Apparatus for Investigating the Reactions of Land Arthropods to Humidity

In recent years, a large amount of work has been done on the effect of humidity conditions on terrestrial invertebrates, especially on the rate and manner of water loss in insects (Buxton, 1932; Mellanby, 1935; Ramsay, 1935); there are relatively few accounts in the literature, however, of work on the reactions of animals to the humidity stimulus (Herter, 1926, 1932; Martini and Teubner, 1933). There is reason to believe that the failure to fill this gap is partly due to the difficulties encountered in attempting to obtain suitable known humidity conditions. In the course of work on cockroaches (Blatta orientalis), locusts (Locusta migratoria) and woodlice (Porcellio scaber), we have devised various sorts of apparatus which enable us to test and to analyse the humidity reactions of arthropods. We have been urged to publish an account of this apparatus and its use immediately, rather than defer publication until the experimental work is completed, so that our experience may be at the service of other workers without delay. In this paper, then, the reactions of animals are mentioned only when they throw light on the properties and use of the apparatus.

An essential part of the apparatus is the hygrometer. In only one paper so far published on the reactions of invertebrates in still air, is there any account of the measurement of humidity conditions (Martini and Teubner, 1933). Measurements must be taken without changing the conditions, so that methods involving the extraction of air or lowering of temperature are not applicable. The most convenient instrument is therefore the type which depends for its action on the variation in length of some organic material with variation in humidity. There are three such instruments available, in which hair, paper, and pine-cone fibre respectively are the essential materials ; and we have found that the paper hygrometer (Edney) is the most satisfactory in use, and the cheapest. The instrument is shaped like a pocket watch, about 5 cm. in diameter, and it gives a direct reading of relative humidity. As received from the manufacturer, it usually reads within ± 6 per cent, of the true value. We calibrate it in a sealed chamber over sulphuric acid of known concentration. Over a long period and on successive testing at one relative humidity, the reading varies irregularly by about ± 3 per cent., and if the hygrometer is placed in saturated air, the Calibration curve becomes permanently and considerably changed, so that frequent recalibration is necessary. This hygrometer is suitable for measuring steady humidities alone. Although it reacts sufficiently quickly to indicate changes of humidity, it cannot be used to measure changes, owing to the slowness with which the paper reaches its equilibrium condition. Thus, a hygrometer removed from a desiccator at 24 per cent. R.H. to still air in a room at 54 per cent. R.H. registered 44 per cent, in 11 min. and 51 per cent, in 25 min. The final value was reached in 50 − 80 min.

If it were possible to obtain a sample of water vapour with a pressure of one atmosphere at room temperature, that sample would have a density of about 0· 65 (air=1·0). Consequently air with any considerable amount of water vapour in it is appreciably lighter than dry air. If a dish of water is placed in dry air in a large closed chamber, the air over the water becomes moister and rises as a column to the top of the chamber. Owing to this convection current, the air at the top of the chamber may actually be moister than the air in other parts, even when the water is at the bottom. If the water supply is removed from the chamber, the water vapour slowly diffuses down and eventually its distribution becomes uniform. Once uniformity is attained, it is permanent.

These facts dictate the form of apparatus required. There must be no currents and no leaks, and differences in humidity must be set up and maintained entirely by diffusion. In order to minimise convection currents, the vertical height of the apparatus must also be small, for then diffusion downwards has the maximum opportunity of neutralising the tendency of the water vapour to float to the top. The only alternative to this arrangement is to have a tall chamber with the water at the top (giving a vertical gradient), but so many arthropods react to gravity that this form of apparatus has no general application.

It is possible to set up another kind of gradient of humidity, in which there is a current, or a number of currents, of air (Shelford, 1913); but this introduces complications, since moving air is often itself a stimulus (e.g. Necheles, 1924), and we have aimed at elimination of such complications. Moreover, if one bears in mind the considerations outlined above, it is a relatively simple matter to set up a diffusion gradient which lasts for a long time, and which can be used for experiments on animals.

In the past, a clear distinction has not always been made between reaction to liquid water (either by contact or by sight) and reaction to. the humidity of the air (Herter, 1926). This distinction can be expressed by using the terms hydrokinesis or hydrotaxis on the one hand and hygrokinesis or hygrotaxis on the other (Wolsky, 1933 ; Savory, 1930, 1934). In studying humidity reactions (e.g. hygrotaxis), no contact with the water source must of course be allowed.

When an animal is placed in a chamber in which there are differences of humidity, and it consistently remains longer in the drier region than it does in the moister one, we say that the animal shows a “preference” for the drier region, and we shall use this term in an objective technical sense without any anthropomorphic implication.

The first problem is to discover whether the given animal exhibits any reaction to humidity at all. For this, the animal is offered a variety of air humidities, to see if it comes to rest in, or restricts its activities to, a certain limited range of humidity. The problem of the mechanism of such a reaction is dealt with afterwards.

The diffusion gradient is in this case set up along the channel of a circular brass trough with a double plate-glass roof sealed on with vaseline. The channel is 9 cm. wide and 3 · 5 cm. high (Fig. 1). The diameter of the circle forming the centre line of the channel is 90 cm. Pockets for hygrometers, water, and the drying agent (calcium chloride, concentrated sulphuric acid, or phosphorus pentoxide) are arranged under the main channel, the whole floor being covered by a carpet of perforated zinc. Thermometers, with only their bulbs projecting into the channel, are fitted through bungs in holes at intervals along the trough. The dishes of water and of drying agent are placed in pits at points diametrically opposite one another.

Since the whole apparatus forms a closed system, the animals are stirred by means of a piece of soft iron wire, which can be moved about from outside the channel with a strong Darwen horseshoe magnet.

This ring apparatus gives, of course, two separate gradients continuous at their extremes. An animal which approaches the drier end, for instance, is able to pass perfectly freely and without turning round into the moister region again.

It is very important to note that if the apparatus is tilted so that the water and drying agent are in the same horizontal line, but one side is higher than the other by as little as 3· 5 mm. (i.e. about 0· 2°), then a convection current is set up which practically destroys the gradient (Fig. 2, curve B). With accurate levelling, a satisfactory gradient is set up (Fig. 2, curve A) after 16 hours, which will remain for weeks. The straight line diffusion gradient shown accords with expectation (Ramsay, 1935). The convection current is not found in the straight apparatus (see below) and has been demonstrated to be due to the existence of two separate routes from the wet pole to the dry.

In the experiments, the position of the animal is recorded at 20-min. intervals. If the animal is found in the same place in two successive observations, that position is regarded as one reading. The distribution over the gradient of these positions where the animal remains for as long as 20 min, gives a measure of the animal’s reaction to humidity. If there is a reaction, this distribution indicates which part of the humidity range is concerned.

While this apparatus possesses the advantage of providing a long straight humidity gradient, whose other physical features are perfectly uniform, the relative narrowness of the channel interferes with the reactions of larger animals.

The ring gradient is also unsuitable for very active animals, or for groups of gregarious animals, since these are found to travel round and round the ring for long periods without stopping. In certain cases, perforated zinc barriers might perhaps be placed in the channel in places where they do not impair observation of the humidity reaction, in order to stop this unceasing travel.

A channel gradient constructed on the same principles as the ring gradient, but in straight line instead of ring form, does not provoke perpetual motion, but for general purposes it suffers from the great disadvantage that there are two places in it—the two ends of the channel—where the animal is inclined to stay rather than in other places, even before humidity differences are provided.

In order to overcome the above difficulties, another apparatus was designed, which gives the animals adequate space in which to manœuvre. Here, instead of attempting to make available to the animal all possible air humidities at once, and to render all parts of the gradient physically uniform, a short range only is provided and the animals’ discrimination between the moister and drier portions of this range ascertained. The principle of the chamber is similar to that upon which Graber (1887) based his apparatus for the investigation of temperature reactions in insects.

The apparatus (Fig. 3) consists simply of a shallow cylindrical glass chamber 22 cm. internal diameter, containing a false floor of perforated zinc, leaving 3− 4 cm. between this and a glass roof. Under the false floor, dishes containing humidity-controlling fluids (sulphuric acid-water mixtures or saturated salt solutions ; Buxton and Mellanby, 1934) are placed in two opposing groups. A hygrometer is placed on the false floor with the paper towards the wall, against each of two opposite walls. A short thermometer is placed in each half of the chamber also. In spite of evaporation in one half of the chamber, the temperature difference between the two halves is never more than 1· 5° C.

Any difference of humidity from o to 75 per cent, difference between the two extremes may be obtained, according to the combination of fluids used and the number of dishes on each side of the median line.

Very active animals may be studied in this apparatus by observing a single specimen continuously for a period of, say, 20 min., and recording on paper the path followed. Groups of animals or less active animals may be studied by counting every 20 min. the number of animals each side of a median dividing line. The difference of humidity shown by the two hygrometers in a given experiment is the maximum difference offered to the animals, and when a reaction appears it may be a reaction to this or to a smaller difference. It is not possible to say exactly to whar humidity difference the animals are responding. After obtaining a clear preference with a given difference of humidity, it is then possible to derive a fair measure of the degree of discrimination of which an animal is capable. This can be done by conducting experiments with progressively smaller humidity differences between the two sides until a point is reached where no significant majority of animals is found in one half.

Having established the existence of a reaction to humidity, the analysis of the mechanisms by which the reaction is carried out may be begun with the following forms of apparatus.

It should be borne in mind, however, that an animal may have shown no reaction to humidity in the forms of apparatus described above, and yet be able to show an avoiding reaction. This reaction must be looked for separately.

It is possible that the animals are stimulated to activity when in moist air, and not so stimulated when in drier air, or vice versa. For the investigation of such a kinetic response to humidity, a modification of Szymanski’s “Aktograph” (1914) has been devised (Fig. 4).

This aktograph consists essentially of a box, pivoted on a knife-edge about its median transverse line. Any movement of an animal along the longitudinal axis of the box tips the box. This tipping is recorded by a lever writing on an ordinary smoked drum.

The box was cast in aluminium, with walls 3 mm. thick, and internal dimensions 20 × 10 cm., 6 cm. deep. A small ledge 2 cm. above the true floor runs all round the inside walls and carries a false floor of perforated zinc. The true floor is divided by transverse partitions 1 cm. high and 1· 5 cm. apart into a number of troughs in which the humidity-controlling fluid or solid is placed. The partitions prevent gross movements of fluid when the box tips. The lid is of glass and is sealed on with vaseline. The writing lever is attached to one end of the box and an adjustable counterpoise to the other end. A rod passing vertically downwards below the centre point of the box carries a weight, which serves to bring the centre of gravity of the whole below the fulcrum, and may be screwed up and down in order to alter the sensitivity of the balance. Below the weight a transverse vertical vane attached to the rod is immersed in a fluid so as to damp the oscillations of the balance.

With suitable adjustment of the lower weight and of the damping fluid the balance will give good records of activity over long (48 hours) or short periods with animals ranging in weight from 30 mg. to 2 or 3 gm. Results are purely relative. The lever records nothing of the movements across the floor, but animals which are active usually travel round and round the box, and degrees of activity can be distinguished from the record (Fig. 5).

The best known example of this reaction is that shown by Paramecium (Jennings, 1906). In passing through regions of differing concentration of certain substances, the animal is seen to stop, back, turn, and move off in another direction. In order to find out whether such an avoiding reaction to drier or to moister air encountered by an animal exists, the animal’s behaviour may be closely observed in the “Alternative chamber” described on pp. 454− 6. Animals placed in this chamber are observed carefully as they traverse the dividing line. The humidity gradient across this dividing line may be rendered steeper by having the roof as low as possible, and by having the floor covered with dishes of controlling fluid. By these means an humidity difference up to 30 per cent, may be obtained between points 2 cm. each side of the dividing line, with a much less steep gradient in the other parts.

It is possible that the animal’s reaction to humidity is an orientated one, either tropotactic or telotactic (Kiihn, 1919, 1929; Fraenkel, 1931). A preliminary apparatus for the study of such a reaction has been devised. It might supplant the channel gradient described on pp. 452− 4, for almost all purposes, for the only advantage known to be possessed by the ring and straight channel forms of apparatus over that to be described is the great length of steady gradient obtainable.

This apparatus consists of a square chamber of plate glass. The internal length of one side is 30 cm., and the internal height 5 cm. The floor and walls are cemented together with de Khotinsky cement, and the roof made airtight with vaseline. Strips of glass are fitted on the floor 15 cm. away from two opposite walls, forming two shallow troughs. Water is placed in one of these troughs and a drying agent in the other. Hygrometers are placed at various points on the floor and a false floor of perforated zinc laid over the whole floor space including the troughs, leaving a height of 3 cm. between roof and false floor.

This apparatus has not been adequately tested, but a preliminary test showed a steady gradient from 25 to 80 per cent. R.H. between the two sides where the water and drying agent were. The large area available should provide ample space for the display, for instance, of circus movements after unilateral extirpation of sense organs.

In order to test for the existence of a reaction to air humidity, to investigate the degree of discrimination displayed in this reaction if present, and to test for an avoiding reaction, a long, regularly falling, humidity gradient is not required. Alternative chambers will do for these purposes.

In order to find an accurate measure of such of these and other reactions as may be discovered, it is desirable to be able to state the exact rate of fall of relative humidity with distance. For this purpose the forms of apparatus (A (1), pp. 452− 4 and B (3), above) which give known, steadily falling humidity gradients are required. The square gradient (of the dimensions given) is cheaper, less unwieldy, and provides more space for manoeuvring than the ring gradient ; but it does not—as the ring gradient does—provide almost the complete range of humidity.