, accumulation of CO2, accumulation of metabolic acids and reduction of oxygen concentration increase variation in nematode growth rates and reproduction. Infrequent sub-culture may inhibit reproduction and introduce a lag-phase immediately after inoculation into a new media (Wilson, 1976). Developmental studies have also shown a direct effect of temperature on: egg laying, rate of development and survival (Byerly, Cassada and Russell, 1976).

Even when nutrients are plentiful, some nematodes do not mature and reproduce at normal rates. Laggardism (Dougherty et al, 1959) is described as a failure to mature and reproduce at a normal rate. Laggards occur in media containing all essential nutrients as well as in nutrient-deficient media. The occurrence of laggardism hampered the evaluation and development of early defined-nutrient media. These laggards can bias measurements of growth rate and generation time. Experimental design must allow for the occurrence of laggards by increasing the number of replicates or increasing the number of samples. The researcher must understand factors contributing to laggardism and recognize its presence. The experiment must be constructed to limit the occurrence of laggardism. Cultures in which laggardism is obvious should be excluded from calculations.

Under normal conditions the 10 % of a population may exhibit laggardism. The use of old eggs or adults to inoculate a culture or the exposure of cultures to high or low temperature may increase the proportion of laggards to 30 % of the population. Laggardism is not necessarily related to the ability of a medium to support growth. Laggards usually increase if previous environmental, or cultural, conditions were unfavorable. Laggardism is an environmentally induced change. It is not a genetic change. However, an injury, or environmental signal, may induce a biological pathway promoting a delay in development and/ or reduction in reproduction. Lower, Hansen and Yarwood (1966) report laggardism in one out of every fifteen larvae under normal laboratory conditions.

Measurements of population change and growth rates are complicated by the lack of a consistent rates of egg production in nematode cultures. The production of eggs varies with culture health and age. Reproductive variation of 10 % per individual was reported in the nutrient studies by Vanfleteren (1975a, b and 1976). Ohba and Ishibashi (1982) investigated population problems related to temperature and pH and reported standard deviations in egg production from 20 % to 100 % within their populations (Hodgkin and Barnes, 1991).

The reproductive potential of a nematode may be inhibited through several pathways. If the intestinal physiology is altered after the J3 stage, the reduced nutrient supply will result in the cessation of egg production and laying (Figure 8). If egg laying is delayed, the eggs retained in the nematode reproductive system will hatch inside the parent and destroy it while trying to exit. This causes the premature death of the hermaphrodite, the possible premature death of the juvenile, thereby, a reduction in progeny produced by a reproductive worm. A reduction in reproduction may also be observed if the sensory structures are blocked. For example, the binding of lectins to amphids blocks chemoreception and may damage the sensory nerves of the nematode (Zuckerman, 1983).

Nerve damage and interference with chemoreception may promote dauer formation. Formation of the dauer form is normal when food supply is low or environmental conditions are poor. Dauer larvae do not produce young until they mature. As long as they remain in the dauer form, they are non-egg producers and immediate population increases are restricted. Lectins may also interfere with nutrient uptake by damaging the intestine. Any damage to the intestine would reduce nutrient uptake and may slow egg laying. Any delay in egg laying may increase the internal hatching of eggs in the parent and increase the "bag of worms" phenomenon.

Figure 8. Factors Affecting Nematode Reproduction Pathways.

In these studies of reproduction, the standard deviation as a percentage of the population tended to increase as the population increased. The Chi-Square test has been used to show statistical significance. Chi-Square was used to show the significance in toxicity tests with Panagrellus redivivus (Samoiloff et al., 1980 and McInnis, 1996). The same analysis used in this study. The Chi-Square test has been applied to the average population data and rate of population growth in relation to the control for each sampling date. This method provides two degrees of freedom based on the categories: greater than control, less than control, and equal to control. The critical value for Chi-Square was 5.991 with an alpha of 0.05.

Smaller rectangular bottles with an internal volume of 16 ml were used in three assays URP experiment # 1, KRS experiment # 1 and # 2 while the square bottles holding a volume of 21 ml were used in all other experiments. This difference in volume translated into an increased surface area for the remaining experiments. This difference may have contributed to the reduced growth observed in these experiments. The improved gas exchange in the other experiments may have provided better growing conditions. This would explain the minor increase of population seen in the control populations in cultures grown in the larger bottles; however, no dramatic increase was seen until KRS-experiment # 4.

The increase in control populations in KRS-experiment # 4 were more likely due to the fresh preparation of the HLE used in stock cultures. KRS-experiment # 4 was the first experiment to use this fresh HLE. The more recent preparation of HLE may have produced a healthier stock culture. Use of a healthy inoculum reduces laggardism and increased baseline reproduction on the defined CbMM.

Anatomical Variations

Post Anal Swelling: The post anal swelling observed in these studies was similar to that seen by Kisiel, Nelson and Zuckerman (1969) and possibly the same form of swelling noted by Anderson (1968) in Acrobeloides. In both instances, swellings were observed in nematode cultures fed on bacteria or bacterial extract. Apparently some bacterial component in the living cells or cell extracts exerts an influence on the morphology of C. briggsae, Acrobeloides and C. elegans. Similar swellings were observed in nematodes grown in cultures containing KRS-1297-5 or KRS-1297-6. This anatomical change may also correspond with the phenotypic expression of lin (abnormal cell LINeage) gene mutations (Wood, 1988). The same aberration was present in all three experiments in which fractions KRS-1297-5 or KRS-1297-6 were incorporated into the CbMM media.

Cuticular Swelling: The cuticular swelling or blistering, seen in these studies, has been reported to occur in nematodes grown on bacterial cultures. The 'blister' phenotype of C. elegans exhibits anatomical deviations similar to those observed in some experimental treatments (Herman and Horvitz, 1980). The cuticular swelling may be a result of the premature cuticle detachment in a localized region. This anatomical change may correspond with bli (BLIstered cuticle) gene mutations.

Vulval Prolapse: Regions of vulval prolapse were commonly observed in all of the liquid culture tubes. The occurrence of the prolapse was not directly related to the presence of any of the test supplements in the culture media. Prolapse of the vulva may be related to the premature cessation of egg laying or the premature hatching of eggs. Alternatively, the prolapse may be the cause of egg retention, internal-premature hatching of eggs or modified cell development in the reproductive system. At this time the cause of the observed prolapse is not known. This anatomical change may correspond with egl (Egg Laying defective) gene mutations or phenotypes or the mab (Male Abnormal) gene mutations or phenotypes.

Midsection Swelling: There was an abnormal increase in the midsection diameter of some nematodes with prolapsed vulvas. The midsection swelling may be related to an increase in the diameter of the larva or due to a blockage of the vulva and the subsequent accumulation of unlaid eggs. If this swelling corresponds with the premature cessation of egg laying, then it would be expected to occur more frequently in cultures with lower fecundity. Data to demonstrate this relationship was not specifically recorded or demonstrated in these experiments. The swelling may also be the result of the abnormal growth of cells in the intestine or improper development of cells in the reproductive system. The importance of the swelling is unclear; but its presence indicates a possible problem with the reproductive system. More studies concerning these anatomical abnormalities should be done to demonstrate the relationship between the midsection swelling and the decrease in reproductive potential. This anatomical change may also correspond with dpy (DumPY: shorter than wild type) gene mutations or phenotypes. Midsection swelling was not observed with nematodes inoculated onto bacterial plates from the axenic stock cultures used in these experiments.

Culture Trends

Although the health of the inoculum was not optimal, in the initial experiments in CbMM media, the patterns of population growth observed in these experiments were representative of C. elegans growth. This conclusion is supported by observations of populations in the control cultures from other experiments. Nematode growth and reproduction in cultures with CbMM only were similar to that reported by Lu and Goetsch (1993). The inclusion of fresh HLE in the stock media appears to be related to an enhanced population growth and reproductive rate of nematodes in both HS-YE-HLE and subsequently inoculated CbMM cultures. Although the initial state of health of the inoculum was low, population growth and reproductive rates still follow data reported by Croll, Smith and Zuckerman (1977) and Lu, Cheng and Briggs (1983).

Synchronization of nematode cultures was accomplished by the method used to prepare C. elegans in HS-YE-HLE cultures for inoculation into CbMM cultures. The sedimentation process is dependent on motility, size and density. Living nematodes move to the bottom of solutions faster than dead nematodes. Nematode body density is not identical for all larval stages. By allowing only 2/3 of the nematodes to settle prior to nematode transfer, the bottom layer of nematodes generally contains only second and third larval stage larvae. This process partially synchronized the nematodes and washed them free of the HS-YE-HLE solution. The partial synchronization of the nematodes helps to standardize the inoculum.

The synchronous cultures do not remain synchronous. For example, if time zero is the point at which the first egg from a single parental nematode is deposited, then by day 5 or 6 all progeny will have hatched. The resultant initial increase in population occurred 7 to 14 days after inoculation (Lu, Cheng and Briggs, 1983). The first F1 nematode will begin to produce progeny at day 5 or 6 at almost the same time the initial parental stops producing eggs. This will result in a reduction of population production but will not allow population production to actually stop. This second growth phase is a result of hatching F1 eggs (Croll, Smith and Zuckerman, 1977). The production of the F2 generation will continue from day 5 to day 15. Day 15 is the estimated point for the last F1 produced to stop laying eggs. The first F2 larva will start to produce eggs by day 10. The last F2 larva will stop producing larvae by day 25. As the F2 generation is producing progeny the parental generation is beginning to die. Between day 10 and day 15 both the F1 and F2 generations will be producing progeny. Nematodes can survive up to 10 days after producing their last egg. This overlapping of generations and uncertain mortality can obscure rates of population increases due to the F2 generation.

Figure 9. Example Rate Chart

This is an example showing the time shift in rates due to the influence of a reproductive inhibitor. The first rate maximum shows a shifted from the control when there has been an influence on reproduction. Chart data is modified from KRS Experiment # 6 concentration series.

Analysis of the observed changes in the rates of population growth can be used to identify bioactive materials and to dissect the response. The later analysis may contribute to a better understanding of the potential mechanism of the active fraction.Figure 9 demonstrates the shifting of growth rates in cultures who's average populations were reduced by a bioactive substance. The rates for A, and E illustrate only a mild rate depression while B, C and D illustrate a higher depression when compared to the control. E appears to show the spreading of the rate peak from day 9 to day 12, but the drop in rate could not be important as the population growth rate was above control levels at day 9 and would appear to show a stimulated growth rate at day 9. This spread could be a result of the F1 population spread between the two data points.

The control, A and E demonstrate a large decrease in population growth at day 16. This decrease in growth could be due to the declining production of the F1 generation or a result of a mathematical aberration caused by the culture population increasing above readable levels. Any population above 1,000 was recorded as 1,000. This would not correctly represent the rate of growth during the final recording interval.

Decreases in population growth rate, below that of the control, indicate interference with reproduction. The stressed or stimulated cultures may have a wider rate peak as compared to healthier cultures. The increased development of the F1 generation, the subsequent production of the F2 and F3 generations may smooth the growth curve and remove characteristic changes in population. This was also seen in the HLE population growth rate study. Increased population of the F1 generation can obscure the mortality of the parental generation. The control and E of Figure 9 demonstrate increased population growth without an observable pause denoting the death of the parental generation. The depressed rates of C and D are a result of population depressions caused by a bioactive additive. Their rates are lower and the initial population growth is observed later than in controls.

Counting Method using a Dissecting and Inverted Microscopes: The dissecting microscope provided better optical clarity, but lacked a moveable stage. The populations of cultures were open to contamination of bacteria and fungi and population counts were lower (Figure 4) than those recorded with the inverted microscope. The inverted microscope did not hamper the sterility and offered reasonable optical clarity. Although, the glass bottle walls varied with thickness and could blur the image, observations were not hindered. The inverted microscopes biggest advantage is based on the ability to count the nematodes in a constant sterile environment. This allowed the population from any single culture to be recorded over a time series. In the end, sterility and accuracy were the greatest factors determining the counting method.

URP Extracts

There was some initial reduction due to the gelling of extract URPPCM#28 with the 0.4 ml addition. Cultures with URPPCM#28 contained significantly lower populations than controls from days 10 to 31. This is not representative of URPPCM#28 (0.2 ml) or either volume addition of URPPCM#29. The observed initial mortality of the inoculum may have been due to premature fatalities caused by gelling of the extract. However, the surviving populations did continue to increase and follow expected, but delayed, growth patterns.

The short-term toxicity test appears to be of little use for this study due to its ability to only detect acute toxicity. Immediate toxicity makes the determination of long-term reproductive modification impossible. Acute and chronic toxicity can be tested using the CbMM media making this type of assay not necessary. No acute toxicity was detected in these extracts. The nematodes surviving at day 20 are most likely dauer larvae.

Dauer larvae form when nutrients are unavailable or a dauer forming hormone is present (Golden and Riddle, 1984). If the dauer forming hormone was present in the inoculum, all of the extract treated tubes would have formed a similar percentage of dauer larvae even with nutrients present. This percentage of dauer larvae would be represented in the percentage of inoculum surviving at day 20. The present wide range of percent mortality seen in the experiment does not fit this prediction and therefore precludes formation of dauer larvae induced by hormone present in the inoculum.

The formation of the dauer form may be inhibited by the presence of food. All of the nematodes were inoculated from CbMM into a non-nutrient M-9. The minimal nutrient transferred into the M-9 may not be enough to inhibit the formation of dauer larva. The cultures containing protein fractions would have extra nutrients available. If the nutrients provided by the extracts were high enough to inhibit dauer formation and yet not high enough to promote growth and reproduction, then the percent survival, or percent of nematodes forming dauer larva, at day 20 would be lower than in the M-9 control. This kind of effect was seen in the nematodes surviving after inoculation. However, dauer formation was not scored in this assay. It is assumed that any nematode alive at day 20 must be a dauer larvae as the normal life expectancy has been exceeded.

KRS Extracts

Fractions KRS-1086-4&5, KRS-1297-3&4 and KRS-3316113 were all of similar composition but were prepared by slightly different methods. The same is true for fractions KRS-1086-6&7, KRS-1297-5&6 and KRS-13256 which were similar but prepared by different methods. Bioactivity of KRS-1086-4&5 and KRS-1297-3&4 was similar in that these fractions inhibited reproduction in a concentration dependent fashion without the appearance of secondary stimulatory activity seen with KRS-1086-6&7 and KRS-1297-5&6. It is unfortunate that the activity of fractions KRS-3316113 and KRS-12356 was reduced over time and was not directly comparable with their related fractions.

All levels of KRS-1086-4&5 added to the media reduced population growth below that of controls suggesting that these fractions contained an inhibitor of development and/or reproduction. The presence of low levels of KRS-1086-6&7 reduced populations while higher levels did not. These fractions apparently contain more than one type of factor affecting reproduction. One is an inhibitory factor while the second stimulates reproduction. The latter must be present in higher levels to be effective. If the levels of the positive effector are high enough, they can overcome reproductive inhibition caused by the inhibitory factor.

The age of the extracts tested in KRS Experiment # 2 may contribute to their inactivity. Only the crude extract KRS-cdsp7518k80c1 reduced populations. The inhibiting activity decreased as the concentration was raised. This is consistent with the presence of both a stimulatory and an inhibitory component in this extract. However, the population increase may have been due to the addition of extra protein (Lower Hansen and Yarwood, 1966). This assay was not always effective when crude extracts were used due to the presence of unknown factors in the extract which may change the population growth of nematodes. There is always the possibility of multiple activities in crude extracts. This assay was developed to be used on highly purified fractions and specifically designed to test for a pure component.

Prior to KRS Experiment # 3 a new stock of HLE was prepared and used in all stock cultures. KRS Experiment # 3 was the first experiment using inoculum cultured with the fresh preparation of HLE. In KRS Experiment # 3, the average control populations were higher and the distinctive pause between different generations was no longer present. It is possible that the initial inoculum contained more or healthier eggs and less developed juveniles than in previous experiments. An inoculum containing juveniles at an earlier stage of development would modify the time line for population growth. The main population increase would shift to a later time and the initial population increase would be a result of the F1 generation.

KRS extracts 1297-5&6 reduced average populations up to 80 % for a period of 7 days. This consistent reduction of population growth shows promise for future studies. Population reductions in cultures with KRS1297-3&4 increased with increased additions of this fraction while KRS1297-5&6 did not. However, the greater activity of KRS1297-5&6, and the distinct presence of protein bands on the SDS-gel, provided the impetus for further observations in experiment # 4 and experiment # 5. The lower level of population reduction observed with KRS1297-5&6 may be due to secondary factor associated with this fraction. The secondary factor may not be active enough at low concentrations to overcome inhibitory activity. At higher concentrations the secondary activity enhances population growth. The general reduction in population associated with all KRS-1297 fractions diminished over time. This apparent loss of activity may be due to the limited stability of this factor or to metabolism or deactivation of the active component.

The increased reproduction in the control cultures for this experiment may be the result of the healthier stock culture used as inoculum for this experiment. Even so, inhibition factors were still effective in reducing population growth with this inoculum. Thus, the ability of these fractions to reduce nematode population growth is more significant. An effective reproductive inhibitor must work in healthy as well as stressed cultures. In these experiments, both healthy and stressed nematodes were used to inoculate cultures. In the later case, the inhibitory substances were more effective than they were with more healthy nematodes. In both cases these fractions were still active. If the bioactive portion of the fraction was irreversibly bound or destroyed after exerting its effect, then a nematode culture could deactivate enough of the inhibitor over time to reduce its concentration below effective levels. Healthier cultures would bind or deactivate the bioactive portion of the extract faster than cultures with lower reproductive potential. Hence, in the earlier experiments the population depression was present for a longer period of time than the later experiments using healthier stock cultures.

The heat treatment did not destroy all of the bioactivity in KRS-1297 but decreased the impact of these fractions on population growth. After further testing, it became apparent that the activity in KRS-1297 increased from fraction 3 to fraction 6. This suggests that the activity may be spread over a range of fractions as is often the case with fractions isolated by chromatographic methods. The greatest activity was present in fractions 5 and 6.

Lectin binding and population inhibitory activity were greater than 50,000 in molecular weight based on separation by Centricon ultrafiltration of KRS-1297-5&6. Stimulatory activity without lectin binding was present in the fraction below 10,000 molecular weight. All Centricon retentates possessed inhibitory activity, while stimulatory activity was seen with the Centricons filtrates. This stimulatory activity apparently has a molecular weight below 10,000. The stimulatory activity does not appear to be a protein due to the lack of detectable protein by Bradford analysis. The lack of detectable protein after SDS analysis was consistent with this interpretation. Further purification of the active fractions is necessary to determine their structure and properties.

The reproductive reduction caused by the addition of concentrated was correlated to the amount of protein in the sample. This population inhibition appeared to diminish over time. This observation was similar to changes in population described in KRS Experiment # 4 and KRS Experiment # 5.

Haemagglutination is a general characteristic of lectins, a group of carbohydrate-binding proteins unrelated to immunoglobins. Lectins may have both toxic and non-toxic effects on animals. Some lectins can influence the behavior of nematodes.

The ingestion of lectins can cause damage to the epithelial cells through an interference with its metabolism. Lectins also show a toxicity toward nerve tissue, and binding affinity toward and inactivation of olfactory tissues (Pusztai, 1991). There are some conflicting reports concerning specific binding and the activity of lectins on nematodes. It is generally assumed that the binding of some lectins to nematodes is species specific (Perry, 1996).

The role of lectin binding in the inhibition of nematode host finding behavior was proposed by Zuckerman (1983). The binding of lectins to chemoreceptors blocked chemotaxis toward a preferred food source in C. elegans. as well as M. incognita. In this way, lectins may interfere with host finding ability of parasitic nematodes (Perry, 1996). In other studies, lectins were unable to inhibit the chemotactic response of H. schachtii males to pheromones released by females of the same species (Aumann, Clemens and Wyss, 1990 a, b).

Spiegely and Robertson (1988) reported a possible species and race-specific relationship of lectin binding to the nematode cuticle. Based on electron microscopy studies, lectins may be bound selectively to amphids, a nematode sensory structure located on the head. Amphids are chemosensory structures containing large negatively charged organic molecules (Croll, 1977). McClure and Zuckerman (1982) used the lectin Concanavalin A and hemocyanin as markers to study the role of lectin binding sites on the anterior head region of nematodes. Lectins were bound on the head from the first to the fifth annule. The labeling emanated from the amphid, but no labeling was noted in the secretions of the amphid. McClure (1988) later demonstrated lectin binding to amphid exudates in Meloidogyne by gold labeling.

In a culture system where the nematode must seek out its food, lectin binding might inhibit chemotaxis and food acquisition. The inhibition of chemotaxis would not be a factor under axenic liquid culture conditions. When the nematode is feeding, any supplement in solution would enter the nematode along with the rest of the nutrient media. If the blocking of sensory sites caused the cessation of feeding activity, then the reduction in population growth might be the result of lectin binding. If this were true, then the blocking of amphids by lectins as suggested by Puszati (1991) might promote the formation of dauer larvae reducing the reproductive potential of the culture. Nematodes with damaged amphids have already been observed to lack wild-type chemotaxis (Croll, 1977). Dauer formation has been induced by destroying amphids with laser ablation. This may be equivalent to the nerve tissue deactivation or blocking by lectins. It is possible the reproductive inhibition is due to the activity of a lectin; however, further testing is necessary to substantiate this hypothesis.

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