Several experiments have been devised for investigating this question. One that amateurs can perform easily is designed to measure the learning response of invertebrates. The procedure is described by John Frost, a graduate student at California State College at Fullerton, as follows:

"An interesting specimen to use for observing learning behavior in invertebrates is the common sow bug, Porcellio laevia. These organisms live in moist places almost everywhere. The adult is about half an inch long. The body consists of seven free segments, each of which bears a pair of legs.

"The animal has no effective biological mechanism for preventing the evaporation of water from its body. In order to survive it must avoid the drying effect of direct sunlight. Hence it has learned to shun light. The experimenter can take advantage of this characteristic to train the bugs, causing each to run a simple maze in a direction contrary to the path preferred by the bug before training.

"Sow bugs can be found under rocks and logs. The insects may be scarce in winter and when the weather is hot and dry. In cities they tend to congregate during all seasons in the damp cellars of apartment buildings under wooden boxes and the like. If search is unsuccessful, try making a trap by hollowing out a potato and placing it under a tree or shrub. Cover the potato with a few leaves and come back after 48 hours. The trap will usually contain several lively specimens. I do not recommend the potato trap for indoor use because it may attract some less desirable organisms. Sow bugs run when they are frightened, which is exactly what one wants them to do in the maze. They may faint when severely frightened, but they soon recover and scurry off.

"Captured sow bugs can be maintained indefinitely in a culture chamber improvised from a one-pound coffee can or a similar container [see Figure 2]. Half-fill the can with a mixture consisting of one part of sand by volume to two parts of leaf mold. On this surface place a peeled raw potato and a damp sponge of about the same size. Replace the potato and moisten the sponge every two or three days. The container should be closed by a perforated cover.

"Specimens must be kept in individual chambers during training. These chambers can consist of test tubes half-filled with peat moss or leaf mold covered with a piece of paper toweling [see Figure 3]. A sliver of fresh potato is placed on the toweling along with the bug. The containers are loosely plugged with tufts of damp absorbent cotton and labeled so that the bug can be distinguished from others. Replace the potato as necessary and keep the cotton moist.

"The maze in which the bugs are trained consists of a simple T, made by cementing cardboard partitions in a box of clear plastic [see Figure 4]. The box should be about three inches wide, 4 1/2 inches long and 5/8 inch deep. The passages should be made about 3/8 inch wide. Rectangular openings that match the cross-sectional area of the passages are cut in the walls of the box at the base of the T and at each end of the crossarm. Two blocks of wood that make a loose fit with the openings must be provided for closing either or both openings of the crossarm.

"The experiment is divided into two phases. First, determine and record the natural turning preference of each bug. Most sow bugs will take a preferred path through the maze. Having crawled up the leg of the T, some will habitually turn into the right portion of the crossarm and others into the left. Some will show no preference. During the second phase of the experiment the bugs are trained to turn in the direction contrary to their natural preference.

"Begin the experiment by transferring five or six specimens from the culture chamber to labeled individual chambers. Then remove a bug from a selected container and, holding it lightly between your thumb and forefinger, let it crawl from your fingertip into the base of the T. Record the direction of the turn, right or left. The bugs can tolerate only about 10 runs a day without suffering ill effects. For this reason the 20 runs needed to establish a reliable estimate of turning preference should span two days.

"Training is then accomplished by running each specimen through the course and punishing 'wrong' behavior. Each time a bug makes a turn in the direction it naturally prefers, immediately plug all exits with wood blocks and hold a 100-watt incandescent light close to the top of the passageway for about 20 seconds. When the bug turns in the direction opposite to its natural preference, plug the exit of the runway for 20 seconds but do not expose the animal to the punishing light.

"The training runs must be spaced a least five minutes apart. Between runs return the subjects to their individual quarters to 'think it over.' The experimenter can conserve his time by training a number of animals sequentially. This practice also tends to increase the reliability of the experimental results and to minimize the statistical effect of the occasional specimen that does not survive the training experience.

"The training period should normally take three to 10 days, depending on how quickly the individual learns. The bugs should be subjected to no more than 10 training runs a day. At the conclusion of the training phase nine consecutive correct turns can be taken as evidence that the bug has learned. A correct turn is defined as one made in the direction opposite to the bug's natural preference as determined by the first phase of the experiment. Statistically it can be shown that nine consecutive correct turns will occur by chance only once in 100 runs.

"The procedure can be varied. For example, the omission of the bright light following a correct turn can be considered a reward. The leg of the T is lighted brightly until the bug reaches t he crossarm; the light is removed if a correct turn is made. The desired behavior can be further reinforced by darkening the passage when the correct turn has been made.

"Much serious work has been done in recent years on the turning behavior of sow bugs as well as on the learning ability of cockroaches and box-elder bugs. The objective has been to clarify the role of reward and punishment in training procedures. I am certain that amateurs who repeat and extend the experiments will be surprised to find evidence of intelligent behavior so low on the scale of evolution. They will also have the satisfaction of exploring animal behavior by means of experimental procedures that do not injure the organism."

Pete Rowe of Los Altos, Calif., is another amateur who enjoys working with small animals. His interest is in freshwater crustaceans and particularly ¹n the physiology of the organisms, including their mechanisms of locomotion. These features are not easy to observe even under a microscope because most such crustaceans are largely transparent and their appendages move too fast to be seen by eye. For this reason Rowe worked out a procedure for recording the anatomical details of live crustaceans by high-speed photomicrography. The equipment he uses is available at prices most amateurs will find reasonable.

"In principle," Rowe writes, "the procedure of making a clear black-andwhite photograph through the microscope differs only in detail from that of making a picture with a conventional camera. The subject should be clean, posed against an uncluttered background and properly lighted. The photographic emulsion should be capable of recording the full scale of gray tones present in the subject without displaying a grainy texture. The exposure should be sufficiently short to record an unblurred image.

"It is possible to make reasonably good photomicrographs with a fixed-focus camera, such as the inexpensive box type, if the subject is adequately lighted and the camera is supported rigidly above the eyepiece of the microscope. The front surface of the camera lens should be placed at, or very near, the point at which the rays from the eyepiece of the microscope converge. That point is called the eyepoint or the Ramsden disk. It can be located by focusing the microscope on an object, placing a screen of ground glass or white tissue paper over the eyepiece and moving the screen up and down until the disk of light that appears on the screen reaches minimum diameter.

In eyepieces of conventional design the position of the Ramsden disk varies between 1/4 inch and 1/2 inch above the top surface of the upper lens.

"To make the photomicrograph place the specimen on the stage of the instrument. With live crustaceans I make the transfer by means of a pipette. Adjust the light for comfortable viewing and bring the specimen to sharp focus by eye. Center the camera over the eye piece. Place the front surface of the camera lens at the eyepoint, darken the room and make the exposure. The correct interval of exposure must be determined experimentally. The magnification of the finished photograph, with respect to the diameter of the specimen, is equal to the visual magnification of the microscope multiplied by the amount by which the negative is enlarged during the printing process and multiplied again by the focal length of the camera lens (in millimeters) divided by 250. The accompanying photomicrograph of a female cyclops, which happened to be carrying two sacs of eggs, was made by this procedure [right].

"My first photomicrographs, including the one of the cyclops, were made with an old Leitz monocular microscope equipped with a condensing lens for directing light through the specimen. The light source was a photoflood bulb. The exposure interval was controlled by the shutter of the camera. The film was Eastman Kodak Plus-X. Although the 35-millimeter camera I was using was equipped with a lens-focusing mount, I adjusted it for universal focus and so it functioned like a box camera. The speed of the shutter had to be kept below 1/100 second because of the limited amount of light available from the photoflood bulb.

"During the brief exposure interval of the cyclops photograph the animal moved enough to blur the image. The intestine appears as a fuzzy dark patch and the image of one swimming appendage fades into the gray background. The gray areas of the original print have a granular texture. The area immediately surrounding the organism also appears gritty. The shallow depth of field makes the image appear out of focus.

"Granular texture can be caused by excessive enlargement of the negative, a small change in the temperature of the solutions used for developing the negative or both. The shallow depth of field was the result of admitting light to the full aperture of the substage condensing lens. The depth of the field can be increased by closing the substage iris, thereby reducing the angle of the cone of rays that strikes the specimen. This expedient, however, would reduce the amount of light available for exposing the film, necessitating an even longer exposure and so aggravating the loss of detail occasioned by the movement of the specimen. The gritty appearance of the area adjacent to the specimen turned out to be a valid image: the organism was dirty.

"In spite of these defects the photograph disclosed several interesting details. Approximately 50 eggs can be counted in the sides of the egg sacs that face the camera. The total was perhaps twice that number. The segmented structure of the swimming appendages at the rear of the animal is clearly visible, as is the branching Structure of the large antennae in front.

"It was apparent that a light source of more intensity would be required for improving the depth of field and reducing the exposure interval. I therefore bought an electronic flash lamp that developed a light output of 150,000 candlepower during a flash interval of 1/2,000 second. The flash tube was placed seven inches from the mirror of the microscope and covered with two sheets of white tissue paper. A green filter was also inserted in the substage to reduce the intensity of the light somewhat. The diaphragm of the camera was set at f/8 to minimize light reflection within the camera. (The setting of the diaphragm has no effect on the exposure because all light enters the camera through the minute Ramsden disk located on the optical axis of the lens.) The shutter was adjusted for an interval of 1/500 second. To prevent room light from reaching the film I coupled the lens barrel of the camera to the eyepiece with an adapter ring. In addition I switched to a camera fitted with an adapter for using four-inch by five-inch film (Polaroid type 55 P/N film packets). These packets deliver both a positive print and a negative that is capable of recording exceptionally fine detail.

"The results were most gratifying. Resolution was substantially improved. The problem of movement was solved by the high-speed exposure, as is evident in the accompanying photograph of a Daphnia, Simocephalus expinosus [see Figure 7]. Observe in particular the fine structural features of the antennae.

"By the time I made this photograph I was using a microscope of better optical quality than the one used initially. Most of the improvement evident in the photographic print, however, resulted from the increased light, the shortened exposure and the use of the fine-grain Z photographic emulsion. With luck I finally succeeded in making a photograph of a daughter hydra breaking away from its parent [see Figure 5]. Observe the cellular structure of the processes and the clear line of cleavage between the parent and the offspring.

"My most recent experiment consisted in adding a 101-millimeter, f/4.5 Graflex Optar lens, complete with diaphragm and shutter, to the standard condenser lens of the microscope and thoroughly washing the specimen before making the picture. In addition I used Eastman Panatomic-X film for the 35millimeter camera in place of Plus-X; the former is slower and finer grained. The new condenser improved the depth of field about tenfold and also increased the contrast substantially. The cleaning procedure eliminated the foreign matter in the vicinity of the organism that not only imparted a granular texture to the surrounding fluid but also distorted the fine details of the specimen. Previously organisms had been transferred directly from the culture to the slide of the microscope by means of a pipette, which also transfers a drop of culture fluid containing bacteria and decaying matter in suspension.

"To clean the specimens I used a microculture slide in the form of a glass plate containing 12 depressions, each approximately one centimeter wide and four millimeters deep. The depressions were filled with distilled water at the same temperature as the culture solution. The organism to be cleaned was transferred to one of the depressions and allowed to swim freely for about 20 seconds. It was then transferred by means of a clean pipette to another of the depressions for 20 seconds and so on through the series of 12 baths. My next procedure was to transfer the specimen to a clean microscope slide, which I then put in position on the stage of the microscope.

"This procedure appeared to eliminate about 95 percent of the foreign matter. The accompanying photomicrograph [Figure 8] of a Daphnia so cleaned reveals many details not previously recorded For example, observe the bubbles that surround the compound eye, the upper intestine, the bristle-like processes on the second branches of the antennae, the body cilia, the thoracic leg stopped on its outward stroke and, most interesting of all, the texture of the shell of the body.

"I do not want to give the impression that careful techniques can compensate for a microscope of inferior optical quality. The quality of the recorded image can be no better than that of the objective lens. Good objective lenses as well as eyepieces are now available at reasonable prices. They can be made to work almost as well on an inexpensive instrument as on one costing hundreds of dollars. The quality of the camera lens, on the other hand, is not so critical. It takes a better eye than mine to distinguish between a photomicrograph recorded by a camera priced at $35 and one by a camera that sells for $350.

"Where does the amateur who is interested in small crustaceans get specimens and how are they preserved? I collect mine from a nearby creek. A pint jar dipped anywhere along the stream contains numerous algae, hydroids, free-living copepods, ostracods and Daphnia. Similar organisms will be found in most freshwater brooks and pools of the U.S., particularly in late spring.

"Cultures can be maintained for some time merely by placing a bit of mud from the brook or pool in the bottom of a five-liter flask, filling the flask with water from the brook or pool and maintaining the temperature. I am experimenting with a prepared culture in which Daphnia are fed bacteria. The bacteria, Bacillus cereris, are cultured on yeast extract procured from Difco Laboratories Inc., 920 Henry Street, Detroit, Mich. 48201. The solution consists of 250 milligrams of yeast extract in one liter of water. The mixture is sterilized in an autoclave, or pressure cooker, at 15 pounds for 15 minutes. When the solution has cooled to room temperature, it is inoculated with bacteria. The crustaceans are added the next day.

"As I write I have kept an adult female Daphnia in this medium for two weeks. The organism appears likely to have a normal span of life. I shall be pleased to communicate with anyone interested in the outcome of this experiment and others that can be done with small crustaceans. My address is 24012 Country Club Drive, Los Altos, Calif. 94022."

Bibliography

ANIMALS WITHOUT BACKBONES. Ralph Buchsbaum. The University of Chicago Press, 1962.

FRESH WATER INVERTEBRATES OF THE UNITED STATES. Robert W. Pennak. The Ronald Press Company, 1953.

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