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Assignment and Lab Cover Page Title: THE SCIENTIFIC METHOD AND EXPERIMENTAL DESIGN AND the ATTACK OF THE KILLER FUNGUS EXPERIMENT Date of the report submitted: April 1, 2022 Author’s Name: Saloni Ashesh Patel Student ID number: 501122884 Course and section: BLG144-022 TA Full name: Morrigan Everatt Introduction Nematophagous fungi are soil-dwelling organisms with a variety of worm-trapping methods. This provides fungi with food, is beneficial to the environment's ecological balance, and gives agricultural benefits. This review has gathered literature regarding nematophagous fungi and the regulation of worm capture. Arthrobotrys oligospora Background Information Fungi are eukaryotic microorganisms that are abundant across the globe and inhabit many different types of niches. They have a variety of benefits, both for society and the environment. These organisms are consumed for nutrition and used for food preservation purposes, for example through the addition of specific microbes to cheeses (11, 29). They also naturally produce antibacterial substances like penicillin (3). In addition, fungi are essential members of the ecosystem. Mycorrhizal fungi associate with plant roots, which increases the surface area of the roots, allowing greater uptake of water and nutrients. They also protect plant roots from infection by harmful pathogens. Saprotrophic fungi colonize dead material through hyphal penetration of materials such as wood, leaves and manure, which are rich in cellulose. As animals are incapable of metabolizing cellulose, it is essential that fungi can break these items into simple compounds like sugars and carbon dioxide. Fungi also have uses in the agricultural community as potential control agents for parasites in farm animals (9). Although most species of fungi forage on decaying matter, some species, which include the fungi of interest, are carnivorous and use predatory techniques to capture their prey. Of the many predacious species currently under study, Arthrobotrys oligospora holds great promise in the agricultural industry for its potential as a pest control agent for both crops and animals. A. oligospora belongs to the class Leotiomycetes, and the family Orbiliaceae. It is mostly found on compost and decomposing wood, as well as animal excrements and metal polluted soils (9, 12, 38). It is broadly dispersed across the globe, found in both terrestrial as well as marine environments. Moreover, A. oligospora is considered the most abundant predacious fungus in the environment. In nutritious environments, A. oligospora survives through a saprotrophic mechanism (12), by processing dead and decaying matter. However, in low-nutrient environments, this species has an increased potency for capturing prey, which leads to the formation of traps. Caenorhabditis elegans Background Information C. elegans come from the genus Caenorhabditis, a line of nematodes that extends to 23 different species. C. elegans inhabit the soil and eat the microorganisms which live there (16). As an adult, C. elegans follow the typical nematode body plan, which is unsegmented, cylindrical, and tapered at both ends. The body is separated by a pseudocoelom into two tubes: an outer tube that consists of the cuticle, hypodermis, nervous system, muscle system, and excretory system and an inner tube that includes the pharynx, intestine, and reproductive system. Homeostasis is maintained through an internal hydrostatic pressure (2). Images of C. elegans anatomy can be found in the Worm Atlas (2). The two sexes of adult C. elegans are self-fertilizing hermaphrodites (XX) and males (XO). Hermaphrodites possess 959 somatic cells while adult males have 1031 cells, however, adult males tend to be slimmer and shorter. The major difference between the two lies in the latter’s development of male-specific sexual organs in the posterior half of the body. Hermaphrodites, in contrast, also possess an egg-laying apparatus and undergo a 3-day reproductive life cycle. While self-fertilizing hermaphrodites can produce about 300 progeny, those that mate with males’ breed 1200 to 1400 offspring (2). The developmental stages of C. elegans are the embryonic stage, the four larval stages L1 through L4, and adulthood, as illustrated in the Worm Atlas Introduction Figure 6 (2). Egg-laying and subsequent embryogenesis and hatching occurs within roughly nine hours. Development through the four larval stages is triggered by feeding post-hatching. Molting occurs between each stage as C. elegans develop. The time course of this process at 25C is as follows: beginning when the egg is laid, L1/L2 molting occurs after approximately 18 hours, L2/L3 molting after 25.5 hours, L3/L4 molting after 31 hours, and L4/adult molting after 39 hours. In the L1 larva stage, five of the eight classes of motor neurons are developed, along with somatic gonad precursors and the formation of the germ line. At this stage, males are already distinguished. In the L2 larva stage, the nervous and reproductive systems continue to develop. Of note, in the event of unfavorable environmental conditions such as high temperatures, C. elegans at the end of the L2 stage can go into the dauer stage, an arrested state, which is maintained until growth conditions are favorable again. The L3 stage begins the more concrete formation of the parts of the reproductive system in hermaphrodites and males, which is then completed in the L4 stage. The completion of these larval stages at 45 to 50 hours after hatching marks the adult stage and the reproductive life cycle. C. elegans is known as a colonizer of environments rich in microbes, such as decomposing plants (10). While the natural habitat of C. elegans is unknown, this organism is mostly found in human-made habitats: compost, mushroom beds, and garden soil in Europe, North Africa, Asia, North America, Hawaii, and Australia (16). Within its community, C. elegans shares their environment with arthropods, mollusks, and other nematodes. C. elegans find their food source through a complex olfactory chemosensory system that recognizes the by-products of microbes. As such, potential predators include pathogenic microbes that use this system to their advantage (26). Additionally, C. elegans are preyed upon by fungi that adhere to the cuticles of nematodes or uses trapping devices to paralyze its movement and puncture the organism (10). 3. Nematode-capturing Fungi Importance of Nematode-capturing Fungi Nematodes can infect and destroy plant roots, resulting in reduced nutrient uptake and increased susceptibility to diseases. Use of pesticides is only a short-term solution to nematode infestation of agricultural crops and has resulted in a surge of resistant nematodes. Several nematode-trapping fungi have been identified and studied for their potential role in controlling nematode growth. This would provide an alternative to the use of toxic nematicides, which are potentially dangerous to our food supply and the environment. The three primary consumers in the soil food web are bacteria, fungi, and nematodes, and all of these serve as food sources for other organisms (31). Some nematodes, such as C. elegans, utilize bacteria as their food source, and nematophagous fungi can consume these bacteria-eating nematodes. Bacteria and fungi often co-inhabit areas, known as the bacteria-fungus interface. Interestingly, in vitro experiments have shown that the presence of soil bacterial strains increases A. oligospora trap induction (18). This suggests bacteria and fungi may share a symbiotic relationship in controlling the nematode population. There have been two models suggested for explaining the relationship between nematophagous fungi and nematodes and the importance of trapping for these fungi (31). The numerical response model suggests that nematode-trapping fungi are obligate parasites that use nematodes as a carbon and nitrogen source. This plays an important role in the ecological cycle because during the decomposition process, the population of microorganisms increases. This increase in food would result in an increased nematode population, but nematophagous fungi can control growth by capturing these nematodes as their own energy source. On the other hand, the supplemental nitrogen model suggests nematodes serve only as a nitrogen source for the fungus. Thus, fungi are facultative parasites that degrade nematodes for nitrogen to survive in a nitrogen-poor environment while they exploit other organic matter as a carbon source and for energy. Classification of nematode-trapping fungi Nematode-preying fungi are classified as “nematophagous fungi” or “endophytic fungi”. Endophytic fungi grow within plant tissue without causing diseases and are important for preventing parasitic nematodes from growing on plant roots. On the other hand, nematophagous fungi, a diverse group of fungi, colonize and parasitize nematodes for exploitation of nutritious substances (21). Nematophagous fungi are subcategorized into facultative parasites or obligate parasites. Facultative parasitic fungi utilize trapping structures and secrete antimicrobial and nematicidal compounds. They produce adhesive spores or develop specialized hyphae to penetrate the nematode. Trapping structures are usually complex three-dimensional nets with the branches covered with adhesive material. The adhesive knobs are composed of adhesive polymers produced on the apex of a slender hyphal stalk. The trapping structure also consists of constricting rings that swell to trap the worm. Toxins are secreted to immobilize the nematodes before penetration of the hyphae through the nematode cuticle. Scanning electron micrographs of A. oligospora traps can be found in Nordbring-Hertz et al. (1986). Obligate parasitic fungi release spores that are ingested or adhere to the nematodes. Ingested spores germinate inside the intestine or adhere to the cuticle of the nematode and sporulate on the surface, generating an infection on the nematode. Zoospores are also used by parasitic fungi to infect nematodes that inhabit inside of the surface of plant roots as cysts or root knots. Classification of nematodes Nematodes are generally classified as “free-living nematodes” or “plant-parasitic nematodes”. Free-living nematodes can move freely through the soil and rhizosphere and are captured by fungi that form trapping networks of constricting rings or adhesive hyphae. Plant-parasitic nematodes are sedentary and characterized by the formation of cysts (egg masses). They localize near the roots of plants, form a root-knot and feed and reproduce permanently on the roots of the infected plants. The larvae that grow from the egg masses can infect the plant roots and draining the plant’s photosynthate and nutrients. Fungi that prey on plant-parasitic nematodes colonize the rhizosphere and grow into cysts on the nematodes (15). The C. elegans we are using are classified as free-living nematodes that are preyed on by nematophagous fungi. Actions of A. oligospora following worm capture A schematic of the events described below can be found in Figure 18 of the article by Veenhuis et al. (1986). After contact with the nematode cuticle, A. oligospora utilizes a fibrillar matrix, approximately 0.1?m thick, to attach the nematode to the hyphae (8). This adhesive is not found on vegetative hyphae. The fibrillar matrix becomes reorganized in one direction targeting the site of capture on the worm, and indentations of the nematode cuticle begin forming (34). Then, the hyphae begin penetrating the nematode at the site where the fungal cells have the greatest adhesion to the cuticle, which usually occur within 2 to 4 hours after capture. This begins by the accumulation and association of small vesicles at the cellular membrane of fungal trapping cells. A new cell wall begins forming on the cytoplasmic side of the fungal cell, and the original nematode cell wall begins to degrade. This leads to the release of small vesicles into the fibrillar matrix and emergence of the newly formed cell wall, creating a penetration tube that indents the nematode cuticle. The cuticle begins thinning and eventually, the fungal hyphae penetrate the nematode creating an infection bulb with large numbers of electron- dense microbodies. New trophic hyphae then develop from the infection bulb inside of the nematode within a few hours of capturing (36). Mycelium growth outside of the nematode body is not observed until 10-24 hours later. This mycelium only develops from trapping cells that had captured the nematode. Trophic hyphae are rarely seen growing and developing outside of the nematode, so this indicates that trophic hyphae are mainly utilized for the digestion of nematodes to support the growth of vegetative mycelium. Approximately 6 hours after penetration, low levels of lipid droplets and microbodies accumulate in the trophic hyphae (35). Lipid droplets are used for storage of nutrients derived from 13 the nematode. In the later stage of infection, lipid droplets begin increasing in number and fusing into larger droplets while microbodies also develop and undergo fission. Microbodies were found to contain catalase and thiolase and are often associated with the lipid droplets. Eventually, the lipid droplets begin disappearing while growth of vegetative mycelium accelerates. Veenhuis et al. suggest that the catalase and thiolase from the microbodies provide enzymes for the ?-oxidation pathway of fatty acid metabolism to provide the carbon source that supports the mycelium growth (35). These organelles eventually disappear, and only the nematode cuticle, filled with trophic hyphae, remains after digestion. IV. Regulation of Nematode Capture by Nematophagous Fungi Below are results accumulated from a variety of published articles that examined fungus-dependent worm capture. This is by no means an exhaustive list of the available literature. Time dependence of trap formation After introducing the nematodes to the fungal plates, researchers have attempted to determine the timescale of trap formation. The traps were observed at three-hour intervals over a 27-hour period in a study by Nansen et al. (24). Another approach was to observe the traps at 17 hours after worm addition (37). A third study reported that no traps were observed until 24 hours, and after that, trap formation ceased within 5 days (28). Thus, there seems to be variability on the observed timing of trap formation. This may be due to differing factors such as media used, strain of the fungus, or temperature of incubation. Furthermore, the age of the traps seemed to have no impact on their ability to capture the worms, as young developing traps had the same efficacy as already existing traps (35). Larval density There is a correlation between increasing concentrations of nematode population and the number of traps formed (28). Researchers added 50, 100, and 200 worms to fungal plates, and observed a much higher number of spores and rings in the first day in the 200-nematode condition. This was also seen in another study testing suspensions containing 50 to 3200 L3 nematodes, as increased nematode trapping was seen at higher larval densities (22). 14 Temperature Incubating the fungal plates with nematodes at multiple temperatures from 7 to 37C elucidated the optimum temperature required for trap formation with the nematode Heligmosomoides polygyrus. A. oligospora was found to have a peak growth rate between 20 and 25°C. Predacity of the fungus was highest between 25 and 28°C. The fungus was found to be significantly slower in capturing nematodes at much lower temperatures (22). Media conditions Morgan et al. tested the effect of using corn meal agar diluted with water. Increased trapping occurred on plates with a lower CMA concentration implying that nutrient poor conditions forced the fungus to obtain more of its nutrients from the worms. Scholler and Rubner analyzed carbon and nitrogen sources and concluded that the fungus is more predacious when either is lacking (27). Additionally, they found that a specific concentration of carbon and nitrogen was sufficient to prevent the fungus from forming traps at all. It has been concluded that nematodes may not even be necessary for trap formation and that a combination of a low nutrient medium and small peptides will be sufficient to induce traps to form (25). Learning Objectives: 1. Perform dilution calculations 2. Use micro pipettors with confidence 3. List benefits of fungi in nature 4. Describe how fungi and C. elegans are maintained in the lab 5. Develop a testable hypothesis 6. Design an experiment to test hypothesis 7. Data analysis Hypothesis The addition of Pseudomonas influences the trap creation of A. oligospora and the survival of C. elegans. Results Variable 1 Variable 2 25 26 13 42 45 22 12 28 20 37 19 44 18 29 11 33 30 21 15 19 28 23 31 41 41 34 15 27 24 36 t-Test: Two-Sample Assuming Unequal Variances Variable 1 Variable 2 Mean 23.13333333 30.8 Variance 106.6952381 64.74285714 Observations 15 15 Hypothesized Mean Difference 0 df 26 t Stat -2.267767591 P(T<=t) one-tail 0.015942945 t Critical one-tail 1.70561792 P(T<=t) two-tail 0.031885891 t Critical two-tail 2.055529439 P(T<=t) two-tail is 0.031885891 which is smaller than 0.05. Control - 6 hrs - ratio of surviving worms on fungus plate/worms on non-fungus plate 16 degrees - 6 hrs - ratio of surviving worms on fungus plate/worms on non-fungus plate 27 degrees - 6 hrs - ratio of surviving worms on fungus plate/worms on non-fungus plate Plate 1 1.158 0.931 0.756 Plate 2 0.961 1.185 0.904 Plate 3 1.032 0.818 0.263 Plate 4 1.132 1.042 0.877 Plate 5 1.126 1.365 0.931 Control - 24 hrs - ratio of surviving worms on fungus plate/worms on non-fungus plate 16 degrees - 24 hrs - ratio of surviving worms on fungus plate/worms on non-fungus plate 27 degrees - 24 hrs - ratio of surviving worms on fungus plate/worms on non-fungus plate Plate 1 0.112 0.357 0.076 Plate 2 0.198 0.387 0.057 Plate 3 0.324 0.327 0.078 Plate 4 0.214 0.421 0.065 Plate 5 0.351 0.435 0.06 50% CMA - 6 hrs - ratio of surviving worms on fungus plate/worms on non-fungus plate 2% Mannose - 6 hrs - ratio of surviving worms on fungus plate/worms on non-fungus plate P. aeruginosa - 6 hrs - ratio of surviving worms on fungus plate/worms on non-fungus plate Dark - 6 hrs - ratio of surviving worms on fungus plate/worms on non-fungus plate Plate 1 1.099 1.577 2.217 0.597 Plate 2 1.114 0.873 2.411 0.809 Plate 3 1.348 1.483 2.319 1.561 Plate 4 0.601 0.804 0.952 1.689 Plate 5 0.855 0.637 1.493 0.091 50% CMA - 24 hrs - ratio of surviving worms on fungus plate/worms on non-fungus plate 2% Mannose - 24 hrs - ratio of surviving worms on fungus plate/worms on non-fungus plate P. aeruginosa - 24 hrs - ratio of surviving worms on fungus plate/worms on non-fungus plate Dark - 24 hrs - ratio of surviving worms on fungus plate/worms on non-fungus plate Plate 1 0.397 0.782 1.47 0.096 Plate 2 0.755 0.605 0.544 0.132 Plate 3 0.537 1.102 0.375 0.154 Plate 4 0.378 0.853 0.954 0.099 Plate 5 0.29 0.959 1.404 0.066 Class data exploring the influence of several variables on C. elegans capture by A. oligospora. One week before the experiment, A. oligospora was struck on CMA plates and cultured at room temperature. The worms were cleaned off the CMA plates at t = 6 and t = 24 hours after worm addition and computed % survival. The data is provided as a percentage survival ratio in the variable condition versus the control condition. For each variable, data from at least 5 groups were averaged, and the standard error of the mean (SEM) was calculated. Discussion The protocol involves the addition of C. elegans to plates with and without fungus. The “no fungus” plate is a control condition to take into consideration worms that die naturally during the experiment. At set times, worms are rinsed off the control and fungus plates and are counted. From these values, the percent of worms that survive the fungus is calculated. Percent Survival = Worms on Fungus Plate/ Worms on No Fungus Control Plate Prior to adding the worms to the fungus plate at the beginning of the experiment, a rough count is taken to approximate how many C. elegans are added to each plate. Equal numbers will be added to the fungus and control plates. This experiment supports the initially mentioned hypothesis that is “The addition of Pseudomonas influences the trap creation of A. oligospora and the survival of C. elegans”. Conclusion The purpose of the experiment is to measure the fungus dependent capture of C. elegans after 48 hours of co-incubation and how a specific variable alters the ability of A. oligospora to capture C. elegans. Percent survival is calculated as worms on fungus plate divided by worms on no fungus plate. This value is calculated with both the control and variable conditions. Independent variable is presence of pseudomonas. Dependent variable is survival of C.elegans. 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