Nematodes were transferred using a plastic Pasteur pipette carefully trying to count 50 nematodes under the low-powered light microscope.
The methods of controls used were to protect the environment, the worms, and the agar Petri-dishes, and the scientist involved; by simply not allowing contact between the scientist and the substances via gloves. Any concentrations of the chemical substances were poured down the sink (towards a septic tank) and washed well following usage.
In order to set-up a nematode chemical avoidance assay on the agar plate, set distances were allocated, and gave the worms time to avoid any substances or chemo-tax to them. The movement of the worms is very similar to that of a snake. The worm brings the posterior part of its body forward, and then when it is level with the anterior, it pushes the anterior forward. The posterior segments of the body are then drawn forward again, and the whole process is repeated.
Results (*After Five Minutes)
Table 2, Control
Table 3, 1mM Vanillic acid
Table 4, 1mM Glucose
Table 5, Yeast Extract
Table 6, 1mM drug – anti-nematode agent
Table 7, Extract of dead Panagrellus nematode worms
Table 8, Secretary Products of Panagrellus nematode worms
Table 9, Basic, Acidic and Neutral Solutions
Discussion of Results
An adequate way of displaying a control is used against chemical related results. From this experiment, nematodes forage / loop in the absence of both attractants and structure, however, within structure the looping behaviour is replaced by more random movements. In the presence of attractant gradients, emanating from the bacterial source, more linear and directed movements were observed. This model considers only one of the three main factors affecting nematode movement: chemo taxis. Random movement (diffusion) and foraging strategy are noted but are not distinctly seen because of the closeness in relation to chemo taxis. In conclusion, it is found that structural heterogeneity impeded both attractant diffusion and nematode movement. Furthermore, like higher mammals, the ability to alter their foraging behaviour to random movements and again to biased linear movements will allow them to find food and avoid predators in a variety of structural environments. However, in very dense structures (e.g., sand) this mix of strategies cannot be exploited, resulting in the nematode being unable to find a food source.
The results obtained through testing Panagrellus redivivus’s reaction to 1mM Vanillic Acid shows as, more worms went to the higher concentrations: ratios of 2:2 (50% of the worms), and 3:1 (36% of the worms) with 50% of the worms going for 4:0. From these results it is conclusive that this species of Nematode worm prefers stronger chemo-attractant substances to the lesser ratio (3:1) or less easy to chemo tax to concentrations.
The results obtained through testing Panagrellus redivivus’s reaction to 1mM Glucose display that the worms can detect the food source once the food source has been concentrated into a higher ratio per parts of water (e.g., 4:0). From this result, it is apparent that Panagrellus redivivus’s sensory receptors for this type of chemical is low.
Studying their motion towards the chemical concentrates is difficult since worms are about 1 mm long, translucent, and difficult to see when crawling long distances on the agar. To solve this problem, add a backlight via the low-powered microscope and watch them as closely as possible.
The results obtained through testing Panagrellus redivivus’s reaction to Yeast extract show that yeast is a complex food source that can be chemo taxed to easier than other food sources. The higher the concentration the easier Panagrellus redivivus chemo taxes to the yeast. Even in the lowest concentration, (1:3) 14% of the worms chemo taxed to it.
The results obtained through testing Panagrellus redivivus’s reaction to 1mM Drug reveal that the drug can be easily chemo taxed to when in small concentrations. This reveals that larger concentrations are sensed and deter the worm from moving towards them. Most worms were noted (after recording results in the 1:3 areas of the Petri-dishes) as being dead.
The results obtained through testing Panagrellus redivivus are reaction to Basic solution, acidic solution, and neutral solution display that the nematode prefers more alkaline conditions to acidic conditions. The worm also prefers more neutral conditions to acidic conditions. Several dead worms were noted after recording results in the acidic region of the Petri-dish. Also, no assumption can be made to how acidic the region would have been, as the original pH values were not stated.
The results obtained through testing Panagrellus redivivus’s reaction to extract of dead nematode worms reveal that some elements of cannibalism occurred in the ratios 2:2, 3:1, and 4:0, whereas in 1:3 the worms did not even move towards this concentration.
The results obtained through testing Panagrellus redivivus’s reaction to secretary products of a Panagrellus worm display that on the ratio 4:0 the worms could detect their own types. The lower concentrations barely attracted any worm movement.
There were several experimental drawbacks, which could have affected the results: light / dark, room temperature, and vibrations through the tables from movements within the room. Ideally, the assay would work better if it could be carried out under various light / dark and temperature climates in a non-motile environment thus preventing vibrations.
There is a high importance that free-living Nematodes should sense chemicals within their environment because they need to evade predators and survive by reproducing, eating, etc. This can be compared and contrasted through the sensory receptions of an invertebrate nematode with that of a mammal in some aspects. Like mammals, nematodes respond to many senses like touch, temperatures, vibrations (by sound, movement), chemicals (e.g., drugs, and acidic conditions), and light.
Had there have been more time, to properly plan an experiment, several preliminary experiments could have been ran (to best judge how to conduct the final experiment and obtain more accurate results).
Behaviour Physiology in the Flour Beetle (Triblium confusm)
Introduction
There are three common beetles that infest stored grains, flour, cake mixes and other flour products. These are the saw-toothed grain beetle, the red flour beetle and the confused, flour beetle. This experiment uses the flour beetle. All life stages of these "bakery bugs" can be found directly in the food they infest. Infestations are usually discovered when an infested package is opened for use, or when small brown beetles are discovered in the kitchen near containers of stored grain products. The flour beetle is a serious pest of milled and processed grains, especially flour. They may also infest beans, peas, dried fruits, shelled nuts, chocolate, spices and tobacco. Adult flour beetles are small (1/7 inch), reddish-brown and have a smooth-sided thorax. They are shinier and more convex than the similar saw-tooth grain beetles. The larvae are yellowish-white, worm-like, and have a two pointed structure on the tail end. The life cycle (egg to adult) generally takes 6-8 weeks. There may be up to 5 generations per year. Adults generally live for a year.
Taxis refer to the act of orienting towards some external stimulus, or combination of stimuli. Spatial orientation, aided by different sensory modes, is described by the corresponding term e.g. relative to light (phototaxis), smell (chemo taxis), sound (phono-taxis), or gravity (geotaxis). If orientation is towards the source, it is called positive taxis, and away from the source a negative taxis. In such instances, individuals move in a directed fashion along a particular stimulus gradient until they reach a perceived optimal range. In contrast, kinesis refers to non-directional orienting reactions in the presence of a particular sensory stimulus. Animals which suddenly find themselves in an unfavourable environment (e.g. with regard to humidity, temperature, or salt concentration) may change direction by trial and error.
Also, chemo-sensation allows animals to detect food, predators, potential mates, and other key stimuli in their environments.
Animals identify chemicals as attractants or repellents and modify their behaviour accordingly. Multi-cellular animals sense chemicals using specialised cells in the nervous system. Free-living Beetles are sensitive to numerous environmental chemical stimuli, and will chemo tax to an attractive (water soluble or volatile) compound, and avoid noxious compounds. These animals will also modify their movement, egg-laying, feeding, defecation, and their developments based on the detected food available.
Materials
Petri-dishes / Filter Paper / Flour Beetles / Hot water / Ice / Plastic gloves / Broad Forceps/scissors (from dissection kit) / food source (oats) / black paper / cardboard / large plastic box / desk lamp / mobile phone (with vibrate feature).
Aim
Working in groups of two, and using the materials available, students must design an experiment to address what chemical the nematode senses in its environment. Each member of the group should have an active role in the design and execution of the experiment, before writing up the practical in a suitable report format.
Methods
Cover half a Petri-dish in black paper – sealing it around the edges to prevent light coming in. Place 5 beetles into the Petri-dish. Position a desk lamp over the Petri-dish so that light is more intense over one side of the Petri-dish. Allow five minutes for the beetles to settle. Record the results noting the beetles’ reaction and, movements to a light/dark chamber.
Animals generally exhibit preferences for certain habitat types. Habitats may vary with respect to useful resources, such as protection from predators, and animals are expected to discriminate between alternative places to forage, sleep or rear young. This experiment can only mimic habitat variations. Set-up a Petri-dish, to function like a simple obstructive maze, by placing card in various positions. Record how the beetles reacted, and make observations. Measure whether the beetles would walk over, around or under the obstructions.
To measure reactions to vibrations, use a mobile phone with a vibrate feature. Place the mobile phone under the large plastic box. Allow time for the beetles to settle. Activate the phones vibrate feature. Measure two beetles, reactions to the sudden change.
To measure gravity reaction, up-end the plastic box on a 75 degree axis and then watch how the beetles reacted to this.
To measure reactions to cold use an ice tray. Place the ice tray under the large plastic box. Allow time for the beetles to settle in. Measure two beetles reactions to the cold temperatures.
To measure reactions to heat at 37 degrees Celsius, use a tray of hot water. Place the hot water tray under the large plastic box. Allow time for the beetles to settle in. Measure two beetles reactions to the cold temperatures.
Results
Figure 2A, Beetle Control Results
Figure 2B, Beetle Control Results
NB: On the above graph the timescale is labelled 1 to 6. Times this number by 30 to get the true value.
Figure 2C, Beetles’ Reaction to Heat
Figure 2D, Beetles’ Reaction to Noise/Vibrations
Figure 2E, Beetles’ Reaction to Gravity
Figure 2F, Beetles’ Reaction to Obstacles
Figure 2G, Beetles’ Reaction to Other Beetles
Figure 2H, Beetles’ Reaction to Light / Dark Chambers
Figure 2I, Beetles’ Reaction to a Food Source (Oats)
Discussion of Results
The experiment revealed that beetles sometimes explore external physical and tropic interactions such as light, dark, heat, cold, noise, vibration, other beetles, obstructions, food, and gravity.
Therefore, it becomes possible that beetles have instinctive and modified physiological responses to external physical and tropic interactions. Evidence for this, lies within, the results. There are often huge differences between some results (notably temperature related results, i.e., ice and heat) to that of the control results. Another result, apparent is that beetles have a fixed action of patterns to phototaxis. They are clearly negatively phototaxic.
These beetles seem to learn a few things from the experimental situations they were placed in but rarely asserted it. They seemed to be based on chemotaxis, phototaxis, phonotaxis, or geotaxis. Orientation towards the source (positive taxis) was recorded (e.g., towards a food source).
Do beetles learn not to respond to specific stimuli or are they simply reflex bound small animals incapable of learned behaviour?
From my experience keeping flour beetles for feeding reptiles and tarantulas is relatively easy. Culture of confused flour beetles is simple. To a well covered container such as a gallon size plastic ice cream tub with holes punched in the lid for ventilation, add an inch or so of flour, powdered milk, or a combination of the two, and a starter of adult beetles. Put on the cover, set in an out of the way place, and ignore for a while. The cultures require little care beyond occasional freshening of the culture medium.
Bibliography
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Animal Physiology Mechanisms and Adaptations, David Randall et al, Fouth Edition, W.H.Freeman and company, New York, 2000.
- Potato Cyst - Nematode Management, P. P. J. Haydock of Harper Adams College, Wellesbourne: The Association of Applied Biologists, 2000.
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Electrophysiological analysis of nematode responses, R.N. Rolfe, UWA, 1999.
- Animal Physiology – Introduction and Guide, Open University, Milton Keynes, 1999.
- Comparative Animal Physiology, C.Ladd Prosser and Frank A.Brown, Second edition, Saunders Press, London, 1961.
- Brandmayr, P., den Boer, J. and Weber, F. (Eds) (1983) Ecology of Beetles: The synthesis of Field Study and Laboratory Experiment. Wogeningen. Centre Agric. Publ. Doc. 196pp
- Dickens, J. C. and Payne, T. L. Chemical messengers and insect behaviour, pp. 201-230. In Mandava, N. B. (ed.) Handbook of Natural Pesticides: Methods, Volume 1: Theory, Practice and Detection. CRC Press, Boca Raton, FL. 534 pp. 1985.
