The Feeding Responses and Nutrition of the Larvae of the Beetle Phaedon Cochleariae F

The feeding responses and nutrition of the larvae of the beetle Phaedon cochleariae F.

Thesis presented for the Degree of Doctor of Philosophy in the Faculty of Science, University of London.

by Michael Thomas Tanton.

Imperial College, Silwood Park, Ascot, Berks. October, 1960. -1-

ABSTRACT.

The stimuli effecting the orientation and feeding of the mustard beetle, Phaedon cochleariae F., were investigated, with a view to incorporating these stimuli in an artificial diet and inducing optimum feeding. The response of the larvae to the mustard oil, allyl iso-thiocyanate, which occurs in the natural food-plants, the Cruciferae, was tested in an olfactometer. Oil solutions of 0.5 to 250p.p.m. w/v concentration attracted the larvae, but concentrations above 500p.p.m. were repellent. The oil alone was not sufficient for complete orientaion° work with coloured paper cones showed that colour was also essential. Kodak 'Wratten' filter combinations were used to transmit known wave-length bands at equal energy° the response of the larvae to these gave a curve with a peak at yellow-green (530-560W. Using an 'energy curve' for the lights, and also a constant energy method, true colour vision and colour attraction were proved. Then the larvae reached the source, the mustard oil stimulated contact chemoreceptors and elicited a biting response: delayed biting also occurred as a hunger response. Continued feeding ensued if the glucoside sinigrin was present to give a suitable taste. Sinigrin was tested in an agar gel, and concentrations of 5,000-10,000p.p.m. were the most effective. A photo-electric method, reliable and accurate, was used to assess the small areas eaten. Increased leaf toughness, measured by a Williams type penetrometer, affected continued feeding on leaves, and slowed the growth of the larvae. Physical factors were also important in the formulation of the diet: 5% was the best concentration of an agar gel, but agar did not allow the larvae to eliminate waste, nor did it provide a suitable -2- mechanical otimulus for feeding. For this reason, the formulation of a satisfactory synthetic diet failed, and future work must first find a suitable inert base. ---oOo----

-3- INDEX. Page. ABSTRACT. e..,.... 1 INDEX...... 0....0. 3

I. INTRODUCTION. 000000 7 II. LITERATURE, 10

A. Orientation to the food. el 00 000 60 12

a). Olfactory stimuli. 0•0 00 0•• 12

b). Colour responses. 00•0000• 13

B. The biting response. • 0 • 0 00 00 26 C. Continued feeding. 26

D. Physical factors. CO 0•0 00 0 28

E. Insect nu.etion. 06.0..0• 30

a). Mineral requirements. • 00 ..... 30

b). Carbohydrates and fat. 00000000 32 c). Proteins and related compounds. 33

d). Accessory growth factors. 0•••000• 37 F. The nutrition of phytophagous insects. 42

III. REARING THE LARVAE. •• 000 00 0000 600 00 46 IV. ORIENTATION OF LARVAE TO THE FOOD PLANT. 50 A. Orientation to the food. The presence of an olfactory response. 50

B. The biting response. 00006000 53

C. The feeding response. 00000000 56 D. The effect of physical factors...... 59 E. The nature and optimum concentration of the olfactory stimulus...... 68 a). Use of a Varley and Edwards type olfactometer. 68 b). Confirmation of olfactometer results:

the 'Split chamber'. ....•. .0 81 F. The effect of colour on orientation. 84 a).The use of coloured paper cones. 84

b). The use of coloured light sources. 000 100 i). Use of filters for the light source. 100 -4-

ii).A preliminary experiment to test response to equal energy lights. ... 107 iii).Effect of illuminating each end of the chamber with different colours

of equal energy. ****** 00•00•0000 113 1v). Energy curve experiment to demon- strate colour vision. 117 v). Constant energy method for checking presence of colour vision. •• a 0 • • • 119 V. THE FEEDING RESPONSE OF THE LARVAE. .... 126 A. The method of getting readings. 126 B. The effect of sinigrin on the feeding responses. 128 a). Tests with different concentrations of sinigrin. 128 b). Effect of concentration of sinigrin and presence of colour. 132 c).Effect of mustard oil in agar containing sinigrin. OOOOOOOOOO 137 d). The effect of agar concentration. 0000 140 C. The main factors in feeding revealedby the preceding work. 142 VI. THE PRELIMINARY FORMULATION OF A SYNTHETIC DIET. 000000000,0000000 144 A. The formulation of the mixtures. .. 144

a). The amino-acid mixture. • O• 0 00 0 a 144

b). The vitamin mixture. 0••0 00 0 145 c).The fatty-acid mixture. 146

d). The salt mixture. O OOOOO 0 0 147

e). The carbohydrate. O 00 0 a a 00 148

f). The composition of the basic diet. • 40 148 B. The effect of varying the proportions of the main constituent nutrient groups. 148 C. The technique for aseptic culturing. 152

a). The sterile chamber. a OOOOOO 152 b). Sterilisation of the glassware. 153 c). Surface-sterilisation of the eggs. 153 d). The feeding chambers. 155 D. A preliminary experiment to test growth under aseptic conditions. OOOOOO 155 E. Comparison of growth of larvae from surface-sterilised, and from untreated, eggs. •.••00.0•0•00 157

VII. DISCUSSION. 4000000.00000000 160 A. The orientation of the larvae of Phaedon

to the food. 000000000.000000 160

a). Odour. OOOOOO 0000000000 160

b). Colour. OOOOOO 000000.00 164

B. The biting response ose00000sopp0000 174

C. Conttinued feeding. OOOOO 000.00 OOOOO 175

a). Taste. 000000.000000000 175

b). Physical factors. 000000000.000000 177 D. The main factors in orientation and

feeding. 0000000.00000000 178 E. The importance of the orientation and

feeding stimuli in nature. OOOOO 000 179

F. The formulation of the diet. OOOOO 000 180

Suggestions for further work. OOOOO 000 184

VIII. SUMMARY. OOOOOOO 000000000 186

Acknowledgements. 000000000.400000 189

IX. REFERENCES......

Table. Page. Table. Page.

1 OOOOO 00 54 6 000000. 64

2 57 7 00.0000 65 3 57 8 op..... 67 4 58 9 . . 77-78 5 0000000 62 10 ...... 83 Table. Page. Table. Page.

11 87 22 OOOOO 0 . 116 12 92 23 119

13 93 24 • •0 00• 124 14 96 25 130

15 .O..... 97 26 130

16 101 27 • ••..•. 135

17 10+ 28 • 00 00•0 135

18 105 29 0000•0• 139

19 106 30 • 0••0•0 139 20 111 31 141 21 114 32 141

Figure. Page. Figure. Page. 1 52 8 OOOOO .. 80 2 52 9 89 3 OOOOO .. 61 10 108 4 ...... 71 11 OOOOO 0. 121 5 73 12 131 6 73 13 136

7 ...... 80 14 .. OOOOO 143

Graph. Page. 1 66 2 94 3 98-99 4 103 5 OOOOO 112 6 125

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I. INTRODUCTION.

So far, relatively little work has been done towards elucidating the many factors that must influence the feeding of a phytophagous insect; mostly, this work has been directed at showing the importance of chemical substances in affecting the feeding responses of but a few insects. This work on chemical attraction arose mainly from the primary study of Verschaeffelt (1910). Food plant/selection initially involves the location of the specific food plants, and Verschaeffelt did some of the earliest work on chemical factors influencing the selection. His study on the response of the larvae of Pieris rapae to the mustard oils of their food plants initiated the idea that some plants are subject to the attacks of insects because of the presence of some chemicals, such as essential oils and glucosides. Verschaeffelt's work did not immediately stimulate further research into the problem of host plant selection, but since his time a good deal has been revealed by the work of a few people. The investigation of these factors is of some importance for studies of phytophagous insects, because of the bearing they have on the formulation of satisfactory diets for these insects. When the importance of this group of insects is considered, the number of papers on the subject are relatively few: those insects th6t have been most fully investigated in nutritional studies have had a diet which lent itself to replacement by artificial mixtures. The present work attempted, firstly, to define the factors affecting the feeding behaviour of the larvae of the Mustard beetle, Phaedon cochleariae F., and, secondly, to show how these factors could be applied to the formulation of a satisfactory synthetic diet. -8-

The larvae of Phaedon cochleariae were chosen as test subjects for the following reasons. The beetle is very easy to rear throughout the year: it has a life-cycle of only three weeks, which is usefully short for nutritional work: and the larvae are small, easy to handle, and require relatively small amounts of food. A final reason for the selection of these larvae was that they feed on various cruciferous plants, and might be expected to show a similar response to chemical factors as that shown for certain lepidopterous larvae by Verschaeffelt and Thorsteinson. The presence of such a response would probably make a synthetic diet very much more acceptable if the attractants were included in it. The present work with the larvae of Phaedon was started on the assumption that chemical factors were affecting their orientation and feeding behaviour. Preliminary experiments with a chamber olfactometer showed that the leaves of turnip had an odour attractive to the larvae. Choice experiments, as described in the experimental section, showed that olfactory and gustatory stimuli were present: these stimuli were identified as those shown by Verschaeffelt and Thorsteinson to be attractants for lepidopterous larvae which feed on Cruciferae. Optimum concentrations were determined, and during this work it was noticed that relatively few larvae reached the test spot, although some came very close to it. An investigation 'of this effect showed that a colour stimulus was also necessary for orientation. The results could now be applied to the formulation of a synthetic diet. Before this could be successful, it seemed likely that the physical texture of the diet would be important. The leaf toughness, and its effect onilarval growth, was determined, and the findings were applied to the -9- formulation of the initial diets. The final work, the formulation of a successful synthetic diet, was not taken as far_as was originally hoped. This was because of the discovery of the colour response, which required full investigation. It is believed that this is the first record of colour vision in the larvae of a beetle. ---o0o---

-10-

II. LITERATURE. A Review of some of the literature about feedin res.onse and nutrition in insects.

Active selection of a food-plant by an insect pre- supposes a means of recognition. Whilst adult insects are at liberty to select their own diets, many larvae are restricted to the food-plant chosen by the female; but these larvae are still able to identify a preferred diet, a feature essential if the larvae are to get back to food from which they have been dislodged. While vision, phototaxis, and hygrotaxis probably play a part in directing the insect, close range forces operating in the recognition of preferred plants must be mainly chemical. (Dethier, 1947, 1951; Painter, 1936). The behaviour pattern which culminates in normal feeding can be broken down into - a.). orientation to the food, b). a biting response, and, c). continued feeding. These categories are clearly shown from. the work of Verschaeffelt (1910), and Thorsteinson (1953). Because these works provide classical examples, and because the present work with Phaedon proved similar, they are described now in more detail. Verschaeffelt worked with the larvae of Pieris brassie- ae and P. rapae, which feed on Cruciferae. He found that these insects were attracted to the food plants by glucosides, which produce mustard oils (alkyl iso-thiocyanates) by hydrolysis. Not all species of the Cruciferae were attacked, nor were the leaves of certain Caricaceae, which contained mustard oils this was attributed to the presence of repellents. Pieris larvae would immediately accept the -11- normally unacceptable leaves of Apios tuberosa if these were smeared with the juice of Crucifer leaves. Wetting the leaves with the glucoside sinigrin also caused feeding. From these results, Verschaeffelt concluded that the mustard oil and the glucoside caused attraction and feeding, but he did not distinguish clearly the effects of the two substances. Thorsteinson (1953) repeated the work, using Pieris brassicae larvae, and the larvae of Plutella maculipennis. He showed that the orientation to the food was caused by the mustard oil given off by the leaves of the Cruciferae. Continued feeding was then caused by specific taste attractants, which were the glucosides sinigrin, sinalbin, and gluco-cheirolin; in the absence of these glucosides, even with the mustard oil present, continued feeding was blocked. Effective concentrations were as low as 0.0002%. Since mustard oil plus sinigrin enhanced the amount of feeding over that obtained with sinigrin alone present, he concluded that a biting response was initiated by the olfactory stimulation of the oil. But if the oil was absent, biting would occur as a hunger response, and feeding continued in response to the stimulation of the glucosides. It is interesting to note that recently (1960) Yamamoto and Fraenkel have reported that a glucosidic substance stimulates feeding by the Tobacco Hornworm, Protoparce sexta, on Solanaceous plants. This stimulus is reinforced by the presence of complementary substances in the plant, such as sugars. The attractant has also proved effective with Leptinotarsa. The olfactory and gustatory stimuli for the larvae of Phaedon closely followed the pattern found by Thorsteinson for his larvae. Further literature on orientation and feeding by insects is given below. -12-

A). Orientation to the food. a). Olfactory stimuli. The importance of plant odours for orientation was first shown by Verscheeffelt (1910), with Pieris larvae (see above). Since that time orientation by odour has been demonstrated by McIndoo (1926, 1928)9 with larvae of the Cotton boll weevil, and of the Codling moth; and by Dethier (1937, 1941), with a number of lepidopterous larvae. Dethier showed that larvae of Danaus plexippus, during 'screen tests', became agitated when they passed over the leaves of the food-plant, but not when passing over unattractive leaves. Unattractive leaves could be made attractive by coating them with Milkweed latex. The odour of methyl alcohol on the leaves over-rode the attractiveness. Sim- ilarly, he demonstrated that the larvae of Papilio alax were attracted by certain common oils in Umbellifers, and inferred that the change of food-plant from Rutaceae to Umbelliferae took place because identical attractants were present in both families. Citrus medica, which has the oil giving the odour of rue, was not attacked because this odour was masked by that of citral. Similar experiments with olfactometers and feeding tests have confirmed the presence of attractive olfactory stimuli acting on other insects. Chin (1950) showed that Leptinotarsa was attracted to its food-plant by an olfactory attractant; Hesse and Meier (1950) claimed that this attractant was acetaldehyde. Cuille (1950), and Dethier (1947) showed that the Banana weevil was attracted by odour: Azal (1954) found that Stegobium paniceum was attracted by the odour of tobacco: the larvae of Bombyx mori were attracted by the odour of mulberry leaves (Balazs, 1952), and Watanabe (1958) showed the attraction to be by/3-11- hexenol and 04,42- hexenal; recently (1959), Hamamura confirmed this result, and he also showed that two other distinct -13- compounds caused biting and feeding. Amines and oxalic and formic acids secreted by the hairs of the food-plant attracted Chloridea obsoleta (Lozina-Lozinskii, 1946) and Ripley and Heerden (1939) found that odour attracted the Wattle bag-worm, Acanthopsyche junodi. In a less well known part of his paper, Verschaeffelt (1910) states that the larvae of the leaf wasp Priophorus padi attacked plants containing amygdalin, which substance gives off hydrocyanic acid gas; the gas acts as an olfactory attractant. He also thought that the beetle Gastroidoa viridula might be attracted by oxalic and/other organic acids in the leaves of Rumex. Of course, not all insects are attracted by odour to their food-plant;' those that are attracted in this way are mostly monophagous and oligophagous types. Williams (1954) found that odour was not important for the primary attraction of grasshoppers. But Slifer (1959) reported that the grasshopper Romalea microptera (Beauvois) responded to strong essential oils. The effective range over which odours are attractive for leaf feeders has boon shown to be short, but a few millimetres (Chin, 1950), but within this range the behaviour of the insect changes perceptibly and characteristically. Orientation by the Banana weevil is also caused by an odour acting over short distances; another distinct substance stimulates contact chemo-receptors on the antennae, and this directs the beetle to the correct site on the plant for feeding (Cuill'e, 1950).

b). Colour responses. A large number of workers have reported a response to colour by different insects; this is often associated with the finding of food. Many have made observations incidental to other work, and conditions were not controlled. Some -14- workers observed the reactions of insects, and others trained certain species to come to particular colours for food; in most cases coloured surfaces were used for training. A disadvantage of this method is that coloured surfaces often reflect wave-lengths other than those which give the physiological sensation of colour in the human eye; it is therefore difficult to say to what wave-length the insects were attracted, and so the results of much of this work are invalidated. Yet other workers used known wave- lengths of light under controlled conditions, but even then many neglected the external source of the colour sensation, which is physical, and described their results in terms of human colour vision, which is physiological. A small number of workers used known wave-lengths, light sources of known transmission, and equal energy output for each wave- length: surprisingly, the qualitative results of these experiments compare closely with those obtained by other workers using less precise methods. More recently, electro- retinograms, instead of the behaviour of the insects, have been used to judge the response. Frequently the conclusions drawn from different experimental set-ups have been the subject of controversy. The main difficulty has been that of determining if the discrimination shown by the insects is due to the intensity of the colours, or to their wavelength. It can be seen from this introduction to the types of work undertaken that colour responses in insects have often been studied as a pure research problem, and the relevance of this work to the orientation of the insect to food is not clear: This applies mainly to the work where known wave-lengths of light were used. In other cases there is a definite connection between the response reported and the orientation of the insect to the food. A review of the literature is given below. Weiss -15- (1943) gives a fairly extensive review to date. Lubbock (Avebury, 1929-reprint) established that bees were able to distinguish one colour from another, and could be trained to associate the finding of food with blue or orange coloured papers. Forel (1908) obtained the same result with paper flowers. This work did not show if the insects were responding definitely to the colours, or to the brightness of the light reflected from them- Hess (1910) projected a spectrunhon to a parallel-sided glass chamber, and in this he tested adults and larvae of Vanessa urticae, and also bees. The insects went to the yellow- green colour zones in preference to the other colours.. Hess stated that Avebury, Forel, and others were mistaken n attributing the response that they obtained to colour vision, and he concluded that, just as totally colour-blind persons see yellow-green as the brightest part of the spectrum, his insects were colour-blind. In addition, Hess determined the shades of grey which exactly matched the luminosities from screens of certain colours, and he then found that his insects showed no discrimination between a colour and its corresponding grey shade. From this he again concluded that various colours are of the same relative brightness to insects as they are to totally colour- blind persons, and therefore that insects lack colour vision. Von Frisch (1924) succeeded in training an asiatic species of honey-bee to come to a given colour for food, and to pick out that colour from amongst others when no food was present. He used a series of 15 shades of paper, from white, through various greys, to black, and a blue paper which was associated with food by the bees. He found that the bees in every case gathered over the blue, and not over the greys. Von Frisch also found that bees conditioned to red or black did not distinguish between these two colours and dark grey, and he decided that they -16- were blind to red. He also discovered that bees conditioned to yellow failed to distinguish between yellow, yellow-green, and orange; and that bees conditioned to blue went also to violet or purple. He finally concluded that bees can distinguish all colours except red and certain greens, which appear as darker or lighter greys, and therefore that their colour vision was identical with that of partially colour-blind persons. Turner (191Q) also did training experiments with bees. Haan (1928), on the other hand, stated that bees trained to go for food to papers of different colours made their selections:.by absolute brightness of the light reflected from the papers. use Ocmia,tc137) showed that when certain butterflies were offered gaily coloured paper flowers, they preferred these to grey ones. Pieris, in search of food, preferred blue and purple papers and, to a lesser extent, red and yellow; it paid no attention to green, blue-green, and grey. For egg deposition the female chose emerald green to greenish blue papers. So far, it is apparent that the various authors believed that insects make their selections of colours on their brightness; that insects are totally colour-blind; that colour vision is the same as that for partially colour- blind persons; or that insects prefer particular colours. Little is known of the wave-lengths with which these authors worked; the results are expressed in terms of human colour vision. In addition, some authors covered their coloured papers with glass to reduce differential reflection of ultraviolet light, but this glass itself could have reflected the colour of nearby objects. Lutz (1924) tested the papers used by von Frisch and found that some of his greens and blues reflected ultraviolet, and his yellows and greens reflected red and blue. Workers now investigated the effect of wave-length, -17- using a range from 250mp to 700m).1 (ultraviolet to infra- red). Their results showed that insects were most stimulated by the shorter wave-lengths, as will be seen from the survey below. Lubbock (Avebury, 1929-reprint) showed that ants were negatively phototactic to ultraviolet light, and that they would move from a region illuminated by this light to one illuminated by red light. This was confirmed by the later observations of Forel (1908). Hess (1920, with various arthropods; Lutz and Richt- meyer (1922), with Drosophila; Marshall and Hienton '(1938), with the Codling moth; Mayer and Soule with larvae of Danaus archippus; and Kahn and Pohl (1921), with Apion; all found that their test insects were positively phototactic to ultraviolet light, and moved to this region rather than to regions illuminated by longer wave-lengths. Hess, and Lutz and Richtmeyer, believed that the response might be caused by fluorescence in the tissues of the eye, but Lutz and Grisewood (1934) could find no evidence of fluorescence with ultraviolet light down to 254mp. Kahn and Pohl (1921) trained honey-bees to come for food in a narrow trough illuminated by ultraviolet light of wavelength 366mp. After training, the food was removed, and the entire spectrum was projected upon a shoot of white paper; the bees collected on the area lit by light of 365mp. These authors concluded that four regions of the spectrum are distinguished by bees; the yellow and green; the blue- green; the blue and violet; and the ultraviolet. Kahn (1927) stated that the vision of bees extended at least to 313mia, but this wave-length was not recognised as distinct from light of 36592. The use, by Kuhn, of spectral colours to test the response of the honey-bee enabled him to confirm von.?Frisch's conclusion about the bee's response to red light, and to show that bees do not respond to wave-lengths -18- longer than 650141. He defined the four regions distinguished by bees as, 313-400111 (ultraviolet); 400-480mp (violet-blue); 480-500141 (blue-green); and 510-650my (red, yellow, and green). Lutz (1924) found that although many insects which visited flowers were responding to red, green, yellow, or blue, most preferred to go to ultraviolet rather than to these colours. In addition, Lutz (1941) trained honey-bees to come to a white card which was reflecting certain ultraviolet wave-lengths. Abbott (1927) used four light intensities and four colour filters of known transmission to test the effect of colour on the ant Formica dakotensis specularis. Yellow had the greatest stimulating effect, and white was second; the results with green and red were very variable. In his paper on the reactions of arthropods to monochromatic light of equal intensities, Gross (1913) reported that blow-fly adults (Calliphora erythrocephala Meig.); fruit-fly adults (Drosophila ampelophila Loew.); larvae of the Wood Leopard moth (Zeuzera pyrina); and a Noctuid moth (Felthia subgothica Haw.); were all photo-positive to colours. The order of attractiveness was blue, (420-480m0; green, (490-550mp); yellow, (570-620mp); and red, (630- 655W. He also found that the cockroach, Periplaneta americana Linn. was photo-positive to blue light, photo- negative to green and yellow, and indifferent to red. Bertholf (1931, 1932) investigated the relative efficiency of different regions of the spectrum in stimulating Drosophila. He found that the efficiency of the longer wave-lengths was very low, but it started to rise at about 575m)1 (Yellow), and increased to a maximum at 487mg (blue- green). From 4871p it declined to 425mg (violet), and then rose to a peak at 365mia (ultraviolet), after which it declined again to 230141. Bertholf (1931) also tested the -19-- response of honey-bees to the visible spectrum, and found that the spectrum for the insect extended from 431 to 67791, with two peaks of stimulation. One peak was at 553mp (yellow-green), and the other was at 365mu (ultraviolet); the peak at 365mp was about 4.5 times that at 553mp. He also investigated the ability of the honey-bee to distinguish between colours of the same brilliance, and his results confirmed those of Lubbock, von Frisch, and Kahn. During these experiments it was found that the bees were able to differentiate between two illuminated areas when the brightness of one was reduced to at least 70% of the other. Von Frisch and Kuhn had concluded that only enormous differences in brightness were distinguished by these insects, and Hess thought that the acutance of bees was similar to that of man. Bertholf kept the energy of, the test wave-length constant and varied the standard (white) light until the lights were equally attractive. He also tested each wave-length against a fixed intensity of white, and after finding the ratio of attractiveness he substituted a white of variable intensity for the coloured light, and determined the intensity which gave the same ratio of attractiveness against the standard light. Sander (1933) made the standard and the test wave- length equal in intensity, and used the number of test insects attracted to each as a measure of the relative efficiency. He also adjusted the intensity of the test wave-lengths until they were equal in attractiveness to a white of fixed intensity and quality. When light of equal energy was used at all parts of the spectrum, there was no trace of the high maximum at 365mp. Sander believed that his method was better than that of using light of unequal energy over different parts of the spectrum, and then calculating the relative effect of equal energies. Bees responded to two maxima of stimulation, one at about 570mp (yellow), and the other at 460mp (blue), with a minimum at about 520mu (green). -20-

From 460m31 there was a steady decline into the ultraviolet. Cameron (1938), in his work on the reactions of the house-fly to light of different wavelengths, reported that the fly was more strongly attracted by ultraviolet light of 365mu than by any other wavelength in the range 302-578mp. Cameron applied to his results the three methods of analysis used by previous workers, and found that qualitatively all three methods gave the same result in showing the peak attraction at 365mp, with a rapid decline on both sides of this wave-length. Gullet al. (1942), in their work on the response of insects to colour, intensity, and distribution of light, found that all colours attracted insects to a greater or lesser degree, with an order of most to least attractiveness of blue, white, yellow, and red. Hallock (1936) reported that lights of short wave- length, near the violet end of the spectrum, were most attractive to the Asiatic Garden beetle. Dirks (1937), in his study of the Maine moths, found that lamps which transmitted light in the ultraviolet region attracted the most insects. Herms and Ellsworth (1934) used coloured lights to trap the Clear Lake gnat, Chaoborus lacustris. Lights claimed to be of equal intensity were used these were 60 watt bulbs transmitting a range of wave-lengths from 350-7009.1. Equal numbers of the gnat were attracted to each light, with no peak response. But of two blue lights, which had an intensity ratio of 30:8, the former was the most effective. This was a brightness effect. In a later work with the same insect, Herms (1937) found that of two red discharge tubes with an intensity ratio of 301, the brighter one attracted three times as many gnats as the other. However, the so- called 'red' was a physiological colour seen by the human eye; it may have masked other wave-lengths that were -21- transmitted, and which may have stimulated the insects. Headlee (1937), in a study of the attraction of mos- qiiitoes to sources of radiant energy, determined that the attractive power of red light (580-717mp neon linos), as measured by the numbers of mosquitoes caught per microwatt of energy, was 6 times the number that would have been taken per microwatt in white light (400-800mp). The number per microwatt to green-yellow (546.1, 579mp mercury lines) was 12 times as many as would have boon taken per microwatt of white light; and 21.5 times as many were taken per microwatt by blue light (404.7, 435.9-546.1mp mercury lines). Headlee stressed the importance of wavelength and intensity, and stated that it appeared possible to make any wave-length that was attractive at certain intensities repellent by increasing the intensity beyond a certain point. He also pointed out that the wattage of a lamp was not an accurate measurement of the energy emitted - a point disregarded by many workers. Kelsheimer (1935) reported that when his filters were arranged in ascending order of wave-length, and with equal energy transmitted, the European Corn borer was attracted more to the shorter, blue wave-lengths than to the longer, red wave-lengths. Ultraviolet attracted the most insects, but the peak was not pronounced. This was probably a result of the wide transmission band of the filters used. Also using the European Corn borer, Ficht and Hienton (1941) demonstrated that increase in the intensity of a white light gave a proportional increase in the number of adults caught. There was no increased attraction to a light source emitting ultraviolet light below 320mi, but greater numbers came to lamps radiating most of the visible energy in the violet and blue bands of the spectrum (380-500mp) than to lamps radiating most of the visible energy in the orange-red band (600-760mp). Weiss et al. (1941, 1942) tested the group behaviour -22--

of over 50 species of insects. Most belonged to the Coleoptera, but some were Orthoptera, Heteroptera, Diptera, and Lepidoptera. Monochromatic filters were used, and the physical intensity (as distinct from human visual intensity) was regulatdd- b3.r altering the distance of the light source from the filters. The insects were exposed to 10 wave- length bands of equal intensity, covering a range of wave- lengths from 360-740mi. When the insects were put one foot from the filters the peak response took place at 470-528mi (blue-blue-green), and the response declined on each side. But at six feet from the filters the peak response was at 365-366m}1 (ultraviolet), and the response to the longer wave- lengths fell off sharply: sometimes a large or small secondary peak occurred at about 470-528mia (blue-blue-green). The spectrum from 555-720mp was significantly unattractive. In other words, as the introductory intensity was lowered, there was a shift in response towards the ultraviolet. The possible theoretical importance of this observation on a response shift at lower light intensities was not seen by Weiss and his colleagues. Some interesting new work has been done by Fingerman (1952), and Fingerman and Brown (1952,1953). Fingerman obtained highly suggestive evidence of colour vision in wild type groups of Drosophila; there was also an indication that the spectral response curves changed shape as the intensity of the monochromatic light stimulus was decreased. As long ago as 1922 Hamilton demonstrated the probable existence of a dichromatic system of blue-violet and violet- green photo-receptive processes in the compound eye of Drosophila. This was accomplished by fatiguing the insect to one of two wave-lengths, when there was no apparent fatigue in response to the second. HanstrOm (1927) demonstrated both long- and short- axoned retinula cells in the compound eye of several arthropods; the long axoned cells probably gave colour vision. Then Power (1943) discovered -23- long and short visual axons, similar to those found by Hanstrom, in the optic lobes of the brain of Drosophila; these could account for Hamilton's results. On these results, Fingerman and Brown (1952) undertook some further work, and found a 'Purkinje shift' in the response of the compound eye of Drosophila, similar to the Purkinje shift that occurs in the vertebrate eye. As the intensity of the monochromatic light stimulus decreased, there was a gradual shift in the over-all sensitivity of the eye towards the shorter wave-lengths. This was the first adequate demonstration of two such receptive mechanisms in the compound eye of an insect, one predominating in bright light and corresponding to the cones of the vertebrate eye, and the other predominating in dim light and analogous to rods. In their work of 1953, Fingerman and Brown used Hamilton's fatigue technique to confirm the result. They used red and blue light, and concluded that colour vision operated at high light intensities but not at low. Red light of 700mp was seen. Further work by Weiss et al. (1942) showed that when the Potato beetle and the Japanese beetle were offered a choice between two lights, the behaviour varied according to the introductory intensity of these lights. When the physical intensity of the ultraviolet was strong most insects went to the blue-blue-green, but when it was weak they went to the ultraviolet. Additional tests with the Japan- ese beetle revealed that if red wave-lengths were offered at a relative intensity of 100, they were more attractive than ultraviolet and blue-blue-green at a relative intensity of 3. Weiss (1944) gave the peak response for a large number of insects at 365-366mp (ultraviolet), with secondary peaks at 492mi (blue-blue-green), 515mp (blue-green), and 606mp (yellow-orange). -24-

Crescitelli and Jahn (1939) tested the electrical response of insect eyes to colour, using Melanoplus differ- entialis and Samia cecropia as test insects. They found that the wave-form of the electro-retinograms was a function of intensity, and there was no effect of wave-length per se. Jahn (1946) re-examined the data, and showed that optimum stimulation was obtained at 500mp, with a decrease on either side. The curve for log. reciprocal intensity plotted against wave-length gave a curve which closely resembled the absorption curve for visual purple in the vertebrate eye, when plotted as log. photometric density against wave-length. Jahn and Wulff (1948) used electro-retinograms for detect- ing the response of Dytiscus to wave-length, and found a maximum sensitivity at 530-540mp. Weiss (1946) believed that intensity was more important than wave-length in initiating response to colours. This belief was based on the observation that he could change group behaviour responseS by increasing the brilliance of the colours, and on the work of Crescitelli and Jahn (1939) on the electrical responses of the eye. Although insects exposed to a spectrum of equalised physical intensities beh- aved as though they had colour preferences, such reactions could represent the absorption spectrum of the photo-sensitive pigment of the sense cells, as was inferred by Jahn. Absorption of the light by this substance varies with wave- length, and the production of a given response needs a certain amount of photochemical change, which in turn requires the absorption of a constant amount of energy. Thus a wave- length possesses both a physical and a physiological intensity, and although the physical intensities may be equalised, the physiological intensity may be different because of absorption. Therefore the ability to distinguish one colour from another does not give proof of colour vision. Weiss states that the stimulating efficiency of light increases slightly from zero at 720mp to 570mp, rises to a maximum -25- at 492mg, declines to 464mp, and then reaches a maximum peak at 365mug he then points out that the gi.dataut" - absorption by visual purple is at 365m11, the wave-length at which most insects give a maximum response. Tsuneki (1953) tried to train ants to come to red and blue light, but he found that colour orientation was often masked by a brightness response. However, despite the exponents of brightness response, the work of Schlegtendal (1934) definitely indicates the existence of colour vision in certain insects. He used the optomotor response of the insects to coloured stripes to show colour discrimination by the beetles Chrysomela, Agelastica, and Geotrupes. The plant-feeding Chrysomelids distinguished green tints from one another, as well as yellow and orange from blue-violet and green. Dixippus, and the bug Troilus, on the other hand, appeared to have no colour vision. In summary, it can be said that many insects behave as if they have colour vision. This could be a response to brightness, because although the physical intensities of the light may be equal, the colour sensitivity and absorption spectrum of the sensitive pigment may modify the sensation experienced by the insect. On the whole, the evidence supports the theory that insects have colour vision. Many insects are sensitive in varying degrees to a spectrum extending from 320mla to about 700mil (ultraviolet to infra-red). A primary peak of response occurs at about 3651/41, with secondary peaks at 492mi (blue-blue-green), 515mu (green), or 555mp (yellow-green). Longer wave-lengths than 555mp are relatively unattractive. The response may be somewhat modified by the introductory intensity of the wave-lengths. Many insects will also respond to the shorter of any two wave-lengths of equal intensity offered to them. That -26- is to .ay, they will go from red to orange, from orange to yellow, yellow to green, green to blue, and blue to ultraviolet.

B). THE BITING RESPONSE. A strong biting response is elicited by odour, contact chemical stimuli, or other less specific stimuli. Often the stimulus is provided by the same substance that effected final orientations this was shown by Verschaeffelt (1910), and Thorsteinson (1953), with the larvae of Pieris and Plutella; Chin (1950), with Leptinotarsa; and Dethier (1937), with Danaus and Papilio larvae. That the same substance which effected orientation may not always cause the biting response was shown by the work of Crombie and Darrah (1947), and Thorpe et al. (1945), with Agriotes larvae. In this insect, sugars may cause both orientation and biting, but more generally asparagine and glutamine cause orientation, and fats and polypeptides induce biting. The silkworm, Bombyx mori, is stimulated to bite by a substance different from those which cause orientation and feeding (Hamamura, 1959). Biting may be induced experimentally by the removal of the olfactory receptors, whereas it would be expected to reduce it. This is probably because of the removal of the response to various inhibitory stimuli (Dethier, 1947). Discrimination was not entirely lost during the experiments, because continued feeding was controlled by taste receptors. Chin (1950) obtained similar results with Leptinotarsa. Williams (1954) found, however, that a specific chemical stimulus did not initiate of suppress biting in Acrid- idae.

C). CONTINUED FEEDING. Continued feeding has been shown to be controlled -27- either by the presence or absence of inhibiting stimuli (repellents acting on the sense of taste), or by the presence of specific attractive substances acting on the olfactory or gustatory receptors. Both categories seem important in nature. In some cases the stimulus which effected orientation and induced a biting response also ensures that feeding continues. Dethier (1947) showed that this was the case with larvae of Papilio ajax. Other species require gustatory stimulation by a separate, specific attractant before feeding will take place. This is the case with Pieris and Plutella, where glucosides provide the stimulus (Verschaef- felt, 1910, Thorsteinson, 1953). The larvae of Bombyx mori require a substance distinct from those which cause orientation and biting (Hamamura, 1959). Raucourt and Trouvelot (1936) had shown that the consumption of leaves by Leptinotarsa was proportional to the concentration of an unidentified material; however, Thorsteinson (1953) pointed out that this work was probably misleading, because nutrients were diluted along with the active principles. The degree of acceptability of a plant, based upon the presence of taste stimuli, could be gauged by the app- earance of biting damage (Busnel, 1938,1939); the relative time spent by the insect in eating, wandering, and resting (Chin, 1950; Trouvelot et al., 1935); or the amount of excreta produced (Thorsteinson, 1953). Leptinotarsa adults may lay eggs on plants which are acceptable to it, because these plants contain suitable olfactory and gustatory stimuli, but the plants may be nutritionally inadequate for larval growth (Chin, 1950; Brues, 1940; and Trouvelot et al., 1933). A plant might provide the correct olfactory stimulus to induce a biting reaction, but at the same time possess or lack taste qualities which prevented further feeding -28-

(Dethier, 1937; Chin, 1950; and Trouvelot et al., 1933, with Leptinotarsa). Kuhn and Gauhe (1947); and Kahn and 18w (1947), for example, found that Leptinotarsa would bite Solanum demissum, but would not continue feeding because of the presence of the alkaloid demissin. Pfadt (1949) also concluded that the regulation of food preferences in Melanoplus mexicanus was mainly by the presence or absence of taste stimuli. Finally, there are other larvae which apparently do not need a feeding stimulus once orientation and biting are accomplished. Cuille (1950) demonstrated this with the Banana weevil.

D). PHYSICAL FACTORS Biting and feeding may be inhibited as a result of the physical characteristics of the plant, such as leaf texture, water content, and pilosity. Dethier (1953) considers that an excessive role may have been ascribed to these factors, and gives as examples the work of Chin (1950), and Painter (1936): his view is based on the observation that failure to consume a given plant cannot always be explained satisfactorily on the basis of physical features. There are, however, numerous cases cited in the literature in which physical factors affected feeding. Allan (1943) suggested that certain lepidopterous larvae on emergence must be influenced by differences between the upper palisade tissue of the leaf and the lower spongy mesophyll. He asserts that larvae of Pheosia tremula confined to the upper surface of Black Poplar leaf will die of starvation, as they normally crawl to the underside and feed there. B8ttger (1940), working with the European Corn borer, Pyrausta, considered that physical changes which occurred in certain plants under laboratory conditions contributed -29- to unsatisfactory growth. He found that profuse pubescence, thick epidermis, and coarse texture of the fibro- vascular bundles all led to low survivals. Chin (1950) could find no common physical character, such as pilosity or thickness of cuticle, that would account for the susceptibility of plants to attack by Leptinotarsa. But Trouvelot and Thenard (1931), also using Leptinotarsa, noted that feeding by the young larvae on Lycopersicum esculentum, Nicotiana, and Phaseola vulgaris was affected by the pilosity of the leaves. With the Wattle Bag worm, Acanthopszche dunodi, Henkel and Bayer (1932) attributed the repellence of Acacia lasio- pelta to its pubescence, of A. arabica to the dryness of the foliage, and the preference for A. ataxacantha over A. arabica to the hardness of the latter. Ripley and Heerden (1939), with the same insect, showed that leaf water content was important, as wilted leaves were less attractive. They also showed an effect of leaf hardness. The succulence and toughness of the host-plant leaves was important for the feeding of Schistocerca gregaria (Roonwal, 1953). Williams (1954), using a penetrometer to test grass leaves, showed that increasing toughness caused an increase in the duration of feeding in Acrididae. The moisture content of the leaves also influenced feeding. A few cases of the influence of texture in an artificial diet have been noted. Beckman, Bruckart, and Reiser (1953) found that fat levels above the 5% level in the diet improved the physical texture of the medium for feeding by the larvae of the Pink Boll worm. Ripley, Hepburn, and Dick (1939) could not get larvae of Aryroploce to feed on an artificial medium based on an agar gel, but the larvae would feed on writing paper. They suggest the importance of a mechanical, rather than a gustatory, stimulus. The water content of the diet was also important. Stride (1953) -30- stated that the texture of the artificial medium was important for the satisfactory feeding of Carpophilus hemipterus. An improved result was recorded by Noland et al. (1949) when they added 30% Cellu flour, at the expense of carbohydrate, to the diet of Blatella germanica.

E). A REVIEW OF THE LITERATURE ON INSECT NUTRITION. This review is relatively short, because the actual work on the nutrition of Phaedon did not progress far. The literature cited was selected to show the elementary principles involved in the formulation of an artificial diet. Insect nutrition has been reviewed by several authors. Albritton has edited a summary of the food requirements of plants and animals, including insects, and exhaustive tables are presented for the formulation of synthetic diets, and the utilisation of the various groups of substances. The 'Handbook of Biological Data', edited by Spector, contains similar tables and information. The main revues are those of Brues (1946); Chauvin (1949,1956); Craig and Hoskins (1940); Hinton (1956); House (1956); Levinson (1955); Lipke and Fraenkel (1956); Trager (1941,1947); Uvarov (1929); and Wigglesworth (1950). Recent work giving fairly successful diets is that of Lea et al. (1956), with Aedes aegypti larvae; of Sang (1956), with Drosophila; of Singh and Brown (1957), with AOdes aegypti; and of Vanderzant (1957,1959), and Vander- zant and Davich (1958), with the Pink Boll worm. a). Mineral requirements. No complete study of the inorganic components of any one species has yet been made. With the phytophagous insects, a rough indication of the amounts of ions ingested (but not of the actual requirements of the insect) can be obtained from plant analyses. (see Fraenkel, 1953). -31-

The salt mixtures most frequently used for artificial diets for insects have been those used in vertebrate nutrition; for example, those of McCollum and Hegstead. The ions contained are generally those of sodium (Na), potassium (K), magnesium (Mg), Manganese (Mn), calcium (Ca), iron (Fe), phosphorus (P), chlorine (C1), and sulphur (S). A deficiency of phosphorus retards the growth of Tribolium (Nelson and Palmer, 1935), and Lucilia (Hobson, 1935). Like sulphur, it may be supplied by organic compounds, and a mineral supply is not necessarily essential. Calcium is required by mosquito larvae (Frost et al., 19361 Bates, 1939). Aedes will develop only if the medium contains both sodium and calcium (Woodhill, 1936). But Sang (1956), with Drosophila, decided that calcium, as well as iron, magnesium, and manganese, could be eliminated from the diet of this insect, although some may have been present as impurities in the casein used. Potassium, phosphorus, and sodium were essentials. Earlier work by Loeb (1915), and by Rubinstein et al. (1935,1936), claimed that neither calcium nor sodium was required. The caterpillars of Alabama argillaceae Hb. showed a high mortality when fed on foliage deficient in copper or zinc (Creighton, 1938). A requirement for ferrous iron was shown for the flea larvae by Sharif (1937). Magnesium improved the growth of mosquito larvae (Frost et al. 1936; Bates, 1939). Zinc is necessary for egg formation in the silkworm (Akao, 1935). Significant de.=.- creases in oviposition rates of the mustard beetle, Phaedon cochleariae, and of the Large White butterfly, Pieris brassicae, were observed after the insects had fed on water- cress leaves deficient in sodium, potassium, phosphorus, or iron (Allen and Selman, 1955,1957). -32- b). Carbohydrates and_fat. Most insects utilise carbohydrate and fat as energy sources. Insects do not require a supply of fat as such; when a fat is required in the diet, the fundamental need is usually for a constituent unsaturated fatty acid as a growth factor (Fraenkel and Blewett, 1947). Beckman. reported that Pectinophora gossypiella larvae showed optimum growth on a diet containing 20%.fat, but in this case the value of the fat may have been to improve the consistency of the medium. Albritton (1955) gives 40 different carbohydrates which can be utilised by insects. Amongst the insects there is great variation in their dependence on carbohydrates in the food. Many species feeding on flour and grain fail to grow normally in the absence of carbohydrates, e.g., Tenebrio (Lafon and Teissier, 1939), and Tribolium, Ptinus, and Ephestia (Fraenkel and Blewett, 1943)• Addes larvae, on the other hand, can replenish their glycogen and fat reserves on a diet of casein, and their glycogen reserves alone when fed on alanine or glutamic acid (Wigglesworth, 1942). Villee and Bissell (1948), and Sang (1956), showed that a lack of carbohydrate did not have a serious effect on the growth of Drosophila. i). Monosaccharides. Of the natural hexoses, glucose and fructose are the best utilised by insects. The pentoses are poorly utilised (Trager, 1953; and Lemonde and Bernard, 1952, with Stegobium paniceum and Oryzaephilus surinamensis), but Vogel (1931) found xylose and arabinose as efficient as sucrose for the honey-bee. Mannose is utilised by adult Calliphora and Drosophila (Hassett, 1948), but is toxic to Apis and Vespa (Staudenmayer, 1938). ii).Disaccharides. Sucrose is well utilised, and most authors give it as

-33- the best utilised carbohydrate, although for an artificial diet it has the disadvantage of reacting with the amino- acids. iii). Polysaccharides. The larvae of Malacosoma can utilise starch, but larvae of Aglais and Pieris cannot (Evans, 1939). Tenebrio larvae grow well on a diet containing 72% starch, fructose, or mannitol, but only slowly on an equivalent amount of glycogen, glucose, or sucrose (Fraenkel and Blewett, 1943). For Tribolium larvae, on the other hand, sucrose was the most efficient carbohydrate, with starch, glucose, and mann- itose almost as good (Bernard and Lemonde, 1949). Tsutsi and Saito (1953) reported that Chilo simplex could not utilise starch during the first instar, but could do so in later instars.

c). Proteins and related compounds. These fall into two groupsg i). Essential. This group is not synthesized by the, insect, and is a necessary part of the diet. A sub- group, which may be called 'replaceable', includes substances which can be synthesized by the insect from specific essential amino-acid precursors in the diet. The end products are still essential. ii). Non-essential. This group can be synthesized by the insect either from substances in group i), or from non- .amino' nitrogen and bai'bohydrate. All the 19 amino-acids required by vertebrates have been studied in insects. Comparatively few of the published estimates of the amino-acid requirements of insects can be regarded as complete, because the role of gut symbionts in synthesizing some of the materials not originally in the diet has not usually been evaluated. For instance, under bacteria-free conditions, Drosophila requires 12 -34- amino-acids for growth (Hinton et al., 1951), whereas earlier work on this insect claimed that it could be reared on a diet containing ammonium salts as the sole source of nitrogen (Loeb, 1915; Uvarov, 192q). In general, there is a close similarity between the insects in their requirements for essential amino-acids. The list given below was determined by Dimond et al. (1956) for egg production by Addesaegypti. It is interesting :. that this classification is almost identical to that given for the rat (Baldwin, 1952).

Essential. Non-essential. Arginine. Alanine. Cystine. Aspartic acid. Histidine. Glutamic acid. Leucine. Glycine. iso-Leucine. Proline. Lysine. Serine. Methionine. Tyrosine. Phenylalanine. Threonine. Tryptophane. Valine. Cystine has not been shown to be essential for most other insects. It seems to be more important for eggUay- ing, as fewer eggs are laid when it is left out of the diet. The L forms are utilised but, in most cases, the D forms are not (Fraenkel and Printy, 1954; Hinton, 1956). The inhibition noted with DL isomers (Schultz et al., 1946, with Drosophila) may be caused by the increased amino-acid concentration to compensate for the inactivity of the D isomers. For all insects studied under aseptic conditions, general agreement with this scheme has been found. Moore -35—

(1946) found that these basic amino-acids were required by Attagenus; similarly for Tribolium (Lemonde et al, 1951; Frobrich and Offhaus, 1953; Fraenkel and Printy, 1954); Hylemyia antiatua (Friend, 1955,1956); Chile simplex (Ishii and Hirano, 1955); and Calliphora erythrocephala (Sedee, 1958). But for Drosophila and Addes larvae, glycine is also required (Hinton et al., 1951; Golberg et al., 1948). Pseudosarcophaga affinis also requires glycine (House, 1954), as also does Calliphora erythrocephala (Sedee, 1954), and the honey-bee (De Groot, 1953). Dermestes maculatus requires dietary cystine (Gay, 1938), as also does Lucilia sericata (Michelbacher et al., 1932). Aedes aegypti requires cystine for full egg production (Dimond et al., 1956). It may well be that more insects will be found to require cystine when diets are adequate to allow growth to the egg-laying stage. The males of Blatella germanica require alanine, pral- ine, and serine; the females do not require the serine (Hilchey, 1953; House, 1949). Hodgson et al. (1956) concluded that proline was required by Phormia regina, and also glutamic or aspartic acid: cystine promoted growth in this insect if methionine was lacking, which suggested that cystine was usually produced from methionine. All the essential amino-acids must be present at one time, or protein synthesis cannot take place (Geiger, 1947, 1948; Wissler et al., 1949). The provision of any number of the unessential amino-acids to the essential ones in the diet accelerates growth further (Baldwin, 1952; Hinton et al., 1951). This effect was also noted by Fraenkel and Printy (1954) with Tribolium confusum, and by Hodgson et al. (1956) with Phormia regina. It has repeatedly been noted that insect growth on amino-acid diets is inferior to that obtained when peptones and peptides are present: this is shown by comparison -36- between casein diets and amino-acid diets (Hinton, 1951; Sang, 1956,with Drosophila; House, 1949, with Blatella germanica; House, 1954, with Pseudosarcophaga affinis; and Chang and Wang 01958; with Musca domestica vicina Macq.). Friend and Patton (1956) however, found that Hylemyia antiqua larvae grew more rapidly on a synthetic diet containing amino-acids than on the natural diet. The inferior growth in many cases must be caused by incorrectly chosen mixtures (Sang, 1956; Dimond et al., 1956; Moore, 1946). Hinton et al. (1951) pointed out that excess of single amino- acids may be harmful. Sang attributed the inferior growth of Drosophila on amino-acid mixtures not only to the balance of the constituents, but also to the presence of heavy metal impurities in the commercial preparations. He also pointed out that most workers followed Tatum (1939) in using 2% casein or amino-acids in the diet, whereas the optimum is probably about 5%. In an artificial diet for Pseudo- sarcophaga affinis and Choristoneura fumiferana, however, 2% was the optimum level for amino-acids: excess of these, or of dextrose or salt mixture, was toxic (House and Barlow, 1956). Haydak (1953) also found that an excess of casein fore-shortened the life of roaches. A complication was ' found in formulation when it was shown that the larvae of the silkworm, Bombyx mori, had significant differences in the amino-acid requirement at different larval stages (Menedez et al., 1950; Stamm et al., 1950). Beck and Hanec (1958) showed that amino-acids had an effect on the feeding behaviour of the European corn borer, Pyrausta nubilalis. The addition of peptides to an amino-acid mixture improves growth in many cases. Glutathione, a tripeptide composed of cysteine, glycine, and glutamic acid had a beneficial effect on the growth of Drosophila (Schultz et al., 1946); Aedes (Trager, 1948); and Corcyra (Singh, 1955). -37- d). Accessory growth factors. Early attempts to culture insects on purified diets showed that extracts of yeast and wheat germ contained substances vital for growth (Delcourt and Guy-6/1ot, 1911; Bacot and Harden, 1922; Richardson, 1926; Sweetman and Palmer, 1928). Most of the vitamins were discovered from work on laboratory vertebrates, bacteria, and certain ciliates; only one, vitamin BT (Fraenkol et al, 1948), later identified as carnitine (Carter et al., 1952), was discovered from work on insects. i). Water-soluble factors. The vitamins required by insects are almost exclusively those of the 'B' group, and it was long accepted that thiamin, riboflavin, niacin (nicotinic acid), :pyridoxine, pantothenic acid, biotin, inositol, choline, folic acid, and vitamin B12 (cyanocobalamin), were required. Until recently, no insect had been shown to require p-amino benzoic acid or inositol, but recently Vanderzant (1959) has reported a dietary requirement for inositol by the boll weevil, and Forgash (1958) reports an effect of inositol on the survival, growth, and maturation of Periplaneta americana. Recent reviews will be found by Lipke and Fraenkel (1956); Magis (1954); and Hinton (1956). More work with the above vitamins showed that all species tested required thiamin, riboflavin, niacin, pantothenic acid, and choline. Generally, pyridoxin, biotin, and folic acid are also required, but this is not always the case. Pseudosarcophaga affinis does not 'require pyridoxin or folic acid (House, 1954)z Phaenicia (Lucilia) sericata does not require biotin or folic acid (Kadner and LaFleur, 1951). House and Barlow (1958) state that the house-fly, -38-

Musca domestica, does not require folic acid, but Brookes (private communication to House and Barlow, 1957) claimed that folic acid is required by this insect, but its action is 'spared' by RNA. Hinton et al. (1951) expressed doubts upon the requirement of Drosophila for pyridoxin. Whereas the male Hylemyia antiqua needs the major B vitamins, folic acid, and ribosenucleic acid, the female also requires vitamin B12, Co-enzyme A, and 0Z-lipoic acid (Friend and Patton, 1956). The larvae of the rice moth, Corcyra cephalonica, grow better if vitamin B12 is present (Bhagwat and Sohonie, 1955). Carnitine was discovered as an essential vitamin for Tenebrio molitor (Fraenkel and Blewett, 1948). It occurs naturally in yeast, whey, and many animal tissues; but plant tissues, with the exception of wheat germ, are a poor source (Carter et al., 1952). A few species closely related to Tenebrio, such as Ep1211 amILEalaml (Fraenkel, 1951), Tribolium confusum, and T. castaneum (French and Fraenkel, 1954; Frobrich, 1953; Magis, 1954) also require carnitine. It is important that aseptic techniques are used for assessing vitamin requirements. Pant and Fraenkel (1954) showed that intracellular symbionts of Stegobium paniceum and Lasioderma serricorne supplied the species with riboflavin, niacin, pantothenic acid, choline, biotin, and folic acid. Evidence of a vitamin C (ascorbic acid) requirement by Bombyx mori larvae has been found by Gamo and Seki (1954). The synthesis of the vitamin could take place from mannose in the pupal stage. Chang and Wang (1958) found a similar requirement for the vitamin by Musca domestica vicina Macq. Murthy (1954) presents similar evidence for Bombyx mori. Dadd (1957) showed a requirement for vitamin C by Schisto- cerca gregaria. But Day (1949) decided that Blatella germanica did not require a dietary source of vitamin C, but -39- the substance was synthesized in the tissues, and this conclusion was earlier reached by Wollman et al. (1937). Periplaneta americana also synthesizes the vitamin in its tissues (Roussel, 1956), and Sarma and Bhagwat (1942) showed that the same applied for Corcyra. A factor, as yet unidentified, in grass juice, has been reported by Beck et al. (1949,1950), and Beck (1950, 1953). This factor was essential for the growth of Pi. rausta nubilalis. Subsequent workers have found that concentrates similar to those prepared by Beck stimulated the growth of other phytophagous species.

ii). Fat-soluble factors. Vitamin A. Vitamin A is not generally required by insects. Blatella germanica neither synthesizes nor requires carotene or vitamin A (Bowers and McCay, 1940). Bettini and Tentori (1947, 1948) could find no vitamin A present in the tissues of larval or adult Anopheles. But Dadd (1957) reported that pigmentation was lacking, and growth was poor, if carotene was lacking from the diet of the nymphs of Schistocerca gregaria. Chang and Wang (1958) have reported reduced rate of growth with larvae of Musca domestica viaina, in the absence of vitamin A.

Sterols. Insects require a dietary source of sterol. Cholest- erol is the most usual, but other sterols may be utilised. Drosophila can survive on cholesterol, ergosterol, sitosterol, stigmasterol, and phytosterol, but not cn irradiated ergosterol (calciferol) (Van t'Hoog, 1935). A similar result was obtained by Souza and Sreenivasaya (1945) for Corcyra. The larvae of Phormia regina can utilise phytosterol and other sterols of animal origin (Brust and Fraenkel 1955); Musca domestica requires cholesterol (Hammen, 1957). -40-

The flour and grain insects, Tribolium, Ptinus, Sitodrepa, Silvanus, Lasioderma, and Ephestia, utilise a number of sterols, including sitosterol, ergosterol, 7-dehydro- cholesterol, cholesteryl acetate, and(f.artially) cholest- anol (Fraenkel and Blewett, 1945,1946). Dermestes vulpinus, on the other hand, cannot utilise sterols of plant origin, and is restricted to foods containing cholesterol (Gay, 1938). No insect has so far been shown capable of utilising calciferol or the related D-group vitamins, or the steroid hormones (Trager, 1953). But, again, Chang and Wang (1958) have reported a reduced growth rate in an artificial diet for Musca domestica if vitamin D was absent.

Unsaturated fatty acids. Insects do not require fats as such, but do require unsaturated fatty acids. Ephestia larvae will grow but not produce adults if the diet is deficient in fatty acids, such as arachidonic or linoleic acid. Sub-optimal concentrations lead to the development of moths with naked wings, the extent of the stripping bf_Htbe-realobeing"-Trffportional to the degree of the deficiency (Fraenkel and Blewett, 1946). Corn oil has been used in diets for Pyrausta nubilalis (Beck et al., 1949); Chile simplex (Ishii, 1954); and Graptolitha (Matsumoto, 1954). But Fraenkel and Blewett (1947) showed that Plodia interpunctella developed normally in the absence of dietary fatty acids, and that Tenebrio could synthesize linoleic acid. An amelioration in the effects of a biotin deficiency in Addes larvae by oleic acid, lecithin, or an oil from hydrolysed horse plasma, suggested that biotin might aid the synthesis of lipids (Trager, 1948). No such effect with lecithin could be demonstrated with Drosophila (Hinton, et al., 1951). An increase in the fecundity of Leptino- -41-

-tarsa adults fed upon leaves painted with lecithin was reported by Grison (1948).

Vitamin E. The growth of ,Ephestia on a synthetic diet was improved by adding .1.-tocopherol. This effect was thought to be due solely to the anti-oxidant properties of the ot-tocopherol stabilising the linoleic acid. The d-tocopherol could be replaced by ethyl or propyl gallate, which confirmed this conclusion (Fraenkel and Blewett, 1947). Bettini and Tentori (1947) showed that the adults and larvae of Anopheles have vitamin E present in the tissues.

iii). Nucleic acids. Yeast nucleic acid or ribosenucleic acid (RNA) improved the growth of Aedes aegypti larvae (Subbarow and Trager, 1940); Drosophila (Schultz et al., 1946); Scobius granosus (Rosedale, 1945); and Pseudosarcophaa affinis

*(4- Borkb‘z. (House, 195491957). Sedee (1958) also showed that this was true for -CalAlphora:2eiwthrocephala. With Drosophila, hydrolysed RNA gave the same improvement in growth as the intact acid, so that growth promotion must be by the constituent purines and pyrimidines, especially adenine (Villee and Bissell, 1948). Hinton (1956), and Hinton et al..(1951), with a strain of Drosophila, found that growth stimulation only occurred when adenine in some form was present. Whole RNA gave a better response than did any of the constituents tested. Of these, the best was a mixture containing the four bases, the four nucleosides, and the four nucleotides. Of the purines and pyrimidines tested, only inosine gave an increase in the growth rate. Orotic acid and adenylic acid also gave a positive effect. De-oxyribose nucleic acid inhibits the growth and pupation of Drosophila larvae (Villee and Bissell, 194$), -42-

Hinton et al. (1951), and Begg and Robettson (1950), included the purine and pyrimidine bases in addition to RNA, but Sang (1956) stated that when RNA was used at the optimum level for Drosophila the addition of such substances was of no further value, and only tended to slow down the growth rate and reduce survival.

F). THE NUTRITION OF PHYTOPHAGOUS INSECTS.

Accounts of investigations with phytophagous insects form a minor part of the literature on insect nutrition. A considerable number of papers have been published on the relationship between insects and their plant hosts, but there are very few which are of help in formulating an artificial diet. The problem is probably not so much one of finding out and fulfilling the exact nutritional needs of the test insects, but rather one of formulating the diet in such a way that normal feeding will take place. Another problem is the finding of a suitable aseptic method for rearing the insects. The initial considerations are therefore tech- nical rather than nutritional. The more important investigations are those given below.

A. Synthetic or semi-synthetic diets fed under aseptic conditions. 1. Stem and leaf feeders. •Pyrausta nubilalis. Beck et al., 1950.

Chilo simplex. Ishii, 1954,1955. € Graptolitha molesta. Matsumoto, 1954.

2. Fruit feeders. •Dacus dorsalis. Maeda et al., 1953. -43-

2. Fruit feeders (Continued from previous page). HF Dacus cucurbitae, Maeda et al., 1953. NM Ceratitis cajpitata. do. € Graptolitha molesta. Matsumoto, 1954.

3. Fruit and seed feeders. Pectinophora gossypiella. Vanderzant, 1957,1959; Vanderzant et al., 1956, 1958.

4. Bulb and root feeders. Hylemyia antiqua. Friend, 1955,1956,1957.

B. Semi-synthetic diets fed under non-sterile conditions. 1. Stem and leaf feeders. Pyrausta nubilalis. Bottger, 1942. fl ft Beck et al., 1949. Wressel, 1955. Prodenia eridania. Elliot, 1955.

2. Fruit and seed feeders. HN PectinophoraAossypiella. Beckman et al., 1953.

- denotes a requirement for a growth factor present in plant juice. HH- denotes the presence in the diet of a plant oil. + - denotes the presence of brewers yeast.

Beck (1953), with yrausta nubilalis, found that a water-soluble growth factor (*Leaf factor') in grass juice was required. This factor could not be replaced by liver or' liver extract, peptone, the B-vitamins, nucleic acid, adenine, ascorbic acid, or carnitine. Brewers yeast, yeast extract, and wheat germ, had slight activity. This probably explains why Bottger (1942) failed to rear Pyrausta -44- on an artificial diet, because it contained no plant juice. The Beck diet can be considered to be a standard type. Substantially the same medium was used for other lepidopterous species; the Asiatic rice borer, Chilo simplex (Ishii, 1952,1954; Ishii and Hirano, 1955); and the Oriental fruit moth, Graptolitha molesta (Matsumoto, 1953, 1954). Ishii and Hirano (1955) replaced the casein in the diet by an amino-acid mixture, and showed that Chilo simplex did not require an unknown growth factor. But on a medium of similar type, Elliot (1955) showed that the growth factor contained in plant juice extract was essential for the development of Prodenia eridania. The value of a dried grass juice preparation in a diet for Ephestia kithniella was shown to be due to the presence of folic acid (Fraenkel and Blewett, 1947). Beckman et al. (1953), and Vanderzant et al. (1956), reared Pectinophora gossypiella on an artificial medium, and no requirement was found for any unknown growth factors. The original diet had contained carotene and vitamin K (menadione), and a large amount of corn oil. The subsequent elimination of the carotene and the vitamin K, and the reduction of the oil concentration from 20 to about 0.55'4 did not significantly affect the growth rate of the larvae. The dipterous species investigated had similar requirements to those found by Hinton (1951) for Drosophila. They include the melon fly, Dacus cucurbitae; the oriental fruit fly, Dacus dorsalis; and the Mediterranean fruit fly, Ceratitis capitata (Maeda et al., 1953). Hylemyia antiqua, the onion fly, has been reared on a chemically determined diet similar to that used by Hinton et al. (1951) for Drosophila, but containing in addition Co-enzyme A and thioctic acid (Friend, 1956). Friend stated that Hylemyia did not require Co-enzyme A when calcium pantothenate was provided at the rate of 6pgm/gm. -45- of diet. Thioctic acid deficiency caused an increase in mortality. Friend and Patton (1956) concluded that Hylemyia could synthesize thioctic acid, but not at a rate which would permit optimum growth. Beck et al. (1949) used a mould inhibitor, methyl p-hydroxy benzoate, in the diet of Pyrausta, and reported that the substance had a toxic effect. Wressel (1955), however, used the same inhibitor, but did not get any toxic effect. McDonough (1953) successfully used sodium ortho- phenyl phenate as a mould inhibitor in a Drosophila diet, and there was no apparent effect on the growth of the larvae. ---000--- -46-

III. REARING OF THE LARVAE.

Phaedon cochleariae is a very easy species to rear through-out the year. After trying several more complicated methods at the beginning of the work, the following simple procedure was found to give the best results. All the rearing techniques described were carried out in a constant temperature (C.T.) room, at 25°C., 75% R.H., and a 16 hour 'day-length', unless otherwise stated. The food-plant for the first 18 months was turnip foliages it became necessary then to change to cabbage foliage, for reasons given later. The initial culture was obtained as eggs, laid on turnip leaves, from Rothamsted Experimental Station, in January, 1958. Experience during undergraduate work on this species had shown that the percentage hatch of the eggs could be as low as 35-40% when the eggs were left on the leaves. This was attributed to the rapid drying out of the leaves associated with the damage caused by the feeding of the adult beetles. A better hatch, of 80-90%, could be obtained by picking the eggs off the leaves under a binocular microscope, using watch-makers forceps. The eggs could be removed from the leaves at any time after laying and up to hatching without affecting the hatch. The eggs so removed were placed in batches of 200 on moist filter paper in 9cm. Petri dishes, and the paper was kept moist until the eggs hatched. The larvae were reared in these Petri dishes until the second instar was reached, when they were transferred to a Watkins and Doncaster rearing cage containing a 2" layer of moist peat, in which was embedded a 3" flower pot containing a turnip plant. These turnip plants were trans- planted from a garden plot to the flower pot when required. The provision of a 300watt Siemans 'Sieray' agricultural lamp in the C.T. room enabled the turnip plant to -47- remain in good condition for a week to ten days, at the end of which time it was replaced by a fresh plant, and the larvae were transferred to this from the old one. Two weeks after hatching the larvae pupated in the peat at the bottom of the cage, and the adults emerged about one week later. The aim was to get about 200 adults for the stock culture. After feeding for about 3 days, the adults mated, and laying began. This stock culture provided all the necessary eggs for experimental purposes over a period of about 6 weeks, provided that the adults were removed from the C.T. room to a cool room after a period of laying. At about the third week in the life of the existing culture, a new stock culture was started, and the adults of this were emerging when the old culture was about to be discarded. For laying, the adults were put back in the C.T. room three days before the eggs were required. This was especially important during the winter months, as the adults did not lay when the day-length was less than 16 hours. After the adults had been put back in the C.T. room, the number of eggs laid each day increased to a maximum about the third day. Then the old food-plant was removed, a fresh plant was put in, and the adults were allowed to lay for 24 hours before the food-plant bearing the eggs was taken out. About 1500-2000 eggs could be obtained daily in this way. If a further quantity of eggs was required the next day, the adults were left in the C.T. room with a new food. plant fora further 24 hours. If not required, they were put back in a cool room, and the food was changed weekly. It was found worth-while to get very large batches of eggs at any one time, because after these had been picked off the leaves and placed on moist filter paper, the dishes containing the eggs not immediately required could be placed in a sealed tin in a refrigerator at 6°C. for up to three -48- weeks. At any time during the three weeks the eggs could be put in the C.T. room, and a normal hatch of about 80-90% was obtained. Then the eggs picked off for experimental purposes hatched, the larvae were grown to the second instar in the Petri dishes in which they hatched. It was found that detached turnip leaves lasted quite well in the humid conditions in the Petri dish, and the 'Sieray' lamp prevented yellowing. The leaves were changed every two days. The larvae began to enter the second instar after 22 days, and most had completed the change after 4 days: individual variations in growth rate accounts for the duration of the over-all period for ecdysis. To ensure that sufficient second instar larvae of the right size were available for the experiments, those larvae which moulted first were picked off into a large dish containing turnip leaves: the dish was then covered and placed in a refrigerator at 6°C. This retarded development until a suffici, ient number of larvae had moulted, and avoided the use of larvae of widely different sizes in the experiments. This precaution was necessary in these experiments, because growth is very rapid during the second instar. During the autumn of 1959, two consecutive batches reared for stock cultures failed to come through. The cultures had been started with 1800 eggs in each case, but only 10-12 adults were obtained. There was a very high mortality rate amongst the larvae, and an even higher one in the pupae. Larvae pupated to give apparently normal pupae, but very few emerged as adults those that did emerge were smaller than average, and in several cases had deformed elytra. Disease was at first suspected, but the cause of the mortality was finally attributed to a nutritional deficiency in the turnip plants used. The hot, dry summer had result- -49-

-ed in the production of small, stunted turnip plants. The rain which fell in the autumn caused some late growth, and it was at this stage that the plants were used for rearing the two poor cultures. It is believed that the plants had been unable to draw up sufficient nutrients from the dry soil to synthesize necessary substances in the leaves. The soil was known to be deficient in boron, and the dry conditions may have prevented the uptake of any small amounts that were present, and the deficiency in the plant may have affected the growth of the larvae. Such an effect would suggest a boron requirement by the larvae of Phaedon, but this cannot be stated definitely, because so many other factors may have been concerned. A change of food-plant to Brussels sprouts and cabbage tops was made for the rest of the winter, and the spring of 1960, and these substitutes were quite adequate for rearing. Fewer eggs were obtained at first from beetles reared on this food. It was then found that the spread of the lower leaves of the tops covered the peat at the bottom of the cage, which was in darkness, and because of this most of the beetles remained inactive in the surface layer of peat. The use of smaller tops, and the stripping of the larger lower leaves to leave three inches of clear stem, allowed light to reach the peat. All the beetles then became active, and the number of eggs laid increased considerably.

- --o0o - - - -50-

IV. THE ORIENTATION OF THE LARVAE TO THE FOODPLANT.

As mentioned in the Introduction, previous workers have shown that the behaviour which culminates in normal feeding can be broken down into orientation to the food, a biting response, and continued feeding. Preliminary experiments were made to find how these categories applied to the feeding behaviour of Phaedon larvae. These experiments were quite simple, and involved the use of the natural food-plants. In this case turnip leaves were used. The effect of leaf odour was tested in a chamber olfactometer to see if this odour caused initial orientation. Then the larvae were offered the choice of leaf discs of both acceptable and normally unacceptable leaves to see if the biting response and continued feeding were both caused by the odour. When it was found that this was not so, the distinction between the biting response and continued feeding was found by brushing normally unacceptable leaves with the juice of acceptable leaves. The unacceptable leaves were then eaten to some extent. These preliminary experiments showed that the initial orientation was by the odour of the host plant. The biting response was caused by this odour, but the feeding response continued only in the presence of a suitable taste. The experiments are briefly described below.

A. Orientation to the food. The presence of an olfactory response. This was tested in a simple chamber type olfactometer, of dimensions 4"x3ex2". At each end were two gauze- -51-

covered port holes of diameter. The chamber had a push- on lid, which was sealed during experiments with Sellotape (Fig. 1). As shown in fig. 29 the olfactometer was con- nected to a single water-operated suction pump. A T-tube allowed a suction tube to be taken to both outlet ports. Before air was drawn in at the entry ports, it had to pass through separate manometers, which registered the suction pressure. Rubber clips on the rubber tubing allowed the flow of air to each port to be equalised. Glass tubes were fitted between each manometer and entry port of the olfactometer (Fig. 2). When the apparatus was in use, one tube contained the crushed test leaf, and the other contained a ball of moist cotton wool, which gave the control air-stream. To ensure even illumination of the floor of the chamber, a 60 watt light was suspended above the olfactometer, and a diffusion screen was placed just below the light. This arrangement gave even illumination of the chamber, and also kept its temperature at about 25°C. This was the optimum temperature for larval movement. Before each experiment, a new sheet of paper, with a centre line ruled on it, was placed in the chamber. This ensured that scent trails from previous experiments could not affect each set of results. The lid of the chamber was sealed down, and the suction pump was adjusted so that lmm. pressure difference was recorded in both manometers. The lid was then removed. For the control experiments, the two glass food containers were detached, and a ball of moist cotton wool was placed in each. For the other experiments, the one tube contained a ball of moist cotton wool, and the other cones tained the crushed test leaf. Thirty second-instar larvae were used for the experiments. They were starved for an hour, and were then -52—

FIGURE I . THE CHAMBER OLFAC TOME TER .

Lid with glass window and rubber seal at edge.

Partition. Partition. Collar. Gauze-covered exit port. Entry tube (cardboard). Glass tube

Rubber bung. Exit tube (cardboard ). False floor.

FIGURE 2. THE EXPERIMENTAL SET-UP.

Glass bottle.

Light source . Air inlets. Diffusion screen. To suction pump.

Glass V. tube . Rubber tubing.

Rubber pressure Manometer. tubing.

Clip for adjusting pressure. Olfac tome ter. Glass tubing f or crushed leaves.

-53- placed at the centre of the chamber, 15 in each air-stream. The lid of the chamber was then sealed down again. Readings were taken at 5 minute intervals over a period of half an hour of the number of larvae in each air- stream and the number on the respective ports. Six tests were made with each leaf type. The following tests were made

Air-stream 1 (Test). Air-stream 2 (Control). Plain air (Vith moist Plain air. With cotton wool). Turnip leaf. moist Brussels sprout leaf. iP cotton Beetroot leaf. IT tr wool.

The results are given in Table 1. The larvae in the air-streams with the odour of turnip or Brussels sprouts leaf present showed a positive response to this odour, as significant numbers of the larvae entered the test air-stream and reached the inlet port. The larvae became very active, and moved rapidly for short distances; they then stopped and raised the fore-part of the body from the ground, and the head was waved from side to side as if testing the odour. These results indicated that an olfactory response caused the initial orientation of the larvae.

B. The biting response. An experiment was carried out to see if the biting response was caused by the odour which gave the initial orientation. It involved offering the larvae a choice between two leaf discs together in the same tube. One disc was of an acceptable leaf, the othe of a normally unacceptable leaf. The idea was to see if the normally -54-

Table 1. Results of the olfactometer tests.

Larvae in Larvae in No. on No. on Plant control air test air control test tested. stream. stream. port. port.

Turnip 9 21 0 14 leaf. 14 16 0 2 6 24 0 14 9 21 0 11 13 17 0 4 7 23 1 10 Total. 58 122 1 55 )

Brussels 8 22 1 9 sprout 14 16 4 5 leaf. 13 17 2 7 7 23 2 11 14 16 1 8 13 17 0 2 Total. 69 111 10 42

Beetroot 18 12 1 0 leaf. 15 15 1 1 18 12 1 1 , 18 12 1 0 14 16 0 2 11 19 1 2 Total. 94 86 5 6

Control, 12 18 0 0 with moist 14 16 3 1 cotton wool. 14 16 2 1 16 14 0 0 15 15 1 1 18 12 0 1 Total. 89 91 6 4

-55— unacceptable leaf would be bitten in the presence of the odour of the acceptable leaf. For the experiments, 2°x1" corked specimen tubes were used. To standardise the leaf areas offered to the larvae during the tests, 3" diameter discs were cut from the leaves with a cork borer. Where single leaf types were offered to the larvae (e.g., Turnip, or beetroot), two discs of this type were put in each tube, but where choices were offered, one disc of the one leaf was put in the tube with one disc of the other leaf. This meant that an equal amount of food was offered in each tube. Brussels sprouts, turnip, beetroot, and privet leaves were tested individually, and then in pairs, as indicated below.

Turnip. Turnip/privet. Brussels. Turnip/beetroot. in Beetroot. alone. Brussels/beetroot. combination. Privet. Brussels/privet.

Three replicates were used for each test, and each replicate contained 10 larvae initially 5 larvae were put on each disc. The tubes were placed in 4" deep dishes containing of water, and the dishes were closed with a glass plate. This was necessary as it was found that the leaf discs dried out too quickly in the C.T. room unless so protected. The dishes were put in the C.T. room at 25°C. for 24 hours. After 24 hours, the area of each disc which had been eaten was measured by counting squares with a squared eye- piece graticule in a binocular microscope. The area of a square had previously been measured against a graduated scale. Both sides of the disc had to be examined, as the larvae did not bite right through the leaf lamina. -56-

Table 2 shows the results for the leaf types when offered alone. It can be seen that, when offered singly, turnip and Brussels sprouts leaves were eaten, but beetroot and privet leaves were not. This confirmed that these last two types did not contain any stimuli. The results in Table 3 are for the choice tests. Turnip and Brussels sprouts leaves were eaten, but still the leaves of privet and beetroot were not eaten. The experiments did not reveal the relationship between the olfactory and biting stimuli, as had been hoped, because nibbles on the unacceptable leaves, if any, were too small to be seen. The results did indicate, however, that the olfactory attractant present in the turnip and Brussels sprouts leaves did not cause the feeding responses otherwise it would be expected that the unacceptable leaves would have been nibbled in the presence of the stimulant odour in the choice tests.

C. The feeding response. The experiments listed above showed that orientation was by an olfactory stimulus, but this did not cause the feeding response. The experiments did not reveal the nature of the biting response. To try and show the presence of a gustatory stimulant in acceptable leaves, the juice of these leaves was brushed on to normally unacceptable leaves. The juices of turnip, beetroot and privet leaves were obtained by crushing two leaves of each plant in individual 2"xl" specimen tubes, and triturating each with lml. of distilled water. The resultant juice was squeezed from the macerated tissue, and was centrifuged to remove debris. The test leaves were then painted with the juice as indicated below. -57-

Table 2. The amount of leaf disc eaten when each leaf type was offered singly. Amount eaten Food plant. Replicate. (sq. mm.). Turnip 1 64 2 112 3 104 Total. 280 Brussels sprouts. 1 173 2 121 3 83 Total. 377 Privet. 1-3 0 Beetroot leaf. 1-3 0

Table 3. Amounts of different leaves eaten by first instar larvae in food choice experiments.

Replicate. Choice offered and amount eaten. (sq. mm.). Turnip. / Privet. 1 18 0 2 22 0 3 8 0 Total. 48 0

Turnip. / Beetroot. 1 14 0 2 12 0 3 11 0 Total. 37 0

Privet / Beetroot. 1-3 0 0 -58-

Turnip on privet. Control privet. Painted with Turnip on beetroot. beetroot. distilled

Beetroot on turnip. I I turnip. water. Privet on turnip.

The control leaves were painted with distilled water to simulate the wetting of the test leaves. From these leaves the test discs were cut, and the experiment was arranged as described on page 55. First instar larvae were used, and these were placed on the still-moist discs. Results are given in Table 4.

Table 4. Summary of leaf-extract brushing experiments.

Replicate. Amount eaten. (sq.mm!). Turnip on privet. Control privet. 1 1.0 0 2 2.0 0 3 3.0 0 Total. 6.0 0

Turnip on beet. Control beet. 1 1.0 0 2 1.2 0 3 0.8 0 Total. 3.0 0

Beet on turnip. Control turnip. 1 10.0 26.0 2 17.0 19.0 )3 16.0 25.0 Total. 43.0 70.0

Privet on turnip. 1 27.0 2 35.0 3 19.0 Total. 81.0 -59-

Normally unacceptable leaves were nibbled, but feeding was not continued. This suggested that the leaf juice of turnip gave a favourable odour and taste to the leaf discs of beetroot and privet, which resulted in nibbling. But once the leaf tissues of beetroot and privet alone were tasted, without the stimulus, feeding ceased. This accounts for the smaller amounts eaten, as compared with the results for the turnip leaf. Beetroot and privet leaf— juices, when brushed on to turnip leaves, did not affect the amount of turnip eaten. These leaves did not contain a repellent, but were not eaten simply because they did not contain the necessary attractants.

D). The effect of physical factors on the feeding response.

It was found during the above work on the stimuli affecting the feeding responses that the measurements of the leaf area eaten sometimes varied widely in a set of replicates. Such variation usually appeared when the discs were cut from different leaves. The differing toughness of the leaves was thought to be the reason for the variation. Since a really suitable piece of apparatus to test the plasticity and elasticity of the leaf tissues could not be made, the simple type of penetrometer apparatus described by Williams (1954) was constructed. This is shown in Fig. 3k. The penetrometer consisted of a 2"x*" specimen tube, a modified cork, a pin, and a sand reservoir. The silver No. 8 entomological pin passed through the floor of the sand reservoir, at right angles to it. This pin was kept vertical by the length of capillary tube inserted in the cork of the specimen tube: it was of such length that about e of the pin projected below the capillary tube when the sand reservoir was resting on top of this tube. When in use, the specimen tube was clamped in a retort -60.- stand so that the pin, as it projected from the lower end of the capillary tube, was at eye-level. A lamp was shone with its beam at right angles to the tube, and from one side: this gave light which was reflected from the pin, and made it very much easier to see when the pin penetrated the leaf. Strips about 1-1-° long were cut from the test leaf. They were then placed individually across the mouth of the specimen tube, and secured in place by pushing the cork home. At first, it was found that the close-fitting edge of the cork punched a disc out of the leaf strip: to prevent this, two diametrically opposite sides of the cork were cut square (fig. 3B). It was important to see that the strip was firmly fixed across the base of the cork, otherwise the pressure of the pin forced the strip downwards before puncturing occurred. This gave too high a figure for the toughness. It was also necessary to ensure that the surface of the strip was at right angles to the capillary tube, or else the pin entered the leaf obliquely and gave a false reading. When the above precautions had been taken, the pin was placed in the capillary tube and allowed to drop until the point just rested on the surface of the leaf. Dry silver sand was then poured into the reservoir until the pin just penetrated the leaf. The reservoir was removed, and the sand poured on to a previously weighed filter paper disc. This was re-weighed to give the weight of sand required to make the pin pierce the leaf. Ten test readings were made on each leaf strip, and the average figure taken. It was found that the variation in the figures for any one set of 10 readings was not statistically significant. On the basis of the amount of sand required to cause -61—

FIGURE 3A THE WILLIAMS TYPE PENETROMETER.

Sand reservoir

Cork lamina. Pin. Capillary tube Leaf strip.

Specimen tube.

FIGURE 3B. T.S. CORK AND TUBE.

Capillary tube. Specimen tube.

Leaf lamina.

Cork.

Pin.

-62- penetration of the various leaves, an arbitrary 'toughness scale' was established. This ranged from T1 to T12, in which Tl was the least tough, and T12 the toughest, grade. The series is shown in Table 5. All the values for toughness obtained with the experimental leaves were related to this scale.

Table 5. The arbitrary scale of toughness.

Gm. sand required Gm. sand required for penetration. Toughness. for penetration. Toughness. 3.2 - 3.8 Ti 5.3 - 5.6 T7 3.8 - 4.1 T2 5.6 - 5.9 T8 4.1 - 4.4 T3 5.9 - 6.2 T9 4.4 - 4.7 T4 6.2 - 6.5 T10 4.7 - 5.0 T5 6.5 - 6.8 T11 5.0 - 5.3 T6 6.8 - 7.1 T12

The leaves of turnip, kale, and Brussels sprouts were used in the experiments. They were tested for toughness, and leaves were selected to cover the widest possible range. Leaves of each degree of toughness were cut to give ao discs, as described for previous experiments two discs of each type were then put in 2"xl" specimen tubes with 10 first instar larvae. Three replicates were prepared for each leaf of any one degree of toughness. The tubes were then put in a C.T. room at 25°C. and 75% R.H. for 24 hours. At the end of this time the following points were noted

1). The area of leaf eaten. This was measured by counting squares, as described on page 55. 2). The number of nibbles taken. this was to see if there was any correlation between these and the leaf toughness. 3). The number of dead larvae. This could explain -63-

some of the variation in the figures for the amount of food eaten between replicates. It soon became apparent that, because the exact time at which any larva died during the 24 hour period was not known, there was no means of knowing how much it ate before it died. These figures are omitted from the tables.

Table 6 summarises the results of these experiments. No leaves of hardness 6-9 were obtained. Table 7 represents the figures from Table 6. From Table 7 it can be seen that the variation of readings within each toughness group was not significant, as the mean exceeds the Standard Deviation by more than three times. The significant result in the case of the readings for T2 arose because a mould appeared on the leaf discs of one of the replicates, and reduced the amount of food eaten. The results of Table 7 are shown plotted in Graph 1. These results show that leaf toughness affects the amount of food eaten this is brought out clearly in Table 7 and Graph 1. In Graph 1, the curve shows a sharp change in slope at about T3-T4; it would seem that at this point the mandibular power of the first-instar larvae is nearly matched by the elasticity and plasticity of the leaf tissue. This would slow down the rate at which the larvae could feed. The figures for the number of nibbles taken are interesting in this connection. At higher toughness values they show a three-fold increase in number for each square millimetre of leaf eaten. From Tl-T3, the number of nibbles was 2.0-2.4/sq.mm. eaten; at T4-T5 it was about 5/sq.mm.; and at T10-T12 it was 6.6-8.5/sq.mm. It should be noted that this increase in the number of nibbles taken -64-

Table 6. The amount of different leaves of known toughness eaten by 1st. instar larvae. Figures for square millimeters (except those for nibbles).

Food- Repli- Toughness and amount, eaten. (sq. mm.). plant. cate. 1 2 3 4 5 ... 10 11 12 Turnip 1 24 27 16 14 14 15 2 17 23 27 23 11 17 3 24 25 30 24 13 19 Total 65 75 73 7 38 51 No. ,nibbles. 278 307 360 321 365 404 Kale 1 102 139 31 74 11 1:1 2 77 121 55 62 17 & 3 106 103 29 89 10 3.:, Total. 285 363 115 225 38 & No. nibbles. 613 609 345 380 309 17(' Bruss- 1 68 29* 39 29 20 11 els 2 80 72 28 22 19 14 sprouts 3 93 90 41 24 18 12 Total. 241 191 108 75 ' 57 37 No. nibbles. 585 501 246 318 511 307

This tube had a mould on the leaf discs at the end of the

experimental period, and this affected the amount of food

eaten. -65-

Table 7. Re-presentation of the data in table 6.

Tough- Area eaten/ No. nibbles/ No. nibbles/sq. ness replicate Total Mean 'S' replicate. mm. eaten. 1 102, 77, 106, 139, 121, 103, 68, 80, 93. 889 99 22.4 201 2.0 2 29*, 72, 90. 191 64 30.0 167 2.6 3 31, 55, 29, 74, 62, 89, 39, 28, 41. 448 50 17.0 108 2.4 4 27, 23, 25, 29, 22, 24, 24, 17, 24. 215 24 4.36 100 5.0 5 16, 27, 30, 14, 23, 24. 134 22 6.25 113 5.1 10 20, 19, 18, 11, 14, 12. 94 16 3.83 136 8.5 11 15, 17, 19, 14, 11, 13. 89 15 2.83 128 8.2 12 11, 17, 10, 15, 8, 13. 74 12 3.32 80 o.6

'Ss in the above tables is the Standard deviation of the replicates

at any one toughness. The mean exceeds the Standard deviation by more than three times in

the figures, so the variation in the replicates is not significant,

except in the case of Toughness 2. In this test one of the replicates marked * had mould on the leaf discs. -66-

GRAPH I. CORRELATION OF AMOUNT EATEN WITH LEAF TOUGHNESS . Mean readings and maximum variation of all readings ( indicated by arrows).

Area eaten (sq. mm .) In 24 hours.

150 -

140 -

130 -

120 -

HO -

100 -

90 -

80 -

70 -

60 -

50 -

40 -

30 -

20 --

10 -

0 1 I I I I 2 3 4 5 6 7 8 9 10 H (2

Arbitrary units of Toughness. -67- occurs at the same point, about T4, that the amount eaten suddenly decreases with increased toughness. The long term effect of leaf toughness on larval growth was shown in a number of rearing experiments. In these, the insects were reared from egg to adult on leaves of known toughness. There was an increased larval mortality on leaves of greater toughness, and the pupal mortality was high. The rate of growth was also affected: the time taken for 500 of the larvae to reach the second and third instars was extended by 1-1* days, but the effect on the growth of the third instar larvae was not so great, and there was no further set-back in the development time. The adults which emerged from pupae the larvae of which had fed on tougher leaves were, on average, somewhat lighter than those fed on less tough leaves. Typical results are shown in Table 8.

Table 8. The effect of larval feeding on adult weight.

Larval food. Toughness. Average adult weight. mg.). Brussels sprouts. T3 6.00 Turnip. T4 5.96 ti 0 5.72 Kale. T5 5.60 T8 5.30

There was very little variation in adult weight when the larvae were fed with the leaves of different Crucifer- ous plants having the same toughness value, so the effect was not a nutritional one between plants. It would appear from these results that the physical nature of any synthetic medium must be considered in detail if optimum conditions for feeding are to be attained. -68-

E). The nature, and optimum concentration, of the olfactory stimulus.

It was now necessary to identify the olfactory and gustatory stimulants. Prom the previous work of Verschaeffelt and Thorsteinson, with larvae which fed on various Cruciferous plants, it seemed likely that the same stimulants might act as stimuli for the larvae of Phaedon. The mustard oil, allyl iso-thiocyanate, was tried as the olfactory stimulant. Various types of apparatus were used, and all had certain limitations, which are described below. It was, however, possible to show that the mustard oil acted as an olfactory attractant for Phaedon larvae, and the optimum range of concentrations was found. a). The use of an olfactometer to determine the attractant effect of the mustard oil.

An olfactometer was required which would give an easily interpreted larval response, and in which the optimum concentration of the stimulant could be determined. The chamber olfactometer used to show the presence of an attractive odour in the leaves (pages 50-52) was not suitable, for the following reasonsg i). It was difficult to balance equally the air flow on both sides of the apparatus. ii). The chamber was too large for the size of the entry and exit ports, so that some mixing and eddying of the air-streams took place. The slow rate of air flow across the chamber resulted in drifting at the central edges of the air-streams, while almost stationary air must have been present at the sides of the chamber. This meant that an even concentration of a test substance could not be maintained across the air-streams. -69-

iii). With the method of testing used, with 15 larvae in the centre of each half of the chamber, the results and their analysis became rather complicated. This was because a certain, variable, number of larvae remained stationary during the experiments. This was probably because the air in the chamber was too dry.

A new type of olfactometer was constructed, based on a design by Varley and Edwards (1953). It had certain features which made it easier to use2 i). A shallow glass chamber, deep, and 4" long by 3" wide, with tapered integral inlet and exit ports (Fig. 4A, Plan). ii). A single air stream, which, with the long, tapered ports, gave a streamlined flow across the chamber. iii). A pit in the basal glass sheet of the chamber (Fig. 4A, Plan)2 the air flowing over this picked up the test odour. The air containing the test odour then fanned out in a manner which could read- ily be observed by putting a drop of concentrated hydrochloric acid in the pit and drawing ammonia vapour through the chamber. Fig. 5 shows the spread obtained with this particular chamber. iv). Wide variations in the speed of the air flow made little difference to the spread of the test substance, although this factor affected the concentration.

The dimensions of the apparatus can be seen from the plan, fig. 4A. The basal plate was a sheet of glass, cut to shape, and with a 3 6" diameter hole drilled in the position shown. A 1" square piece of microscope slide was fixed over the bottom of the hole with 'Durof ix'. 'A' and 'B' were two separate sheets of glass, matching in -70— shape the tapered ends of the basal sheet: they were separated from the basal sheet by thick by *" wide 'Perspex' strip (Fig. 4B). 'Bostik' 252 was used to fix them, as this also acted as a filler for small cavities. Rubber tubing was embedded with sealing compound at the end of each tapered chamber: this was to take the tubing from the humidifying bottles and the suction pump. Wooden laths, -in wide, and just less than deep, were cut to fit all round the edge of the apparatus. lux*" hardboard strips were screwed loosely on to the laths, and the inside of the 'U' so formed was filled with sealing compound. The jacket was now pushed on to each side of the chamber, and the hardboard was screwed down firmly on to the laths. In this way a good seal was made, as the pressure of the hardboard strips forced the glass sheets tightly against the 'Perspex': the seal was completed by the 'Bostik' adhesive and the sealing compound. The top of the experimental chamber itself was built up of 'Perspex' strip, *" thick (Figs. 4A and 4B). When this had been secured with 'Durofix', a 3"x3*° piece of 'Perspex' was filed down to give a tight, flush-fitting lid to the chamber. Gauze glued between the roof and floor of the chamber made the chamber ends. A wooden support was made for the whole apparatus. Originally, the chamber had a hole in the floor, instead of a pit. The idea was that tubes containing the test substance could be placed against the hole: in theory, the passage of air over the hole would then draw up the test vapour-from the tube into the chamber. It was found, however, that the rate of flow of air in the chamber was too slow to give the desired effect. This may have been because the tube was completely closed; but the addition of an air inlet was not satisfactory, because then most of the air was drawn into the chamber at this -71—

FIGURE 4. VARLEY AND EDWARDS TYPE OLFACTOMETER USED FOR THE EXPERIMENTS.

A PLAN. (Not to scale). The hardboard and wood 'jacket' which kept the glass tight against the Perspex is not shown here.

13" 5.. 04

4. I 4 Perspex strip, i wide. Rubber tubing. Pit for test substance.

Perspex forming seal for lid.

B. SIDE VIEW. (Not to scale).

1.. .1 Perspex secured with Bostik 252. 1.- glass sheet. i Perspex lid. 1.• - Perspex forming seal for lid.

Rubber tube Pit for test substance.

Gauze forming end Glass plate forming wall of chamber bottom of pit -72- point, instead of through the inlet port. A sealed pit for the test solution was ideal. Initial tests on the air-flow in the chamber were made by drawing tobacco smoke through the apparatus. The removable 'Perspex' lid was sealed down with Sellotape. A very satisfactory air-flow was obtained° the taper of the ports, from -" to 3" over a length of 13", gave a streamlined air-flow across the floor of the chamber, as shown in fig. 5. The Relative Humidity (R.H.) of the air drawn through the apparatus varied between 34-40%. For the experiments this had to be humidified, as the larvae responded very slowly, if at all, at low humidities. Two humidifying bottlers (bubblers) were found adequate to raise the humidity of the in-going air to about 90% R.H. Initially, 1, 2, and 4 bubblers were tried in series. The measurement of the humidity increase for each set of bubblers was made with a Gregory humidity meter. A 4"x12" tube was fitted between the bubblers and the apparatus, and the Gregory meter element was put in this tube, the lead passing down a groove in the rubber bung (Fig. 6). Readings showed that at 24°C., one bubbler was sufficient to raise the humidity from 40% to 85%; two bubblers took it to 90%; and four took it only slightly higher, to 93-94%. With four bubblers the suction pressure had to be increased, but this increase was out of proportion to the resistance offered by the water in the increased number of bubblers. This was because a number of small leaks in the apparatus let in a remarkable amount of air at higher suction pressures, and reduced the suction available to draw air through the bubblers. Before the tests, the larvae were placed on moist filter paper in Petri dishes, and were starved for half an hour. During this time, most of the material in the gut

-73-

FIGURE 5. THE SPREAD OF THE TEST SUBSTANCE IN THE AIRSTREAM.

♦ --

AIR Dispersion of test

X • substance indicated •••••••••••••• •••••••••••••• ••••••••••••• • • • • • • • • • ••••• • • • • • • ••• • •••• •••••••••••••••••••• • ••••••••••••••• ••••• • —Test pit. 4-- by ammonium — ••••••••• • ••••••• •••••••••••" • • • • • •••••

X LnLI:": chloride deposit '•••• FLOW

Position of larva

FIGURE 6 THE EXPERIMENTAL SET-UP FOR THE OLFACTOMETER. (Not to scale)

62) 60 watt bulb Diffusion screen

To suction pump Olfactometer

Gregory hygrometer \ / 2 humidifying bottles element in tube Tube to Lollec.t water carried over -74- was voided, which reduced the risk of scent trails being left in the chamber. As an added precaution, the glass and 'Perspex' surfaces were wiped over with a moist cloth after each pair of larvae had been tested. Preliminary experiments had shown that the larvae were at times attracted to the light from the laboratory windows, while at others they were negatively phototactic. No definite explanation can be offered for this behaviour, which could not be counteracted by a 60 watt diffuse tungsten light 8" above the chamber. The only relevant observation was that the negative phototaxis appeared when the larvae were nearing the moult to the third instar. The experiments proper were therefore carried out in the darkroom, using the lamp as described above. The solution of the test substance was put on a small ball of cotton wool in the test pit, and this ball was renewed several times during each experiment. The flow of air was standardised at about 300 bubbles/minute passing through the bubblers. Two hundred second instar larvae were used for each test substance and concentration. One pair of larvae only were used at any one time in the apparatus. They were transferred tothe chamber with a camel-hair brush, but certain precautions were required, because it was noticed during the preliminary experiments that the larvae tended to move away from the side last touched, even with the counter-attraction of the olfactory stimulant. Alternate pairs of larvae were therefore transferred to the chamber with the brush on the opposite side, to annul the effect of the response on the final readings. Of each pair of larvae, one was placed facing up-stream in each of the positions marked 'X' in fig. 5g each position was on the edge of the test air-stream, and 2.5cm. from the actual test spot. The lid of the chamber was sealed -75- down, and the first response of the larvae, and the time taken to elicit it, was recorded. If a larva turned out- ward into the plain air-stream, it was given a score of 'O': if it turned into the test air-stream, it scored '1'. If no stimulant was present in the test air-stream, then the expected values of '0' and '1' for a large number of insects tested would be in a 1:1 ratio. A X2 'Degree of Fit' test was applied to decide whether or not the experimental results differed significantly from this expected ratio. The following substances were tested. It should be noted that the concentration given is that of the drop of liquid used° it does not represent the concentration actually in the air-stream.

Conell. Conch. Substance. (p.p.m.) Substance. (p.p.m.)

Water (control). Crushed pea leaf. - Sinigrin. 125 Crushed pea leaf if 500 plus 0 1000 myrosin. 1000 n 3000 Crushed pea leaf Sinalbin. 125 plus 500 sinigrin. 3000 it 1000 n Crushed pea leaf 3000 plus Myrosin. 200 mustard oil. 250 n 1000 Crushed turnip. - Mustard oil. 1.0 II 2.5 Sinalbin plus 3000 II 5.0 myrosin. 1000 tl 12.5 Sinigrin plus 3000 0 25 myrosin. 1000 II 50 II 100 Mustard oil. 500 n 125 if 1000 if 250 " 2000 II Pure oil.

A control experiment with water was carried out immediately after any experiment in which attraction had -76- been found, to make sure that there could not be any effect on the next experiment. The results are shown in Table 9. (Overleaf). It was found that i). Sinigrin, sinalbin, and myrosin, solutions were not attractive when offered alone. ii). Sinigrin and sinalbin, with the addition of myrosin solution, was attractive. Sinigrin and sinalbin are mustard oil glucosides the enzyme myrosin, which is found in the Cruciferae, brings about the hydrolysis of these glucosides to the corresponding mustard oil. iii). The mustard oil, allyl iso-thiocyanate, was attractive in concentrations from 2.5-500p.p.m. The optimum range was between 25-125p.p.m. Concentrat- ions above 500p.p.m. seemed neither attractive nor repellent. iv). Crushed turnip leaf was attractive, but no more so than the optimum concentration of mustard oil. v). Crushed pea leaf was not attractive to the larvae. Nor was it after the addition of sinigrin or myrosin. The addition of mustard oil of 250p.p.m. concentration was tried to see if the presence of the leaf tissue would increase the response, as compared with that obtained with a similar concentration of mustard oil alone. The figures (Table 9) would seem to support this idea. vi). An analysis of the reaction times showed that the time for larvae moving towards an attractive odour was significantly shorter than for larvae which moved outwards. The apparatus proved satisfactory in that there was

-77-

Table 9. The results of the olfactometer tests.

Reaction Reaction Reaction Reaction Number Number time/larva time/larva time into time away moving moving Substance Cone-.Y1 in left- in right- test air- from test toward away from tested. (p.p.m.) hand col. hand col. stream. airstream. test air- test air- Signif- (secs.). (secs.). (secs.). (secs.). stream. stream. icant.

Water (control), 5.23 5.87. 5.37 5.74 101 99 Sinigrin. 125 5.46 4.86 4.79 5.49 106 SI 500 4,26 5.72 5.28 4.68 103 97 II 1000 6.46 6.73 6.03 7.17 101 99 " 3000 5.00 5.82 6.41 4.45 98 102 Sinalbin. 125 6.05 6.29 6.16 6.18 100 100 i, 500 5.42 6.01 5.30 6.11 99 101 11 1000 5.31 4.69 4.82 5.18 103 97 ii 3000 6.01 6.35 5.88 6.50 104 96 Water (control)1 4.19 5.02 4.30 4.91 100 100 Myrosin. 200 4.91 5.63 4.88 5.67 101 99 it 1000 4.87 5.55 5.53 4.89 102 98 Mustard oil. 1.0 4.19 5.43 5.17 4.46 99 101 (Allyl iso- 2.5 4.92 5.52 4.68 6.03 119 81 thiocyanate). 5.0 4.06 4.52 4.02 4.74 127 73 "12.5 5.18 5.72 5.33 5.62 117 83 25 4.74 5.71 5.00 5.98 134 66 50. 4.60 5.92 4.52 7.18 144 56 100 4.52 4.59 4.01 5.77 138 62 125 3.43 3.89 3.32 4.61 139 61 4.57 5.01 4.22 6.19 142 58 250 6.19 5.66 5.16 7.25 127 73 500 4.49 5.55 4.86 5.30 124 76 SI 4.59 4.43 4.07 5.31 129 71 1000 6.20 5.77 5.62 6.32 96 104 2000 5.08 6.01 5.15 5.97 103 97 il 4.51 4.11 4.06 4.58 104 96 Puro oil 6.15 6.50 6.62 6.06 95 105 Water (control). 5.69 5.70 5.76 5.63 101 99 Pea leaf. 5.27 6.09 5.45 5.94 105 95 r Pea leaf plus sinigrin. 3000 3.26 3.93 3.74 3.45 100 100 Pea leaf plus myrosin. 1000 4.56 4.71 4.57 4.70 100 100 (Cont/d..). -78-

Table 9. (Continued.).

Reaction Reaction Reaction Reaction Number Number time/larva time/larva time into time away moving moving Substance Conc-. in left- in right- test air- from test toward away from tested. (p.p.m.) hand col. hand col. stream. airstream. test air- test air- Signif- (secs.). (secs.). (secs.). (secs.). stream. stream. icant.

Tater (control). 4.28 4.01 4.36 3.93 101 99 Pea leaf plus mustard oil. 250 5.37 5.34 5.14 5.95 147 53 H Water (control). 4.86 5.57 5.84 4.52 106 94 Turnip leaf. 5.06 5.02 4.70 5.90 143 57 H Water (control). 4.24 4.55 4.38 4.42 107 93 Sinigrin plus 3000 myrosin. 1000 4.45 5.60 4.99 5.08 122 78 x Water (control). 5.02 4.76 5.30 4.46 102 98 Sinalbin plus 3000 myrosin. 1000 4.90 6.09 5.99 4.72 122 78 H Water (control). 5.75 5.06 5.26 5.54 102 98 -79- no significant difference between the over-all response for each side of the apparatus, or between the over-all reaction times for each side. Any significant difference in the results must therefore be due solely to the test substance. The apparatus had certain limitations. The test substance was diluted by the air-stream, so that the actual concentration to which the larvae were responding was not known. The optimum concentrations of an attractant would therefore be somewhat lower than indicated in iii) above. An anomalous situation arose where high concentrations of mustard oil were used. Theoretically, these higher concentrations should be repellent, but in the experiments the numbers moving into the test air-stream and the control air-stream were roughly equal. This corresponded with the control experiments with water, and indicated a neutral response. The absence of a repellent effect was due partly to the apparatus and partly to the method of getting readings. Even with a high concentration of the mustard oil, a low concentration of the vapour would be reaching the larvae immediately after the sealing of the chamber, because plain air would have to be drawn from the test area first. The larvae gave a positive reaction to this low concentration, but as it rose to the full level, the larvae moved out of the air-stream. This movement of the larvae is shown in Fig. 7, which is an actual drawing of the movement of one larva. In this case the larva moved into the test air-stream until the concentration of the oil increased it then wandered out along a very winding path to 'B'. At this point the concentration was relatively low, and the larva stopped to raise the anterior part of the body, and wave it in the air-stream. The larva then moved in an arc, BC, stopping repeatedly, until the point C was reached up-wind of the test spot. Finally the larva wandered away into the body of the chamber.

-80-

FIGURE 7. TYPICAL MOVEMENT OF A LARVA IN THE OLFACTOMETER WITH A HIGH CONCENTRATION OF MUSTARD OIL PRESENT.

AIR

FLOW. A Starting point. PC. Wandering of larvae to windward of test spot. ♦--

FIGURE 8 THE CONVENTIONAL TYPE OF 'SPLIT CHAMBER' USED FOR SOME EXPERIMENTS.

Glass plate.

I3cm. diameter dish. Muslin platform. Small dish for water.

Small dish for ' Starting position

test substance of larvae -81-

It would be better, rather than taking the first response of the larvae as a reading, to take the time spent by the larvae in each air-stream, as was done by Varley and Edwards (1953).

b). Confirmation of the olfactometer results.

A number of experiments were carried out to confirm the/results obtained with the olfactometer, and more especially to get a better idea of the optimum concentration of the mustard oil.

'Split chamber' experiments. The conventional type of 'split chamber' was used (Fig. 8). The chamber was a 13cm. Petri dish, containing two small 2"x*° glass dishes cut down from specimen tubes. A pencil line was drawn along a diameter of the gauze to mark off the 'test' and 'control' halves of the chamber. At the centre of the gauze disc, a lcm. diameter circle was drawn, and the larvae were placed within this circle at the beginning of an experiment. The experiments were carried out in the laboratory, away from the direct light from windows. A 40 watt light. with diffusion screen, was placed 12" above the chamber. This light was switched on some 10 minutes before the experiments were started, to allow the temperature to reach equilibrium. This was at 20°C. Water was placed in one of the small dishes, to form the control, and the test substance was put in the other. The two containers were placed in the Petri dish so that the inner edge of each was 2cm. from the perimeter of the central circle in the gauze. Twenty larvae, which had been starved for half an hour, were placed within the area of the central circle, and the lid of the chamber was replaced. -82-

Readings of distribution ware taken at i minute intervals over a period of 5 minutes. The chamber was then turned through 180° , the larvae were re-organised into the central circle, and the experiment was repeated. Three sets of experiments were carried out for each test substance, and a fresh lot of larvae were used for each set. The same experiments as those listed on page75 were performed, with the addition of two lower concentrations of the mustard oil, and some further tests with pea leaves. Results are given in Table 10, below. In this series, two tests were made to see if the presence of leaf tissue enhanced the attraction by the mustard oil. Pea leaf on its own is unattractive (tables 9 and 10), so in the olfactometer tests it was tried with mustard oil at a concentration of 250p.p.m., to see if the number of larvae responding was increased, as compared with the response to mustard oil alone. There was some evidence that the leaf protein did enhance the response in this experiment (Table 9). In the present experiments, a sub- liminal, non attractive concentration of mustard oil at 0.25p.p.m. was added to the pea leaf-tissue, to see if the combination then became attractive. The result was not significantly different from the result for pea leaf alone. Similarly, the result obtained when sinigrin and myrosin solution was added to the pea leaf showed no-increase over the response with sinigrin and myrosin alone. It must therefore be concluded that the leaf tissues do not complem- ent the effect of the olfactory attractant. The rest of the results confirm those found with the olfactometer (see page 76), with the following slight differences: i). With this apparatus, the larvae responded to lower concentrations of mustard oil, from 0.5 to 250p.p.m., instead of the 2.5 to 500p.p.m. recorded in the -83-

Table 10. The results of the 'Split chamber' tests. . No. larvae No. larvae Substance Conc.a. in test in control Signif- tested. (p.p.m.). half. half. icant._ Water (control). - 57 63 Sinigrin. 125 61 59 ii 500 55 65 II 1000 65 55 ii 3000 58 62 Sinalbin 125 68 52 it 500 *,-. 62 58 It 1000 62 58 it 3000 59 61 Myrosin. 200 62 58 It 1000 68 52 Mustard oil. 0.25 61 59 if 0.5 71 49 , K ii 1.0 77 43 H n 2.5 71 49 x il 5,0 72 48 H n 12.5 75 45 R ° 25 70 50 H ., 50 80 40 H II 100 il 78 42 m 125 71 49 R if 250 78 42 R if 500 62 58 0 1000 50 70 HH 2000 62 58 Repellent. Pure oil 41 79 HR Water (control). 59 61 Crushed pea leaf. 68 52 Crushed pea leaf plus mustard oil. 0.25 68 52 Crushed pea leaf plus sinigrin. 3000 67 53 Crushed pea leaf plus myrosin. 1000 65 55 Crushed pea leaf plus sinigrin 3000 plus myrosin 1000 72 48 m Sinigrin 3000 plus myrosin. 1000 71 59 H Crushed turnip. 76 44 H -84- olfactometer. Again, it should be noted that these figures refer to the concentration in solution, and not the concentration in the air-stream. ii). A definite repellent effect was noted with concentrations of 1000p.p.m., and pure oil. 500p.p.m. and 2000p.p.m. gave a neutral response, but it was noticed that the larvae first moved to within 2cm. of the test spot, and then stopped; then, as the concentration built up by diffusion, the larvae moved back to the control half.

The apparatus had one serious draw-back, related to the slow rate of diffusion of test odours in the still air. At low concentrations, it took some time for an attractive concentration to be reached a very low concentration, which would make the larvae respond at very close range, failed to do so over the test distance of 2cm. Again, the larvae moved towards the test area at high concentrations, when the odour first reached them, but they later moved away again as the odour became repellent. In some cases, the larvae just remained stationary in the test half when the odour became repellent. The results showed that the repellent level was at about 500p.p.m.

F). The effect of colour on the orientation response of the larvae. i). The use of coloured tapers. The biting response, and the effect of sinigrin on the feeding response, needed investigation. Two preliminary methods were tried.

The first was a 'Spot test'. A. 9cm. Petri dish and cover was used, with a 40 watt diffused light source above it. A 9cm. filter paper disc, pencil marked in concentric circles of lcm. increasing diameter, was moistened and put

in the dish. A email ball of cotton wool was soaked in the test solution, and then placed at the centre of the filter paper disc. Four larvae were placed on the filter paper, one at each quadrant of the circle, and 2cm. from the test spot. The following responses were noted2 i). Distance moved, and the time taken. ii), Direction - towards or away from the spot. iii). Whether biting occurred. 1v). If feeding continued. The apparatus was then turned through 180°9 and the experiment was repeated with the same larvae. It was found that only one larva in four moved into the test area. Again this was attributed to the rate of diffusion of odours in the chamber. The olfactometer was again tried, as it was thought that this would rapidly carry the test odour to the larvae, and the biting and feeding response could be recorded after the larvae had followed the odour up to its source. Instead of two larvae being placed at the edges of the test air-stream, four larvae were now placed directly in it. It was found that, with mustard oil at 50 and 100p.p.m. concentration, the larvae began to crawl towards the test spot; but then when they had come within 3-4mm. of the test odour source, most of the larvae missed it completely, and went crawling off into the body of the chamber.

A very small chamber was now constructed, made of ain- square pieces of glass sheet separated by le strips of 'Perspex' glued to the bottom sheet. A piece of paper, marked with faint, pencilled, concentric circles of lcm. increasing radius, was stuck to the underside of the bottom sheet of glass. The centre point of these circles marked the test spot. It was thought that in such a shallow chamber there would be less effect on the diffusion of the -86- test odour by the eddying of air found in larger chambers, Four larvae were again used, in the way described for the 'Spot tests'. In this case, mustard oil at 25 and 50 p.p.m. concentrations was used, soaked on small balls of cotton wool. Here again the larvae would move towards the source of the attractant, but once within 3-4 mm., most failed to reach it. They would stop, raise the anterior part of the body, and wave it from side to side. Then they would crawl again in any direction, some reaching the cotton wool more by chance than by definite orientation. The impression was given that a visual response might be important for the orientation of the larvae. Prelim- inary experiments were carried out using balls of cotton wool soaked in dyes, and allowed to dry. Brilliant Green dye, and the Deep Green and Light Green of the Pelikan Graphos range of water-proof inks were tried first. Small balls of the coloured cotton wool were soaked in the test solution, and were then tested in the small chamber. A range of mustard oil concentrations from 1 to 1000p.p.m. was tested. The results are summarised in Table 11 (overleaf). It was found that there was a significant increase of from three to six times in the number of larvae reaching the ball of yellow-green cotton wool, as compared with the number reaching the white cotton wool soaked in mustard oil. The effect was less pronounced with the blue-green cotton wool. It was also found that the colour alone caused attraction, when compared with white cotton wool soaked in mustard oil, or in water. The odour of the mustard oil attracte#he larvae to within 1-2mm. of the cotton wool, where they then stopped and tested the air - or it may have been that they were Table 11. The effect of colour on the orientation of the larvae: a preliminary experiment. Colour. White Yellow-green Blue-green Test- Concr;-1 % Resp- % to- T7E % Resp- % to- %gE % Resp- % to- % at ed onding, wards spot. ondinq. wards spot. ondina. wards spot. spot, spot. spot,

Water - 55.0 45.0 0.0 70.0 46.4 7.1 65.0 42.3 0.0 ii 100.0 32.5 15.0 Must- 1.0 77.5 45.2 3.2 67.5 40.7 11.1 85.0 50.0 11.8 and 2.5 77.5 54.8 12.9 90.0 55.3 30.5 82.5 48.5 9.1 oil. - 92.5 37.8 16.2 92.5 45.9 29.7 90.0 36.1 8.3 5.0 90.0 47.2 5.6 87.5 54.8 31.4 87.5 51.4 11.4 - 95.0 36.8 13.2 95.0 57.9 34.2 87.5 37.1 11.4 12.5 80.0 31.2 9.4 90.0 52.8 33.3 87.5 42.8 17.1 - 77.5 45.2 9.7 97.5 48.7 33.3 95.0 39.5 10.5 25.0 85.0 38.2 11.7 92.5 48.6 37.8 90.0 41.7 11.1 - 95.0 '1'1.7 13.2 90.0 44.4 30.6 97.5 41.0 17.9 50.0 85.0 26.5 8.8 67.5 40.7 25.9 ' 87.5 37.1 20.0 125.0 95.0 39.5 10.5 95.0 37.1 26.3 90.0 22.2 13.8 250.0 87.5 31.4 11.4 92.5 21.6 16.2 95.0 26.3 21.0 500.0 92.5 18.9 5.4 95.0 23.7 21.6 90.0 8.3 5.5 1000.0 92.5 24.3 10.8 97.5 23.1 20.5 95.0 15.8 7.9 -88- trying to locate a stem. With the white cotton wool, a few of the larvae reached it by chance° with a colour present, the larvae moved directly, and without stopping, to the cotton wool. A concentration of between 2.5 and 25p.p.m. of mustard oil, with the colour, was the optimum level.

A more comprehensive series of tests was now planned, using a whole range of Pelikan Graphos coloured inks in conjunction with a solution of mustard oil at 10p.p.m. w‘ concentration. The colours were; red, orange, yellow, yellow-green, blue-green, blue, and violet. The inks were diluted with water, and were then epred onto individual 7cm. filter paper discs and allowed to dry. A series of grey shades was also prepared by mixing Pelikan black and white ink in different proportions, and pouring these mixtures onto filter paper discs. Some idea of the relative brightness of the different colours was needed, because variation in this factor might be more important than an actual colour difference. The discs were photographed together in tungsten light, using Ilford Pan F high contrast film. This was then developed to a high gamma, and the negatives printed on normal grade paper. A copy of this photograph is seen in fig. 9. This treatment gave the maximum separation of the grey tones, which could then be compared by taking spot readings with an S,E.I Photometer. Direct readings of the colours could not be taken with this instrument, as it uses a grey spot for matching against the background, and this spot could not be matched accurately against the colours. The readings were taken by tungsten lighting in the darkroom, as the intensity of the daylight in the laboratory fluctuated considerably. Five readings were taken for each disc on -89-

F4Arre 9. The relative brightness of the coloured papers used for the experimental cones.

Intensity. Log. foot Lamberts.

Grey 1 1.48

Grey 2 1.29

White. 1.54

Orange. 1.39

Yellow. 1.48

Red. 1.31 Violet. 1.34 Yellow- green. 1.40 Blue. 1.29 Blue-green. 1.28 -90-- the photograph, and the average taken. The readings were checked by measuring the light reflected from each coloured disc under standardised lighting conditions, using an Everett-Edgcumbe Auto-Photometer for the measurements. The results were in good agreement with those obtained from the photograph. The order of relative brightness was as follows'

White.›[YellollYellow-green.1>Violet.> Red. Grey 1. Orange. Blue-green. Blue. Grey 2. A standard form was required which would reflect light equally to all four larvae in the small chamber (p. 85). The cone fulfilled this requirement. 5 6" diameter discs were cut from each coloured filter paper, and these discs were cut, folded, and glued by a small tongue, to form cones high and 1" in diameter. For the experiments, a small ball of cotton wool was soaked in 10p.p.m. w/v mustard oil solution, and this ball was put inside the cone to be used for the experiment. The cone was then put at the centre of the chamber. Forty second instar larvae were used for each experiment, and these were used four at a time, as described on page 85. The following responses were noted 1). The distance moved by the larvae, ii). The time taken to move this distance. iii). Whether or not the move was towards the test spot, iv). Whether or not there was a biting response and feeding response, v). The time spent biting. Table 12 shows how the larvae moved, and the figures -91-- of this table are plotted in Graph 3A. The readings for the rates of movement, and the biting response, are shown in Table 13. Graph 2 shows the percentage response and relative brightness of each colour plotted together.

A marked increase in the number of larvae reaching the test cone resulted from the use of certain colours. The following are the main points i). The mustard oil alone, at 10p.p.m. concentration, on a white cone gave a result which hardly differed from the control with water on a white cone. ii). With the mustard oil and colour together, there was a considerable increase over the figures with mustard oil alone. But the results were no better than those with water and colour together. This was attributed to the slow rate of diffusion in the chamber; the larvae had already started moving away from the test spot before the odour reached them. The increased movement towards the test area was caused mainly by the colour alone. iii). There was little difference in the rate of movement between larvae moving towards the test area and those moving away from it. Larvae which actually reached the cone, however, moved much more rapidly than the others in the presence of the stimulant. iv). The larvae that reached the cone when the colour colour alone was present did not show a biting response, whereas those that reached the cone when the mustard oil was present did show the response. v). The response of the larvae could be attributed to the relative brightness of the colours, but it was noticed that a low response was obtained with the two grey shades, when compared with a colour of equal brightness. This can be seen from Graph 2. This -92-

Table 12. The effect of colour on the orientation of the larvae.

Colour Water Mustard oil % Resp- % Moving % Reach- % Resp- % Moving % Reach- once towards ing once. towards ing spot. sot. spot. spot. White. 92.5 37.8 10.8 97.5 35.9 12.8 Red. 97.5 48.7 25.6 97.5 53.8 33.3 Orange. 90.0 61.1 50.0 97.5 56.4 46.2 Yellow. 92.5 51.4 37.8 97.5 5849 38.5 Yellow- green. 92.5 45.9 37.8 100.0 42.5 30.0 Blue- green. 95.0 47.4 26.3 97.5 43.6 23.1 Blue. 95.0 34.2 10.5 90.0 41.7 8.3 Violet. 92.5 45.9 24.3 95.0 42.1 21.1 Grey 1. 97.5 41.0 12.8 97.5 35.9 15.4 Grey 2. 97.5 41.0 10.3 92.5 43.2 18.9 -93-

Table 13. Rates of movement and biting response of the larvae.

Colour Water Mustard Oil Secs/ Secs/ Secs/ Bit- Durat- Secs/ Secs/ Secs/ Bit- Durat- cm.to- cm. to cm. ing. ion of cm.to- cm. to cm. ing. ion of wards reach from biting. wards reach from biting. spot. spot. spot. spot. spot. spot White. 22.3 12.5 16.6 - - 24.1 20.0 31.1 80.0 17.5 Red. 23.0 16.5 19.6 - - 28.7 17.6 18.2 92.3 12,2 Orange. 20.1 19.5 19.1 - - 18.2 17.1 23.4 94.5 5.8 Yellow. 13.1 10.8 15.4 - - 26.4 19.6 21.4 100.0 9.8 Yellow- green. 22.4 19.1 13.7 - - 20.3 17.0 27.1 75.0 16.6 Blue- green. 17.5 12.7 20.2 - - 27.5 28.0 23.7 66.6 14.5 Blue. 21.5 14.2 17.8 - - 26.1 7,7 21.0 66.6 7.5 Violet. 20.7 16.1 '21.0 - - 19.8 15.5 22.6 75.0 7,3 Grey 1. 13.7 13.0 24.6 - - 23.3 12.8 18.2 83.4 5,2 Grey 2. 23.6 14.7 22.8 - - 22.8 10.1 26.7 100.0 5.6 -94—

GRAPH 2. THE RESPONSE OF THE LARVAE TO THE COLOURED PAPERS, TOGETHER WITH THE RELATIVE BRIGHTNESS OF THESE PAPERS.

Reaching Relative brightness Colour. of colours. (log foot lamberts.) 50- -1.6

40- -1.5

-1.4 30- 0

20- 0 -1.3

10- -1.2

0 1.1 R. O. Y. YG. BG. B. V. GI. G2. W.

COLOUR.

The line joins the points indicating relative brightness of the colours. The histogram gives the percentage reaching the colours. -95- would suggest that the effect was due to the actual colour. vi). The orange, yellow, and yellow-green dyes were the most attractive for the larvae.

Because the slow rate of diffusion meant that the larvae responded to the colour before the odour, the experiments were repeated with the olfactometer, using the coloured cones and mustard oil at 10p.p.m. concentration. The olfactometer was set up as described on page 72. Pour larvae were put in the centre of the test air-stream, and 2cm. from the test pit. It was thought that the presence of the cone in the air-stream would not affect the air-flow seriously. Readings were taken as for the previous set of experiments (page 90), and these are given below, in Tables 14 and 15.

The results of this test for the number of larvae moving towards the cone are similar to those shown in Table 12, p. 92. The difference now arises, however, that with the use of the olfactometer the presence of the mustard oil has a more pronounced effect (cf. Graphs 3A and 3B). On the whole, rather more larvae reached the test cone with the odour present than when it was absent. In the small chamber experiments, no difference was found. The colour response and the olfactory response are clearly complementary. Although the time taken for larvae to move to the test substance was shorter than for movements in other directions, (Table 15), this was not correlated with the attractiveness of the cones. No conclusions could be drawn from the times recorded for the duration of the biting response. Again it was noticed that although there was a definite attraction to colour, this did not elicit a biting resp- -90-

Table 14. The effect of colour on the orientation of larvae in the olfactometer,

Colour Water Mustard oil. % Resp- % Moving % Reach- % Resp- % Moving % Reach- onse towards ing spot. onse. towards ing spot. spot. spot. White. 95.0 50.0 8.3 it 90.0 47.2 9.4 100.0 50.7 30.0 Red. 92.5 62.2 32.4 95.0 65.8 31.6 Orange. 97.5 59.0 38.5 95.0 73.7 52.6 Yellow. 95.0 57.9 36.3 95.0 68.4 44.7 Yellow- green. 97.5 53.8 33.3 100.0 47.5 37.5 Blue- green. 95.0 52.6 21.1 95.0 52.6 28.9 Blue. 95.0 36.8 15.8 97.5 53.8 28.2 Violet 97.5 41.0 17.9 97.5 56.4 23.1 Grey 1. 100.0 47.5 20.0 95.0 47.4 26.3 Grey 2. 95.0 39.5 18.4 95.0 57.9 26.3 -97-

Table 15. Response times and biting response for the experiment of Table 14.

Colour Water Mustard oil. Secs/ Secs/ Secs/Bit- Durat- Secs/ Secs/ Secs/ Bit- Durat- cm. to- cm. to cm. ing. ion of cm. to- cm. to cm. ing. ion of wards reach from biting. wards reach from biting. spot. scot. 1 spot .pot. spot spot. . White 57.2 33.3 55.7 - - " 46.9 38.9 50.5 - - 49.4 40.0 51.6 91.7 11.5 Red 44.7 31.2 47.0 - - 40.7 45.8 33.8 91.7 18.9 Orange 36.1 25.3 49.8 - - 26.1 25.2 38.2100.0 16.3 Yellow 26.6 20.6 43.6 - - 23.5 37.6 66.4 94.1 10.0 Yellow- green 41.5 27.1 46.8 - - 30.3 23.1 41.2 73.3 12.9 Blue- green 48.9 31.9 40.0 - - 27.5 20.5 51.7 81.8 22.8 Blue 48.7 28.3 52.1 - - 39.2 26.7 38.3 91.0 13.6 Violet 31.9 26.6 38.2 - - 44.5 28.6 64.3100.0 21.7 Grey 1 47.1 36.3 35.6 - - 29.2 24.5 36.8 90.0 20.9 Grey 2 31.7 20.4 31.0 - - 36.7 25.8 43.4100.0 20.6 -98—

GRAPH 3A. PERCENTAGE OF LARVAE REACHING THE COLOURED CONES IN THE SMALL CHAMBER. Figures from Table 12.

0/0 Reaching Test Spot.

With mustard oil.

F Water only present. Presence or absence of hatching at top of columns indicates which was higher result.

R. 0. Y. YG.BG. B. V. GI. G2. W. Colour -99-

GRAPH 3B. PERCENTAGE OF LARVAE REACHING THE COLOURED CONES IN THE OLFACTOMETER. Figures from Table 14.

0/0 Reaching Test Spot. 60 -

With mustard oil.

Water only present.

50- Presence or absence of hatching at top of columns indicates which was higher result. 40-

30,

R. 0. Y. YG.BG. B. V. GI. G2. W.

Colour. -100-

-onse (Table 15). The presence of the mustard oil did bring about such a response. b). The use of coloured light sources to test the colour response of the larvae.

The dyes used for the above experiments probably reflected a much wider range of wave-lengths than was indicated by the visible colour. This could give misleading results, so that it was desirable to use monochromatic light sources. The response of the larvae to a spectrum was first tried. The spectrum was produced by the usual method of having an intensity lamp, a lens, and a prism in the correct relative alignment. The arrangement used threw a spectrum 1.75" long by 0.6" wide on the floor of the chamber used. The light fell at an angle of 30°. It was found that the larvae moved towards the bright source of the light, rather than to the spectrum. The set-up was altered so that the beam fell vertically on to the chamber, where it was reflected towards the larvae by a piece of paper at 45o The larvae moved towards the spectrum, but nothing could be decided from the results, as these were erratic. A disadvantage was that the different bands of the spectrum varied considerably in brightness, and depended on the emission spectrum of the bulb used.

i). The use of filters for the light source.

A method of presenting coloured lights of equal energy levels, and covering only narrow bands of the spectrum, was required. Messrs. Kodak Ltd. suggested a range of 'Wratten' -101- filters which, used in combination, would transmit narrow wave-length bands over the range of the visible spectrum. These combinations are listed below. (Table 16).

Table 16. Transmission of 'Wratten' filters. Wavelength of Wavelengths Filter maximum trans- Colour. transmitted. combinations. mission. Blue. 440 - 510mp 45 + 32 4901/1 Blue-green. 515 - 530mp. 45 + 12 520mp Yellow-green. 530 - 560mp 44 + 16 540/41 Yellow. 570 - 590mp 73 + 23A 580ma Orange. 600 - 640ma 72B 610mp Red. 650 upwards. 70 660mp up.

To get the light energy transmitted by these filters roughly equal, a series of 'Wratten' Neutral density filters was used. All filters were of gelatine, bound between glass. For the initial measurements of the energy transmitted, a thermopile and moving spot galvanometer were used. It was then found that all the filters transmitted light in the red and infra-red regions, to some extent. As the thermopile was most sensitive to these wavelengths, the readings obtained were not true ones for the energy of the visible colour. A lcm. deep solution of 5% copper chloride was used as a filter to cut out all the red light above 690mp, and also a large proportion of the wave-lengths down to 6701w. It was found that with the copper chloride bath interposed between the light source and the filters, the thermopile gave hardly any reading on the galvanometer) the previous high readings for the filters were due entirely to their high transmission of red light. The effect of the copper chloride filter can be seen from Table 18. Further details are given on page 104. An Everett-Edgecumbe Auto-photometer was used instead -102- of the thermopile. The photometer consisted of a barrier layer type selenium photocell, with a galvanometer having three sensitivity ranges calibrated in foot-candles. The manufacturers supplied a spectral sensitivity curve for the photocell, based on an equal energy spectrum, so by taking a reading on the galvanometer for a particular wave-length, and applying a correction factor for the sensitivity of the photocell to this wave-length, an indication of the relative energy transmission of the filters was obtained; although this was not a measure of true units of energy. The wave-length at which each filter gave maximum transmission is shown in Table 16. These figures were derived from data supplied by Messrs Kodak Ltd. The spectral sensitivity of the photocell was determined for each of these wave-lengths, using the curve shown in Graph 4, and the results are shown in Table 17. The sensitivity curve was supplied by Messrs. Everett-Edgcumbe for the particular photocell used. The possible error arising from taking the maximum transmission wavelength of the filters for the photocell sensitivity amounted to about 1% with the blue-green, yellow, and yellow-green filters; about 10% with the blue and orange filters; and about 20% for the red filter. In practice the error was probably considerably less than these figures, because of the rapid fall-off in transmission of wave- lengths on either side of the maximum. A mask of aluminium foil with a 1" diameter hole in the middle was made for the photocell. A microscope high- intensity lamp was placed 2" above the aperture, and this gave the maximum deflection on the high scale of the galvanometer. (150 foot candles). The filter combinations were cut into li-31 diameter discs, which were then bound between li" square glasses. First, the individual filter combinations were placed -103-

GRAPH 4. SPECTRAL SENSITIVITY CURVE FOR PHOTOMETER. Relative spectral response values for 'Autophotie photo- electric cell. Based on equal energy spectrum.

Everett- Edgcumbe curve No. 19131.

ti)

C ).

O ns — 0

co icro llim i (m h t leng Wave

CTD

0 0

o a a- 0 0 0co 0 0 0 0 0 0 0 0 •Ln 0 CT, rs- %Co • N CC -104- over the aperture of the mask, and the readings were noted. Then the filters were again tested, but with the copper chloride filter bath placed over them. The considerable cut-off at the red end of the spectrum when this bath was used can be seen from Table 18 (cf. column 6 with the figures for transmission through an equal depth of water, shown in column 8). It was now necessary to bring the transmission of all the filters to about the same energy level. This was done by taking the filter of lowest energy transmission. as the standard, and finding the theoretical uncorrected galvanometer reading for each of the other filters so that they would transmit the same energy as the yellow filter. From the ratio of the actual reading for each filter against the required reading, the required percentage reduction in transmission was found. These figures are given in Table 19. The appropriate 'Wratten' Neutral density filter was inserted with each colour filter combination to reduce the transmission by the required amount. Figures are given in Table 19, columns 3 and 4. The results were checked with the photocell figures are shown in Table 19, column 5, with the true energy shown in column 6. Final adjustment for equal energy could be made by altering the distance- of the intensity lamp from the filters.

Table 17. The sensitivity of the photocell to the filter combinations. Filter Wave-length of % sensitivity colour. maximum trans- of photocell. mission, mil. _ Blue. 490 88 Blue-green. 520 95 Yellow-green. 540 98 Yellow. 580 98 Orange. 610 85 Red. 660 25 -105-

Table 18. Transmission figures for the filters. Column. 1 2 3 4 5 6 7 8 Colour. Read- % Sens- Corr- Read- Corr- % Trans- Read- % Trans- ings itiv- ected ings ected miss- ings miss- with- ity of fig- with fig- ion by with 1 ion by out photo ure Cucl2 ures. Cuc12° cm,water Cuc12 cell. water. Red 4.40 25 17.60 0.34 1.36 7.7 4.30 97.7 Orange. 1.62 85 1.91 0.64 0.75 39.3 1.46 90.2 Yellow 1.22 98 1.24 0.55 0.56 45.0 1.10 90,2 Yellow- green. 3.40 98 3.47 1.80 1.88 54.2 3.25 95.6 Blue- green. 1.98 95 2.08 1.00 1.05 49.5 1.74 87.9 Blue. 5.25 88 5.97 4.20 4.78 94.0 5.1 97.1 -106—

Table 19. Readings required to give nearly equal energy transmission.

Column. 1 2 3 4 5 6 7 Colour Reading Required % Trans- N.D. Observed Correct- Relative with reading mission filter reading ed energy required Reading. level. Cucl2 required. with N.D. bath. filter. Red. 0.34 0.140 41.2 3 0.20 0.800 100.0 Orange. 0.64 0.477 74.5 1 0.46 0.541 67.7 Yellow. 0.55 0.550 100.0 none 0.57 0.581 72.6 Yellow- green. 1.80 0.550 30.6 4 0.55 0.561 70.1 Blue- green. 0,90 0.534 59.4 1 0.61 0.642 80.2 Blue. 4.20 0.494 11.8 8 0.47 0.535 66.9 -107- ii). A preliminary experiment to test the response of larvae to equal energy light sources

A series of filters transmitting almost equal energy light was now available, so it was possible to arrange an experiment to test the effect of these colours on the larvae. A small chamber was constructed. The sides were 3"xl" microscope slides, and the 'ends were 1," squAre pieces of glass cut from a slide: these were cemented on to a glass base. Matt black paper was glued round both sides and one end of the chamber, and to the other end was fixed a 1" square of translucent plastic sheet, resembling ground glass. This sheet formed a diffusion screen for the coloured light, and prevented the larvae from seeing the bright light source. For the experiments, the set-up was as shown in Fig. 10. The copper chloride filter was made by fixing together two 3" square pieces of glass with a U-shaped strip of warm Plasticine, and pressing them together until a separation of lcm. had been obtained. The bath was then filled with 5% copper chloride solution. The colour filters were put against the face of the bath nearest the chamber, and the chamber was put about 2" from the filters: this gap was left so that the photocell could be moved into the light beam to get readings of the light intensity. The high- intensity lamp could be moved backward or forward, so that the light could be adjusted as required. It projected a beam *" in diameter, and the floor of the chamber was at such a height that a semicircle of light fell on the end wall. White light was used as a 'neutral' energy reference, and was taken to give 100% response on the photocell. Yellow was taken as the colour of standard energy transmission because, as this filter had the lowest transmission, all the other colours had to be adjusted to it. Neutral FIGURE 10. THE SET -UP FOR THE EXPERIMENTS WITH NARROW WAVEBANDS OF

LIGHT. Light trap box round filters and lamp omitted.

Position of larvae, Lens barrel of 1.5- from end of intensity lamp. chamber.

C9 Bulb of ( lamp.

3 x x I test cell Plastic diffusion with matt black screen. Photocell. Filter. paper on walls and one end. Copper chloride solution.

U-shaped Plasticine seal . -109- density filters were put in front of the white light to get it approximately equal to the transmission through the yellow filter; final adjustment was made by moving the light source nearer to, or further from, the photocell. Successive over-all reductions in intensity were made by putting the necessary neutral density filters in front of the light source. Ten larvae were used for each test. They were placed 11" from the illuminated end of the chamber, on moist filter paper. A new piece of filter paper was used for each replicate test, of which 5 were done for each colour at each energy level. Observations were made at one minute intervals over a period of five minutes of the number of larvae moving towards, or away from, the illuminated end; the number reaching the end; and the number which remained stationary. The following tests were carried out for the experiment. The end of the chamber was illuminated in turn by colours of equal energy. Intensity. Foot candles. i). High intensity light. White reference. 0.81 ii). Light 50% level of i) . 11 11 0.425 iii). Light 8% u u if if ft 0.065 iv). Light 1.5% u IT it u 0.012 v). Light 0.3% .i 11 11 11 u 0.0026 vi). Light 0.03%" if 0 ” 0 0.00026 vii). Chamber in darkness.

These intensities cover a range in which the brightest light is over 3000 times as bright as the dimmest. Results are given in Table 20. The averages for three tests at each colour and intensity are given. Graph 5 shows the curves of the number of larvae reaching the -110- coloured end of the chamber plotted against the wave-length at different energy levels. The figures show that most larvae move towards the yellow and yellow-green regions, with a fall-off towards the red and blue regions. Because the different colours were offered at equal energy levels, these results could indicate a colour sensitivity curve for the response of the larval eye, rather than an attraction by certain colours. The larvae could then be reponding to the relative brightness of the colours on a grey scale, rather than to the . actual colour. This point was checked in later experiments. It is also interesting to see from Graph 5 that there is a double peak at low light intensities, with a low point in the yellow region. There are two possible explanations. One is that there is selective absorption of the yellow light by the tissues of the eye•. At low energy levels this would reduce the relative energy of the yellow seen by the larva this would indicate a larval response to relative brightness, but it is not clear if this would be for a grey scale or a true colour response. The other possible explanation is that the sensitivity curve of the eye is represented by one peak, and the other peak represents a colour attraction superimposed on the sensitivity curve. Thus the peak in the yellow-green region could be that for the sensitivity curve, and the peak in the orange region would then be that for the attractive region, or vice versa. This would indicate a true colour response in the larvae, rather than a grey scale response. At high light intensities, the effect is not noticeable. Similar double peak effects were noted by Bertholf (1932), and by Sander (1933), working with the honey-bee and Drosophila. They found a maximum effect in the yellow and blue regions, with a drop in the green region. Later experiments had therefore to confirm if the -111—

Table 204. The rospon6c of larvae to lights of equal energy, over a range of energy levels. Figures are the average for three sets of experiments. Colour. % toward colour. % reaching colour Reference energy level. Reference energy level. foot-candles. foot-candles. .81 .425 .065 .012 .00 .000 .81 .425 .065 .012 .00 .000 26 26 26 26 .----- 0....^....—..". r-...... r-.....^- "TgrEPTIP. ..„ ,..e..res.... White. 82 89 74 76 57 49 63 64 49 42 30 32 Red. 81 83 41 51 41 48 65 76 26 19 9 16 Orange. 96 95 88 70 37 52 88 79 59 42 13 26 Yellow. 94 97 90 70 62 42 88 79 73 32 42 15 Yellow- green. 97 90 91 70 74 62 83 76 76 52 46 39 Blue- green. 88 84 88 76 73 52 66 62 67 45 46 22 Blue. 88 80 77 64 57 48 63 52 44 23 20 18 Dark. 47 13

-112—

GRAPH 5. THE RESPONSE OF LARVAE TO MONOCHROMATIC LIGHTS OFFERED AT EQUAL ENERGY LEVELS.

Six series of experiments at different overall energy levels. Results from Table 20.

0/0 Reaching Relative Colour. Intensity. 1.c. 100 —

70 —

60 ---

50 / —x— x I 40

30 — /...,...\ ...*--... x — 0.065 x — ,.•./\ •..-.. 20 — 1 o— 0.012 \ ...."' — 0.00026 x-______I 0 — ------x — 0.0026 B. BG. YG. Y. 0. R. 1 t I I i I 0 I 1 1 I I • 450 500 550 600 650 700 Wavelength. mp. -113- response was due to: a). A colour sensitivity curve for the larval eye, b). A grey scale response, or a true colour response, to brightness in a), c). Colour vision and colour attraction, or to a combination of any or all of these factors. iii). The effect of illuminating_each end of the chamber with different colours of esual energy.

This experiment was to confirm the results obtained in the previous experiment. The set-up was similar to that shown in Fig. 10, but now there was an intensity lamp and copper chloride filter at each end of the chamber, and both ends of the chamber were fitted with diffusion screens. At one end the light was white, and of constant value, corresponding to the energy passing through the yellow filter at the highest intensity. Against this standard white other colours were compared at the other end of the chamber. These colours were: white, red, orange, yellow, yellow- green, blue-green, and blue. The larvae were used as described on page 109. Because the energy of this white light matched the energy of the colours, it was expected that, if the larvae had a flat linear response to colour, equal numbers would go to each end of the chamber for each colour tested. But if there was a colour sensitivity curve for the eye, then • for some colours on each side of the peak of the curve, more larvae would go to the white end than to the coloured end: only when the colours at the peak of the sensitivity curve were tested would the numbers of larvae be equal at each end of the chamber. If now there was definite colour vision, and associated colour attraction, more larvae would be -114- expected to go to certain colours than to the white light. The results are shown below in Table 21.

Table 21. The results of the experiments in which coloured lights were tested against a white light of equal energy. Figures are averages for 3 series of experiments.

Colour. % toward % toward % reaching % reaching white. colour. white. colour. White. 54 46 19 20 Red. 66 34 19 7 Orange. 62 38 21 14 Yellow. 52 48 21 21 Yellow- green. 41 59 17 34 Blue- green. 47 53 19 27 Blue. 60 40 18 12

From this table it can be seen that the red, orange, and blue regions were definitely not as attractive to the larvae as the white light. The yellow was no more attractive than the white, but more larvae moved to the yellow- green and blue-green than to the white. These results indicated the possible presence of a colour sensitivity curve in the eye, and also a definite colour attraction response induced by the yellow-green and blue-green lights this response must also indicate the presence of colour vision. iv). Colour comparison tests.

Because the colours had been tested individually, it was now desirable to test the colours one against the other, -115- to see if the order of attractiveness still held true. If this was so, then it would be a valid procedure to continue testing the colours singly in later experiments. A series of tests was made, in which each colour was tested against every other colour, as follows:

Test colour. Tested _ Test colour. Tested against. Red. White. Yellow. White. Orange. Yellow-green. Yellow. Blue-green. Yellow-green. Blue. Blue-green. Blue. Yellow-green. White. Blue-green. Orange. White. Blue. Yellow. Yellow-green. Blue-green. White. Blue-green. Blue. Blue.

The experiments were done at a relative energy level corresponding to 0.81 foot candles on the photometer scale. Results, are shown in Table 22 (overleaf). These figures showed the apparent effect of a colour sensitivity curve in the larval eye, and confirmed the results of the previous experiment. Most larvae moved to the yellow and yellow-green, and these colours were therefore at the peak of a sensitivity curve, if this existed. It can be seen that the colours showed the same relative attraction when tested one against the other, as when tested singly, so the method used previously, of testing colours singly, was a valid one. The very strong effect of the yellow-green points to a definite colour attraction. The experiments so far have shown that the response curve of the larvae may be due to a sensitivity curve, in which case the larvae could be responding to relative brightness. But the indication of an attraction to Table 12. The movement of larvae when two colours of equal energy are compared.

Figures are averages for three sets of experiments.

Test % towards % towards % reaching % reaching colour. reference test colour. reference test colour. colour. colour. Reference colour. Reference colour. Reference colour. Reference colour. R. 0. Y. YG. BG. B. R. 0. Y. YG. BG. B. R. 0. Y. YG. BG. B. R. 0. Y. YG. BG. B. White. 16 47 50 91 52 24 84 53 50 9 48 75 0 15 18 53 13 0 19 19 14 3 16 5 Grange. 12 - - - - - 88 - - - - - 7 - - - - - 75 - Yellow. 12 35 - - - - 88 65 - - - - 5 20 - - - - 72 37 - - - - Yellow- green. 7 21 35 - - - 93 79 65 - - - 3 9 21 - - - 70 53 39 - - - Blue- eI) green. 14 41 45 83 - - 86 59 55 17 - - 5 13 19 45 - - 52 35 21 8 - - cL

Blue. 15 32 44 93 63 - 85 68 56 7 37 - 2 7 12 56 22 - 50 28 14 1 10 - ei

Z `Z ( • -117- certain colours pre-supposes the presence of colour vision, and the response curve could arise from a differential response to wavelength, with relative brightness unimport- ant. The matter is dealt with fully in the Discussion. The following experiments were devised to try and confirm the presence of true colour vision, and also to show definitely the attractive effect of certain colours. v). An 'Energy Curve' experiment.

Instead of offering the larvae colours of equal energy level, in thes experiment the colours were presented with the blue and red regions at very high energy levels, and the yellow and yellow-green regions at very low energy levels. Intermediate colours were at intermediate levels. Each colour was compared against a standard white light at the other end of the chamber. From the curves in Graph 5 (derived from Table 20), it can be seen that the red light at relative energy levels of 0.81 and 0.425 gave a larval response as good as that obtained with yellow-green light at 0.055 'units. This was a factor of 6.4-12.5 times for the increased red energy level, over that for the yellow-green. For these experiments, therefore, increase of the energy level of the red light over that of the yellow-green light by this factor should give an almost equal larval response to the two ends of the chamber, assuming that the larvae respond to a scale of brightness. The presence of a true colour response would be indicated if the larvae still gave an unequal response to the colours. The same approach was applied to the blue light. At a relative energy level of 0.81 units, the larval response was similar to that obtained with a yellow-green light at a level of 0.065 units. This was a factor of 12.5 times for -118- the blue light. The energy levels of the red and blue regions were increased by at least these factors for the experiments. The use of a white reference light at the other end of the chamber, at an energy level above that of the yellow- green, but well below that of the red and blue, meant that if the larvae moved towards the white rather than the red or blue, the response was due t o a negative response to the colour. It could not be a response to brightness, because then the white light and the colour would appear equally bright, as any colour sensitivity curve of the eye had been compensated for by the increase in energy level of the red and blue regions. This argument applies equally to the other colours used. The yellow-green was only a third of the energy level of the white light, to compensate for the apparent sensitivity curve. It would therefore appear leas bright than the white on a brightness scale, so that any extra larval movement towards this colour would be due solely to a definite colour attraction. The results of the experiments are given in Table 23. From the above argument, it is clear that the figures indicate a larval response to wave-length, and not a response to relative brightness. The red was 16 times the energy level of the yellow-green, and the blue 12 times the energy, and yet the yellow-green attracted 34 times as many larvae as the red, and 4 times as many as the blue The figures in Table 23 are very similar to those shown in Table 21 (page 114), where the energy level was equal for each colour. Because of the similarity, the differential response of the larvae to different colours at equal energy levels could not be due just to a spectral sensitivity curve for the larval eye. Otherwise, with the red and blue at very much higher energy levels, more larvae would have moved to these colours. Both the brightness on -119- a grey scale, and as a colour, would have increased considerably. Clearly, the larvae were showing a colour selection, with a preference for certain colours, which acted as attractants. This effect could still be superimposed on a colour sensitivity response curve for the larval eye.

Table 23. Larval movement in the 'Energy curve' experiments. Figures are averages of three tests. White reference light = 0.65 foot candles.

Relative %toward % toward %reaching %reaching Colour. energy. white. colour. white. colour. Corrected. ___ White. 0.65 54 46 6 7 Red. 4.00 95 5 42 1 Orange. 1.31 64 36 22 16 Yellow. 0.56 45 55 17 29 Yellow- green. 0.25 39 61 18 34 Blue- green. 0.49 45 55 11 16 Blue. 3.19 69 37 6 9

vi). Checking the above conclusions that there is a colour response and colour preference shown by the larvae.

Confirmation was now required for the conclusion that the larvae were reponding to the colours. An experiment was carried out making use of the apparatus used for the earlier experiments, and described on pages 107 and 113. A third lamp was now incorporated, at 90° to the axis of the other two, and throwing a beam horizontally on to a silvered cover glass set at 45° in the beam of one of the other lights. This third lamp was to project the light of the colour filters, while the other two were for the white light only. -120-

The set-up is shown in Fig. 11. Initially, each end of the chamber was illuminated by a semi-circle of white light r in diameter, and of equal intensity at each end. The interposition of the mirror in one beam cast a shadow region in the middle of the semicircle of light on the end screen of the chamber. The area of this shadow region was measured, so that the area relative to that of the white part of the semi-circle was known. The side lamp had a bulb with a 'square coil' filament, so that the image on the diffusion screen was also square With the apparatus set up, the reference white light at the one end of the chamber was adjusted to a pre-determined energy level. The interrupted light source at the other end was then adjusted so that the energy level per unit area of the outer part of the semi-circle was equal to that of the reference light. The light was then switched off, and the side light was put on: the energy level of the coloured area on the screen was balanced to the same energy level per unit area as that of the reference light. Now, with both the white and coloured beams swiched on, the energy per unit area of the whole semi-circle was equal to that of the opposite reference semi-circle of white light. Bodies'which transmit equal amounts of energy per unit area appear equally bright, no matter what their size. The only effect that size could have would be to change the angle subtended by the body at the larval eye. This did have an effect, which is mentioned later. With the outer circle of light at the test end on, and also the reference light at the other end, movement of larvae should be equal to both ends, regardless of the dark area in the centre at the test end. Also, if the larvae were responding to the colours on a brightness scale, the number of larvae moving to each end would still be equal when the FIGURE I I PLAN VIEW OF THE SET-UP FOR THE EXPERIMENTS TO CONFIRM THE PRESENCE OF COLOUR VISION IN THE LARVAE .

Lamp providing coloured centre light at test end.

CuCl2 bath. I Colour filter. 141

eutral density filters. CuCl2 bath.

416

Neutral density filters.

CuCl2 bath. Lamp providing Lamp providing Chamber with Silvered cover- white light at white reference diffusion screen slip at 45°. test end. light. at each end. -122- coloured light was .on as well, because of the brightness of the outer area of white light. Only if the response was due to a true colour effect would any difference be expected in the response. The presence of a colour attraction would be shown if significantly more larvae moved to the test end than to the reference end, when both the outer white and inner coloured light regions were on. Fifty larvae were used for each test, and three tests were made for each colour. The results are given in Table 24, and these results are also plotted in Graph 6. The points which arise from these results are: i). With the centre of the test end dark, but with the peripheral circle of white light on, the results were no different from those obtained with a complete semi- circle of white light. This was expected, because the area subtended at the larval eye was the same for both ends, and the energy level/unit area was the same for both. ii). With the peripheral semi-circle of light off, but with the central area illuminated with white light, of the same energy/unit area as the reference end, more larvae moved to the reference end. This indicated that the angle subtended by the light area at the larval eye was important. iii). With the centre region coloured, and the peripheral region white, the number of larvae moving towards each end of the chamber was the same, except for the yellow, yellow-green, and blue-green regions, where more larvae moved towards these colours. The numbers actually reaching the colours showed a sharp increase over the range from orange to blue, with a peak response in the yellow-green region. This can be seen from Graph 6. These numbers were higher than -123-

those obtained when the central area was illuminated by white light. This could only mean that the larvae were responding to the colour, and not to bright ness. If the response had been one to brightness, due to a colour sensitivity response curve for the eye, most of the colours would have appeared less bright than the peripheral region of white light; equal numbers of larvae would have been expected at each end. It was confirmed that the larvae responded to the colours as such. iv). With the peripheral white light switched off, the number of larvae moving towards the colours was less than the number moving to the reference white, in the case of the red, orange, blue-green, and blue lights. Yellow, yellow-green, and, to a slight extent, blue- green, had more moving to the colour than to the reference white. This can be seen from Graph 6, where the curve for the colours crosses that for the white light at the points 1 2. It was concluded that, because the yellow, yellow- green, and blue-green attracted more than the white, despite the small angle subtended, these colours must be very attractive to the larvae. The results of all these colour experiments have shown three things i). That there may be a colour sensitivity curve for the larval eye. ii). That the response is to the colour itself, and not to brightness, which would be dependent on a colour sensitivity curve for the eye. iii). That the larvae are attracted to certain colours, because the response figures for certain colours were much higher than would be expected from a response to colour brightness.

-124- These results are discussed more fully later.

Table 24. The results for the experiment where coloured light was surrounded by a semi-circle of white light of equal energy per unit area. Figures are averages for three sets of experiments.

Test colour. % toward % toward %reaching %reaching white ref.test end reference test end. light. white. Centre white, Edge white. 46 54 10 13 Centre dark, Edge white. 49 51 8 10 Centre white, Edge dark. 70 30 31 7 Centre red, Edge white. 55 45 10 11 Centre red, Edge dark. 90 10 55 1 Centre orange, Edge white. 58 42 11 17 Centre orange, Edge dark. 76 24 40 9 Centre yellow, Edge white. 43 57 10 27 Centre yellow, Edge dark. 45 55 18 31 Centre yellow-greer, Edge white 28 72 4 34 Centre yellow-green, Edge dark. 39 61 12 32 Centre blue-green, Edge white. 41 59 6 23 Centre blue-green, Edge dark. 55 45 20 15 Centre blue, Edge white. 51 49 6 18 Centre blue, Edge dark. 91 9 37 2

---o0o---

-125-

GRAPH 6. THE RESULTS FROM TABLE 24, PLOTTED GRAPHICALLY.

0/0 Response.

6 0 —

1 5 0 — 1 1 1 1 \ 4 0 — \0 I 0 I \ \ Reaching reference \ f _white. Edge test 30 — \ / / end dark. \ /o \ / I 10- \ / Reaching colours. i o/ Edge test end i \\ / 20 — / \ P white. 0 // /A \- 4 2 \\\ / x Reaching reference / \ / white. Edge test 4,1 _ o \\ end white. I0 — (1------r ------. \ x / / \ .,,--;;— / Reaching colours. / .------. \ / Edge test end / V dark. x1 0 1 I I I I j R. 0. Y. YG. BG. B. W. Colour. -126-

V. THE FEEDING RESPONSE OF THE LARVAE.

The gustatory stimulant. So far, the olfactory and visual stimuli have been investigated. The nature, and optimum concentration, of the gustatory stimulus still had to be determined. The mustard oil glucoside, sinigrin (potassium myronate), was used at various concentrations in an agar gel, to test the effect of this substance on the feeding response. The compound had already been shown to be a feeding stimulant for lepidopterous larvae which feed on crucifers, in the work of Verschaeffelt and Thorsteinson.

A. The method of getting readings. A reliable method was required for getting readings of the amount of agar eaten in the tests, and preliminary tests were made to find such a method. The agar was offered first to the larvae as a thin layer coated on strips of black filter paper, after which a photographic reflex-copying method was to be used to get a print of the amount eaten. It was found, however, that the thin film of agar was too transparent to give an image suitable for reflex copying. A square cover glass, coated with a thin film of agar, was then tried. The larvae were allowed to feed for 24 hours on the agar. The coated cover glass was then put in a photographic enlarger, and the image was projected onto a sheet of cartridge paper to a size of 10" square. It was easy to see where the agar had been eaten away, and these areas were drawn round in pencil, after which they were filled in with black Indian ink. To get readings of the relative amounts of food eaten, the 10° squares of paper were cut into strips 1*" wide, and these strips were moved in 1" steps over a 1" square area -127-

of an Everett-Edgcumbe Auto-photometer. The light had previously been standardised to give a known transmission through a 1" square of clean white cartridge paper: each reading from the inked strip was subtracted from this standard reading to give a figure for the light cut off, in foot candles, by the inked portions. The readings so obtained, when aidded up for each 10" square, gave a measure of the relative amount of agar eaten. The readings could quite easily be converted back to give the actual area, in square millimetres, eaten on the cover slip, because 1 foot candle reduction in light transmission by the inked parts of the cartridge paper corresponded to an actual area of 0.1 sq. mm. on the cover glass. Care was taken to pick a paper of uniform thickness, so that the transmission of a standard area, before inking in, was the same for any sample piece. While readings were being taken of the results of the experiments, the transmission was repeatedly checked with a reference piece of white paper, because it was found that there was some variation in the mains voltage to the lamp. Slight changes in the voltage quite appreciably affected the light output of the lamp. The above method was very successful and accurate, although rather tedious. The cover glasses were coated by first pouring a standard amount of the molten agar on to a glass plate, with a standard area marked out with Plasticine. The quantity of agar was such that it gave a thickness of imm. over the standard area, which was sufficient for five replicates. i" squares were cut from the agar, using the edge of a i" square rover glass; the squares could then easily be slid onto the cover glasses. -128-

B). The effect of sinigrin on the feeding response of the larvae. a). Tests with different concentrations sinigrin.

This was the first set of experiments to test the effect of different concentrations of sinigrin on the amount eaten by the larvae. A series of sinigrin concentrations was made up in a 3% agar gel, containing mustard oil at a concentration of 10p.p.m. w/v. Control tests were made with 3% agar gel alone, 3% agar containing turnip leaf juice, and 3% agar containing mustard oil at 10p.p.m. The sinigrin concentrations were 5, 25, 50, 100, 250, 500, 1,000, 3,000, and 5,000 p.p.m. The turnip leaf juice was prepared by crushing thee'. leaves with a little distilled water, and then squeezing out the juice. The solid particles were filtered off under reduced pressure, and the clear juice was quick- frozen, and stored until required. Five replicate 2"xl" tubes were prepared for each concentration tested, and a cover glass, coated in the way described above (p. 127), was put in each tube, together with a small ball of moist filter paper. Ten second-instar larvae were put on the agar film in each tube. The tubes were put in the C.T. room at a temperature of 25°C., and 80% R.H., for 24 hours. The light in the room was on for 16 hours during this time. At the end of the test period, the larvae were removed from the tubes, and the amount eaten from each agar film was measured by the method descibed above (pp. 126-127). The results are given in Table 25. The readings have been converted from the 'foot-candle index' to a true figure for sq. mm. eaten. The results are not comparable -129- horizontally, across the columns, as different batches of larvae were used. Representative photographs of the amount eaten at each concentration are shown in Fig. 12. These results showed that with mustard oil present in a control test, there was no increase in the amount eaten over that eaten in the control test with agar and water alone present. This result was expected, as the mustard oil was shown earlier to be an orientation and biting stimulus, and not a feeding stimulant. As a general trend, the sinigrin caused a greater amount of feeding with increasing concentration. Signif- icantly more food was eaten than in the control tests, and the greatest increase came with sinigrin concentrations above 500p.p.m. The largest amounts eaten, obtained with sinigrin at 5,000p.p.m., approached the amounts eaten when turnip leaf-juice was used. As 5,000p.p.m. was the highest concentration of sinigrin tested, and this gave the highest figures for the amount eatenm it was desirable to test even higher concentrations. This was done in the next set of experiments. Below a concentration of 500p.p.m. sinigrin, the results were rather variable, and not very different one from the other. The rather different results obtained with the first series of experiments (see summary of stat- istical results, Table 26) were attributed to experimental error in getting readings for the small amounts eaten A behavioural response was noticed in these tests. Whereas the leaf juice agar, and the agar containing the lower concentrations of sinigrin, showed relatively large areas eaten out, at concentrations of 3,000p.p.m. and above very much smaller areas were eaten, although the total amount eaten was greater. The effect can be seen from the photographs in Fig. 12. An explanation for this effect was found in the next series of tests. -130-

Table 25. The amount of agar eaten with sinigrin present at various concentrations. Figures averages of 5 replicates.

Amount eaten, (sq. mm.). Test substance. Series?, 1. 11. 111. Control. Agar only. 2.6 2.1 12.5 Control„ Agar plus mustard oil, 10p.p.m. 2.0 2.3 10.2 Turnip juice in agar. 4.4 55.7 46.2 Sinigrin. 5p.p.m. 1.9 3.5 16.6 It 25 " 2.3 4.3 14.6 1 50 " 2.7 6.4 11.9 0 100 if 2.1 6.7 20.5 It 250 " 2.1 5.6 21.3 it 500 " 1.6 9.3 27.5 it 1000 It 2.4 22.5 21.3 II 3000 " 4.2 14.2 29.1 S' 5000 " 6.7 20.4 49.4

Table 26. Summary of the Analysis of Variance carried out on each of the above tests.

Significance. Items. Series 1. 11. 111. Between groups. S S S Within sinigrin. S S S Control vs. sinigrin. N.S ST ST Turnip vs. sinigrin. N.S S S Control vs. control plus mustard oil. N.S N.S N.S

H But note figures at 5,000 and 3,000 p.p.m. S Difference significant. N.S Difference not significant. First named lower than second. + First named higher than second.

-131- Figure 12. Representative photographs of amount of agar eaten with various concentrations of sinigrin present.

:4' ‘fiWire

s., 4 • 7. Water •.11›. Sinigrin • • . • control. tW• 4 j 1000ppM.

• • •

*AV ' • ••• IP • " " v , - • IWater r ' - !plus Sinigrin !mustard 5000ppm. toil.

• *: • 14'. r; .4

• . • • Turnip Sinigrin 41% Gr. .7, leaf 150 ppm. • juice.

Ai. - IT • • • • ft • • 14; ft ft i•!ft a ' 1 • Sinigrin • _ s For details of experiment 250ppm. see page 128. -132- b). Tests with agar containing different concentrations of sinigrin, and a colouring agent.

In the previous experiment, the replicates in some cases showed a rather wide variation. Despite the presence of the mustard oil as an attractant, it appeared that the larvae still wandered away from the agar, and did not get back to it. Generally, at the end of the experiment, only 2 to 4 larvae were still to be found on the agar in each tube. In the present series of experiments, higher concentrations of sinigrin were tried, and also a colouring agent was included. The effect of colour, a proved orientation stimulus, was tested to see if this would increase the amount eaten, or possibly make the results of each set of replicates less variable. A new range of sinigrin concentrations was made up, at 40,000, 20,000, 10,000, 5,000, and 1,000 p.p.m.. Mustard oil was incorporated at a concentration of 20p.p.m. w/v, and also agar to give a 5% gel. Each concentration was divided into two equal partsg to one lot was added a green dye, and to the other lot a quantity of distilled water equal to that of the green dye. Also prepared as 5% agar gels were water (as a control), water plus colour, water plus 10p.p.m. mustard oil, turnip leaf juice, and turnip leaf juice plus colour. The new agar concentration of 5% was adopted because the larvae did not seem to ingest the 3% gel so well. The detailed preparation of the test concentrations was carried out as follows. The sinigrin solutions were not made up until just before they were needed. This reduced hydrolysis at the higher concentrations. For convenience, the sinigrin was dissolved in distilled water already containing mustard oil -133- at 20p.p.m. 5c.c. lots were made up for each concentration. Two series of tubes were now prepared, each series containing the full range of concentrationsg each tube of a set contained 2c.c. of a solution, and 100mg. bacteriological agar. To each tube of one series of concentrations was added one drop of Boots 'Besyet' green food colourant. The average size of this drop was found to be 0.067c.c., so to each tube of the other series was added one drop of distilled water, of the same size. The dilution given by the addition of the drop of liquid to the 2c.c. already in the tube meant that the theoretical figures for the concentrations were reduced by 3.35%. The tubes were autoclaved for 10 minutes at 10 pounds pressure. The small quantity made up for each concentration was sufficient for five replicates. The cover glasses for these replicates were coated by the method used in the previous experiment. Coating of the cover glasses was quite straightforward up to a concentration of 1,000p.p.m. rin, but at 5,000p.p.m. the agar only just set, and at higher concentrations the agar remained as a very thick fluid. The gels had been made up several days earlier, and had gelled properly after autoclaving at that time. Since they were prepared, they were kept in a refrigerator at 5°C. It was after the second melting down, prior to coating the cover glasses, that the agar failed to set. After this, all gels were prepared just before they were required for use, and they were poured immediately on to the standard- area cooling plate, ready for cutting. It was realised that this failure of the agar to gel at high concentrations of sinigrin was probably the reason why, in the previous experiment, the larvae ate out large areas of agar at low sinigrin concentrations, but small -134— areas at concentrations above 5,000p.p.m.. The changed texture of the gel made it impossible for the larvae to nibble and ingest it satisfactorily. Five replicate tubes were used at each concentration. Ten second instar larvae were put on the film of agar in each tube. Drying of the film was prevented by putting a small ball of moist filter paper in each tube. The tubes were put in a C.T. room at a temperature of 26°C. and 80% R.H., and the larvae were allowed to feed for 24 hours. A light was on for 16 hours of this period. Results are shown in Table 27. Table 28 gives a summary of the results of the Analysis of Variance of each set of experiments. The general results of these experiments show that with sinigrin present in the agar, significantly more agar was eaten than in the control tests. The amount eaten with sinigrin present was not significantly different from the amount eaten when turnip leaf juice was present the exception, in the series 11 experiment, was attributed to the dilution of the attractants during the preparation of the leaf juice sample. Again, the control test with mustard oil present was not significantly different from the normal control, and this showed that the olfactory stimulant did not affect the feeding response. The small amount eaten in both these tests must have been a result of a hunger response. The comparison of the results for the green and the colourless tests showed no significant differences colour must therefore be an orientation stimulus, and not a feeding one. A test for the homogeneity of the variances was made between the results of the replicates of the green and the colourless tests, to see if the addition of the colour affected the variability amongst the replicates. There was no significant difference in the variability of the two sets -135-

Table 27. Results for experiments in which larvae were offered both colourless and coloured agar, containing sinigrin at various concentrations. Figures are averages for 5 replicates.

Colourless. Coloured.(Green). Test substance. Amount eaten.sq.mm. Amount eaten.sq.mm. I II III ----4.-•I --r-II III Water (control). 7.5 18.8 14.5 6.2 25.4 7.9 Mustard oil, 20p.p.m. 9.6 '17.9 17.0 4.9 7.2 14.8 Turnip leaf juice. 47.7 25.1 36.0 28.1 21.6 30.4 Sinigrin, 40.000ppm. 10.9 34.0 13.8 13.6 37.5 13.3 ii 20,000 " 22.6 37.9 28.1 34.4 37.6 28.7 II 10,000 ° 51.3 26.5 34.8 53.2 39.0 28.4 ii 5,000 " 23.0 24.0 30.4 49.1 53.1 35.7 if 1,000 " 16.0 31.4 19.6 16.6 33.7 32.8

Table 28. Summary of the Analysis of Variance of the results of the experiments on the effect of sinigrin and colour.

Significance. Items. I 'II III Between groups S S S Green vs. colourless. N.S N.S N.S Within sinigrin. S N.S S Turnip vs. sinigrin. N.S S- N.S Control vs. sinigrin. S- S- S- Control vs. mustard oil. N.S.N.S N.S Turnip vs. control. 8+ N.S S + Other total differences. N.S N.S N.'S

S . Significant difference. N.S = Not significant. + = First named higher value than second. - = First named lower value than second.

-136

Figure 13. Representative photographs of the amount eaten of agar containing various concentrations of sinigrin.

T---

• .-• - .7.:1 ‘•;':' . et. '',,,;,..:',' l'.1,4 ( •••• `4.. . , . 4,. ' 'i.• • . • . . . • - • . I - •: • ..1, '. w...< ..• .`e- ••/,.r.-= • • ',., N.3.. . • ,. : .. ,. '• - . 0_..,•,:7-•.:1?..... 4P" ItO,N.1,. Water •'•••••'. •• ,!..:- , -.7, ,.., , . . 10-•• ... Sinigrin •:I: . -,•,, ..1,..i• .-' •,, ;•-:.1' • .••.;,, 4...,id,... , control. ,-:••••• • ,, / to ... 20000ppm. ::.-..',.; - -e..::„v 410.',. .. . -V; • . ,;.. , • ,...... ;"sf'''•," ,.,..•,;•!..:::..ra„ve,."-- a i. ;, ... .e• ::: • ' •-• ..s, •-•E •‘. ::-!, .,,,s-..•••• ;59, . ,...../.... 1.. 4 f " - .. .44 \J."' r,' '.1.-.4. . ' ' • • - 1 ....

1:17.01 • . .... • w ..• ••• .. - e• ...... ,. •; ;. .• • • '..•••• : ,:...t c A. Water -i....- ••,..• ,, 0..., - - • , ••:, q.,- ti?J". .. plus . ,. . .,... .,,,, Sinigrin ' . • .t •lb.....• ....----! . , -3! mustard , . 40000ppm. .„ , ) - ....I. ,,.. p. .%'3,14' .;;; :. • 'i oil ' 911;1• -7:. -4-•"7-k.. c, ,. _ , r•. . -•:' • . •' ';'-• ... Vra .•'

. • :.:: ... "el ..... '444, nn : -.i. •e• C;'F',,...'' ::4., ''.,... `.'7.:5. : 4:-41, • 1- , i t.:. •• ii-,b,t,, : •.; •,;-, Sinigrin. ,.., , . . • , ,,,:. „;.••• S inigrin .." . alk;14,77e": ...',... ' . ., • . N .••• " ...'• • ••• V.e.1 • 1000ppm . i z „,,,.,,, 7, 1 40000ppm. 3'"•;.;.-...-. ,.! • -3 . :•••••:.,:,.'*g.":...„1:;-:, -,);. • -fiss, .. .} 1 • • . -4...... - , -1, a; * ;• ' '-'.'.' , • •.". N. .* ' . (repeat) •. .. .;. '•. . . • • .... - .4 ..' 4,•' t ' - , .7 ::f; • . 1... ,.• ' .•.' ',..'''..vTilr'.e* ' 1. 1:" . • i .."' .

''. i7". r`.. • ge, ...... ,-.:,...;....t.1 1., _ (1 •* . , ,,1"..., s.. -,:tv :Z.;•••.:.-...::.•-1;,•••1e4 lieNI.: • ? 4 1 :„ le i :: : . . .:,--. -0-...,if .1 ...! ...* ,,*0.".:':-.,1*11?Irf11-4v-,•2 ..:. ..r,..., . Ili' 4. " i, • • .d., ' 1 . 4..; • 4. • ..17 par FAN,: dr:Z‘,1: :: •••, .0.v. ?...... -.. ..31. Turnip •- :•t :iv ff..f.',..1.;;1.4.7.e.3.'F,•L'tre...... : Sinigrin ' -fery:'•7.3,4."..i.e.4,4•.:!...,..vi r .:,' .4-''.:4-!!!.-;.2...j.: leaf :-.46Atie:r• ".r. 'to ••••,',J ..-:: .4...,41-', 5000ppm. '•;••.i',.',...7•9k..e..z:I'' • ••:, ...... r. . .1 -"p','4. • • - V:: • .i. 4r, t.• . .t."..•,,, .-4 5,4 414 juice. ,.....: ...! )... -4 v.-4 1 • 4 4i,f -,.., ty .. •...... §, • .;:ia ":•.•,44-sA:-.-..,. ;7,A-4 iz•s t,A, :;...0. ' -;...... •A.,. 3 -T.)„,••••• ._.., .--"::;.... %;",iy:!. ,...... 4 . ....,.. , -...i 4 s 4' • i l'iii•it%:". '''' ; '..,i1.4. •,.., 4: . • ,:•$ ....,,.., 1, ft. p ?. ,,,o ,,,..,..• • .....- . i . " .•

'. Iti7;: 4...'1;1.rit--Pg.;'4%.%.;'-,•••• •

k*.4...,.*,:::!" i IL% ..'•.„Ti' ; t, A..1 ?,.4...:.t....- - .iii 4 ' Sinigrin •.• ./.. L,reft4A.--e.-44-- .-•-,..z! - ,... -. ,...-.•:,./q,i r 2,-...... For details of experiment 10000ppm. -lit , r,.,.aft...a.r. see page 1...,, 1;•-• • •-t. ....• P..... 132 ...... ,...... 1:...,,,i, 9 i:,::,,

•••••": I ! ....:;! % i ...2 •." ...t:14:, -137- of figures. The Analysis of Variance showed that there was significant variation within the sinigrin results. From the results in table 27, it can be seen that most agar was eaten when sinigrin was present at a concentration of 5,000 to 10,000p.p.m. The amount eaten increased with increasing sinigrin concentration, up to this optimum level, and then decreased with concentrations up to 40,000p.p.m. At these higher concentrations, very much smaller areas were eaten out at a time, and the agar seemed to become rather unpalatable (see photographs, fig. 13). This effect was- not caused by the change in gel texture which had been observed in the first experiment, but seemed to be a repellent effect by the high sinigrin content of the agar. A Tukey test was carried out on the results for the sinigrin tests, to find the least significant interval between the ranked means. The actual differences between the means were compared against this least significant figure, and it was found that the differences between the ranked means were greater than this figure. This meant that the amount of agar eaten increased or decreased by a significant figure for each increase in the sinigrin concentration. Although the larvae could be induced to eat an amount of agar equal to that when the agar contained turnip leaf- juice, the medium was clearly unsatisfactory. The larvae quite soon wandered off round the tube, although they probably returned to the agar for a time during the 24 hour feeding period. 1iVhen this period was completed, only 2 or 3 larvae remained on the agar. This might be a result of an unsatisfactory physical texture, or, more probably, the lack of an adequate leaf-like taste to supplement the stimulatory effect of the sinigrin. e). The optimum concentrations of mustard oil in agar containing an optimum concentration of 8,000p,p.m. sinigrin.

This test was carried out to see if the optimum concentration of mustard oil, determined in earlier tests, was still the most effective for use in a diet containing sinigrin, which itself gave off mustard oil by hydrolysis. Although the mustard oil was shown not to affect the feeding response, it might indirectly affect the amount eaten by enabling the larvae to find their way back to the food after they had wandered away from it. The 5% agar was made up with sinigrin solutions at a concentration of 8,000p.p.m., and containing mustard oil in concentrations of 5, 10, 25, 50, 100, and 200p.p.m.. The experimental method was the same as that used in the previous experiment. The solutions were again divided into two series, one of which was coloured green, so that the effect of colour could be tested again. The results are given in Table 29. Table 30 gives a summary of the results of the Analysis of Variance for each test. The results show that the addition to the food of mustard oil solution, over the range of concentrations 5- 200p.p.m., had no significant effect on the amount eaten. The control, containing sinigrin but not mustard oil, showed just as much eaten as in the tests with the mustard oil present. This confirms that the mustard oil acts as an orientation and biting stimulus, but plays no part in feeding. Clearly, the idea that an increased number of larvae might find their way back to the food, with the orientation stimulus present, was not valid.

-139-

Table 29. The effect of different concentrations of mustard oil in a diet of 5% agar, containing sinigrin at a concentration of 8,000p.p.m. Figures are averages for 5 replicateb.

Colourles-e. Coloured.(Green..-. Test substance. Amount eaten.sq.mm. Amount eaten.sq.mm. I II III I II III Agar + sinigrin, (control). 25.7 61.2 43.8 36.7 41.5 37.3 Turnip leaf juice. 16.4 18.1 21.6 14.0 36.1 27.3 Sinigrin plus Mustard oil, 5ppm. 33.7 44.0 41.8 29.6 55.3 45.3 ° 10 39.9 64.3 44.1 39.8 65.2 52.7 it " 25 0 30.1 61.5 43.7 40.1 46.6 29.9 0 " 50 " 35.3 45.0 57.4 33.0 63.0 29.9 II ° 100 " 35.0 47.0 47.1 46.8 45.6 46.6 1, " 250 " 26.3 49.7 33.4 36.4 38.8 44.0

Table 3 Summary of the Analysis of Variance on the mustard oil tests.

Significance. Items. I II III Between groups. S S S Green vs. colourless. N.S N.S N.S Control vs. mustard oil. N.S N.S N.S H - H - Turnip vs. mustard oil. S S HS Within mustard oil. N.S N.S N.S Other differences. N.S N.S N.S

S = Significant difference. N.S = Not significant. = First named lower value than second The significant value of these readings for turnip leaf-juice vs. mustard oil is attributed to the dilution of the attractants during the preparation of the juice.

-140-

Again,the addition of a green dye to the agar did not give a significant change in the amount eaten,. nor did it affect the variability of the figures for the replicateb this variability was checked by a test for the Homogeneity of Variances. The conclusions drawn from this experiment were that, for an artificial larval diet, the addition of sinigrin alone would be adequate, because the small amount of mustard oil produced by the hydrolysis of the sinigrin would be sufficient to elicit a biting response. It was felt, however, that it would be advisable to incorporate the mustard oil and colour in a preliminary artificial diet, because• these proved orientation stimuli might supplement other, as yet unknown, feeding stimuli found in the natural leaf. d). The effect of a ar concentration on the amount eaten by the larvae.

This experiment was carried out to find the optimum concentration of agar for incorporation in an artificial diet. It had already been found that 5/ agar gave a better result than the 3% concentration used in the first test, but the optimum was not known. Agar gels were used at concentrations of 1.5, 2, 3, 4 5, and 6%. Each concentration contained sinigrin at 5,000 p.p.m., and mustard oil at 25p.p.m. w/v. Each concentration was divided into two equal lots° to one lot was added a drop of green dye, and to the other lot was added a drop of distilled water, of the same volume. Five replicates were used at each concentration, and each replicate contained 10 second-instar larvae. Three series of experiments were done, using a different batch of larvae for each. -141-

The results are given in Table 31. Table 32 gives a summary of the Analysis of Variance for each test.

Table 31. The effect of agar concentration on the amount eaten by the larvae. Figures are averages for five replicates.

Colourless. Coloured.(Green). Test concentration. Amount eaten.sq.mm. Amount eaten.sq.mm. I II III I II III Agar 1.5% 2.28 3.10 9.42 3.63 3.52 11.95 2.0% 5.82 7.21 10.63 5.48 5.90 9.32 3.0% 16.32 10.51 14.97 10.51 4.78 14.97 4.0% 9.99 7.27 27.87 16.94 6.53 25.81 5.0% 26.44 12.24 32.78 25.87 11.47 50.19 6.0% 26.46 18.33 58.96 18.57 21.94 48.70

Table 32. Summary of the Analysis of Variance on the figures for the effect of agar concentration and of colour.

Significance. Items. I II III Between groups. b S S Green vs. colourless N.S N.S N.S Within agar. S S S Other differences. N.S N.S N.S

S = Significant difference. N.S = Not significant.

There was a significant increase in the amount of agar eaten with each increase in concentration. This increase can be seen from the photographs in Fig. 14. It was confirmed by the Tukey test, in which the difference between the ranked means was tested against the calculated least -142- significant interval between the means. An agar concentration of 5-6% seemed to be the most satisfactory. Higher concentrations were not tested because of the method of getting the agar films concentrations higher than 6% could not be poured evenly over the cooling plate.

C. The main factors in feeding revealed by the preceding work.

The work so far has shown that, for an artificial diet, orientation would be given by the mustard oil at a concentration of 20p.p.m. w/v, and by a suitable colour, in this case green. Because the larvae are usually put on the food, and do not have to search for it, the presence of the orientation stimuli is not essential - but the presence of these stimuli may be advisable in case they supplement unknown feeding stimuli. The biting response is initiated by the mustard oil, but the response may be elicited by hunger, in which case feeding will continue normally if sinigrin is present. Continued feeding is caused by the mustard oil glucoside, sinigrin, at an optimum concentration of 8,000p.p.m. The extent of this continued feeding may be modified by the texture of the food, which, in this case, is caused by the concentration of agar. A concentration of 5-6% is about the optimum level for second instar larvae. These results could now be applied to the formulation of an artificial diet for the larvae.

---o0o---

-143- Figure 14. Representative photographs of amounts eaten at different agar concentrations. n. 0 011C— . White. Green-coloured. I It .•-• • .:•-• . • • . . ? " --;:' •••;/, ••,: • ... .1.-..-

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:„kr••• ft^. ....4•• ,,z •1 ' •a:71 ." 0* 't.f '....-•'c'-'-' ,"`-01 .V.". • •• 4 Ps.' 1,.".,•'.• -,0"'•• ..- :'':•:,;, ,,p4f,.., ':;rko "t•-: • • 04.,1-' ';.1; ,..", ..,f • ••.: ie ••,A,I-,, ., '' 4•;:,:: f ",* kill, ••-' '-;.:•••• .'fi...t. or. -.— .e....46;• 1,....„,„ ,. , , ' ' :A l'''' lik.it'' ' "'0',,i':;••,',ii,.. ''„ '''''ICS,, *le 6.0% •;'',..iir...r.. YE i :.../ . '4."' ' ... `' .4,0:::i: V..,e;:,,, ..' .!....,...: :.,1- .7'.... .: 4:14. .S.Ij.:•42.,....;...ic.fi 1 ; 'n'41.. Jt.#4,144 '• . k....%::''.. ' .'1F.}:.;i:';%: cR..ilc.:.{,...,••• , • .4 . %.'i. • 1F-:.. - I- :••• : . ';'•. • '''z-4.,•1:: •P3'...o•it e'," . -144—

VI. THE PRELIMINARY FORMULATION OF A SYNTHETIC DIET FOR THE LARVA-T, OF PHAEDON COCHLEARIAE F.

The formulation of a synthetic diet for the larvae was now attempted, prior to working out the nutritional requirements of the insect. It was thought that the information gained about the various stimuli would be a help in getting the larvae to feed on a synthetic diet. Theoretically, the larvae should feed on a food mixture which contained the sources of the stimuli - especially those of the feeding stimulus. In practice, it was found that the larvae were put off by the taste of the rest of the ingredients in the mixture, despite the presence of the optimum concentration of the feeding stimulus. No satisfactory growth was obtained on the diets used, because of factors which will be described later. But, from the time for which a few larvae managed to survive on some of the diets, it is believed that a satisfactory diet could be formulated for the larvae of Phaedon. At the end of this work, certain modifications to the diet are suggested, which would probably improve its acceptability. Time alone prevented trials of the modifications from being made.

A. The formulation of the mixtures. a). The amino-acid mixture. Many earlier workers started by using casein in the diet. In this case, it was decided to start off immediately with an amino-acid mixture. Different authors give various proportions for the amino-acid mixture these proportions were determined mainly by trial and error. Although amino-acid balance has been shown to be of some importance with certain species, it -145- was decided that in this case all the amino-acids would be given at the same level in the preliminary diet. By doing this, the effects of physical factors, and of the proportions of the main classes of nutrients, could be investigated first. Later, chromatographic analysis of the larval tissue could be made. By comparing these chromatograms against similar ones from larvae grown on fresh turnip leaves, one could get some idea of necessary alterations to the proportions of the amino-acids in the mixture. This approach seemed better in that one was hardly likely to have a serious deficiency of any one amino-acid during the preliminary experiments, and any modifications required would come to light later in the chromatograms. The following amino-acids were mixed, in equal quantities, and the mixture was ground thoroughly for half an hour in a pestle and mortar.

DL-Alanine, L-Histidine, L-Proline,

L-Arginine, L-Hydroxyproline 9 L-Serine, L-Aspartic acid, L-Leucine, L-Threonine, T-Asparagine, L-Lysine, L-Tryptophane, L-Cystine, I-Methionine, L-Tvrosine, L-Glutamic acid, DI-Ornithine, L-Valine.

Glycine, L-Phenylalanine 9 b). The vitamin mixture,

The content of this mixture was based mainly on the formulations given by previous workers; but in some cases the proportions were altered to bring them closer to those found from plant analyses. Even then, the quantity of each vitamin was higher than the figure recorded from such analyses, to allow a safety margin. The weight of each vitamin per gram of dry diet is -146- given below. The actual mixture was made up in bulk, in these proportions, except for the 0(-tocopheryl acetate and choline chloride, which were added to the diet separately. The mixture was ground thoroughly for half an hour in a pestle and mortar.

The amount of each vitamin in the diet.

pg,/gm. pg./u. (Green Vitamin. dry diet. dry diet. Beet). Thiamine. 60 14 Riboflavin. 100 20 Nicotinic acid. 200 58 Calcium pantothenate. 70 14 Pyridoxin hydrochloride 60 3.6 Folic acid. 7 20 Biotin. 1 0.26 Carnitine 2 p-amino benzoic acid. 0.5mg. Inositol. 4.5mg. 0.2mg. 3ECholine chloride. 1.0mg. 3Ek-tocopheryl acetate. 1.0mg. 10.0mg. K - Added separately to the diet.

The mixture gave the above concentrations when used at the 0.1% level in the diet. (1% level dry weight.). c). The fatty acid mixture, and cholesterol.

The sterol, cholesterol, was included in this mixture. The bulk mixture was Cholesterol. 1.5gm. Linoleic acid. 2.5gm. Linolenic acid. 1.0gm. 5.0gm. -147-

The mixture was used at the 0.5% level in the diet. (5% dry weight.). d). The salt mixture.

The average figure for the ion content of six plants was taken (McCance and Widdowson - quoted by Fraenkel, 1951). A salt mixture was then devised to match these figures as closely as possible. This method, of course, had the disadvantage that it did not take into account the ion content of the other nutrients used in the diet. The ion balance is shown in the list below. Average ion Devised content for mixture. six plants. (mg./100gm. Ion. (mg./100gm.) diet). Na. 26 26 K. 284 252 P. 52 75 S. 64 52 Cl. 62 64 Ca. 82 73 Mg. 15 9.8 Mn. - 8.1 Fe. 1.3 4.0

The salts, and the quantities required to give the above proportions of ions in the mixture, are given below. The mixture was ground thoroughly in a pestle and mortar, and used at the 0.1% level in the diet. (1% dry diet.).

Salts in mixture. Weight.(g m. Sodium carbonate, Na2CO3 6.0 Ferrous sulphate, FeSO4.7H20 2.0 Calcium chloride, CaC12 10.0 Magnesium sulphate, MgSO4.71120 10.0 Manganese sulphate, MnSO4.H20 2.5 (Cont/d). -148-

Salts in mixture. Weight. (gm.). Potassium dihydrogen phosphate, KH2PO4 10.5 Potassium phosphate dibasic, K2 HP0 4 12.5 Potassium sulphate, K2SO4 17.5 Potassium carbonate, K2003 16.5 Calcium hydrogin phosphate, CaHPO4 12.5 100.0 e). The carbohydrate.

Finely powdered sucrose was used as the supply of carbohydrate, as most previous workers had shown that this was the best utilised of the sugars. f). The composition of the basic diet.

The proportions of the constituent groups in the basic diet were as follows gm. Amino-acid mixture. 3.400 Sucrose. 5.000 Vitamin mixture. 0.055 Choline chloride. 0.035 4-tocopheryl acetate. 0.010 Fatty acid mixture + cholesterol. 0.500 Salts. 1.000 10.000 dry weight. Agar. 5.000 Tween 80 0.140 Distilled water 100.000m1.

B. The effect of varying the proportions of the main constituent groups of the diet.

This experiment was tried to see if a major modificat- ion of the quantity of any particular constituent group -149— would give better growth than the basic diet. The basic diet given above was taken to contain unit quantities of each constituent group of nutrients, as followsg A. Amino-acids. 1 unit = 3.4 gni.%. B. Sucrose. 1 unit = 5.0 gm.%. C. Salts. 1 unit = 1.0 gm.%. D. Fatty acids + cholesterol. 1 unit = 0.5 gm.%. E. Vitamins. 1 unit = 0.1 gm.%.

The above groups were then mixed in different proportions, so that any one group of nutrients was offered at four times, twice, and one half the usual amoumt. Tests were also made in which the amount of all the ingredients were quadrupled, doubled, or halved. The tests are listed below. The five groups of nutrients are in the order A,B,C,D,E.

Test. Combination. Test. Combination. 1 11111 11 11411 11111 2 22222 12 11211 3 22222 13 llill 4 44444 14 11141 5 41111 15 11121 6 21111 16 11121 7 21111 '17 11114 8 14111 18 11112 9 12111 19 11112 10 lilll 20 Cabbage leaf. (Control).

The mixtures were made up as follows. 250 mg. bacteriological agar powder were weighed into a 3"xil" test-tube. This amount was to give a 5% gel with 5m1. of solution. All the quantities given previously -150- for the nutrients were adjusted proportionately for the preparation of only 5 ml. of solution. To each tube was added the correct amount of amino- acid mixture, fatty-acid plus cholesterol mixture, and salt mixture. The choline chloride, which was hygroscopic, and the ot-tocopheryl acetate, which was a viscous liquid, were added at this stage, instead of later in the vitamin mixture: the vitamin mixture was then kept as a dry powder. To each tube was added 5 ml. of sinigrin solution at a concentration of 8,000p.p.m., and containing 25p.mm. of mustard oil, and 1 drop of green 'Be6yet' food colourant. The Tween 80 (Folyoxyethylene sorbitan mono-oleate) was also dissolved in the solution,to give 7 mg./5 ml. The Tween 80, besides acting as a wetting agent, had a surface active molecule which aided the dispersal of the fatty acids in the mixture. The mixture now in the tubes was stirred well, after which the tubes were plugged with cotton wool. They were autoclaved for 10 minutes at a pressure of 101bs./sq.in., after which, when the tubes had cooled to 55°C., the required amount of sucrose and vitamin mixture was added to each tube. The mixture was thoroughly mixed with the aid of an agitator- stirrer, which was a piece of glass rod with a flattened end. The tubes were allowed to cool, after which the cotton wool plug was covered with a piece of plastic sheet to prevent the diet from drying out. The tubes were stored in a refrigerator at 5°C. until required. This relatively simple method of mixing gave a surprisingly homogeneous medium, although for later work it would probably be advisable to get all the components into solution first. For the first experiment, 5 replicate 2"xl" tubes were -151- used for each mixture. To keep the humidity high in these tubes, a ball of wet filter paper was pinned to the cork. Aseptic methods were not followed. A piece of diet about 2mm. thick and 5x4 mm. in area was put in each tube, and 10 newly hatched larvae were put on it. The tubes were put in large covered dishes containing I" of water° this water was an added precaution to keep the humidity high, so that the food would not dry out. The dishes were put in the C.T. room at 25°C., with a light- on for 16 hours in every 24 hours. The tubes were examined every 24 hours, and the number of dead larvae was noted. The food was changed every 48 hours. The results were poor. Within a few hours mould growth had started on the diets, and this made the surface slimy. The larvae asphyxiated in this slimy surface layer, and after the first 24 hours a number of larvae were dead in each tube. After this time, most of the larvae had wandered away from the food, which smelt quite objectionable from the growth of the mould. The food and the tubes were changed, but the mould growth very soon started again, and most of the larvae were dead after 48 hours° only a few survived until the third day. The control test, with the larvae feeding on cabbage leaf, showed very low mortality amongst the larvae, and normal growth. The figures for the tests are not given, because it is not certain how much these were affected by the mixtures used, and how much by the mould growth. Clearly, aseptic culture techniques were required. There were, in fact, two requirements for the next set of experiments° one was a fully aseptic culture method, and the other was for a suitable very small chamber in which, if the larvae wandered away from the food, they could very soon get back to it. -152-

C. The technique for the aseptic culturing. of the larvae. a). The sterile chamber. A conventional type of glass-fronted inoculation cabinet was used f/or making all transfers of food and larvae. The only opening permitting the entry of air into the cabinet was a sliding door at one side, through which materials were put into the chamber. All sterilisation and culture operations were carried out with the hands in a pair of elbow-length post-mortem gloves. The gloves were sealed to a rubber panel let into the frame of the cabinet just below the observation window this panel allowed much greater freedom of movement than a rigid one. The rear wall of the cabinet was a sheet of glass, which admitted light to the chamber2 no internal light was fitted. Bosman, (Univ. London Thesis, 1956), with this particular chamber, had found that spraying the interior with a solution of 'Dettol' gave adequately sterile conditions. For the present work, a solution of 'Ibcol' was used to spray the interior, and the floor was wiped over with absolute alcohol. A Petri dish containing potato-dextrose- agar was exposed for one hour in the chamber, and a similar dish was exposed for one hour to the laboratory air. The dishes were incubated for 5 days at 25°C. It was found that, although there was no bacterial growth on the agar exposed to the air in the chamber, the growth of fungal colonies was no less than on the plate exposed to the air in the laboratory. A Phillips 150 watt mercury vapour ultra-violet emitting bulf, type MBN/U, was fixed to the roof of the chamber. This lamp had a quartz glass colour filter, so that it transmitted ultra-violet light but not visible light, except for deep purple. The chamber was exposed to radiation for an hour every day, and also for an hour after any sterile glass-ware had been put in the chamber. The lamp -153- was not used when larvae were in the chamber, because of the danger of injury to them; or when diets were inside, because some of the vitamins would have been broken down by the ultra-violet light. Tests with potato-dextrose-agar in Petri dishes showed that after irradiation with U.V. light, the air in the chamber was quite sterile. Plates exposed for one hour in the chamber showed no colonies at all after 5 days incubat- ion at 2500., whereas plates exposed to the laboratory air were heavily infected. b). Sterilisation of glass-ware.

All glass-ware was wrapped in tissue paper and sterilised by autoclaving for 15 minutes at a pressure of 151bs./sq.in. After the sterilised items had been transferred to the sterile chamber, the interior of the chamber was irradiated with U.V. light for an hour. Pipettes, spatulas, forceps, and paint brushes were kept inside the chamber in covered dishes containing 90% alcohol. c). Surface sterilisation of the eggs.

A number of authors have described methods for the surface sterilisation of insect eggs: these methods are summarised below. Some authors have put forward very much more complicated treatments: for example, Sang (1956), with Drosophila, used de-chorionation of the eggs as a suitable technique. For the present work, a 0.1% solution of hexyl resorcinol in distilled water was used, followed by 70% alcohol. The hexyl resorcinol acted as a wetting agent and disinfect- ant; the wetting properties helped clean frass from the surface of the egg. After a rinse in distilled water, the eggs were immersed in 70% alcohol. Alcohol was used in preference to mercuric chloride because of its volatility:

-154- whereas the eggs had to be washed very thoroughly after immersion in mercuric chloride, only a rinse was needed after immersion in alcohol. Any residual alcohol carried over to the Petri dishes had evaporated by the time the larvae hatched, so these suffered no ill-effects.

Sterilising solutions. Sterilisation time. minutes). Author. a). Ethyl alcohol, 70%. 45 Hinton, (1951), Drosophila. b). Mercuric chloride, 0.1%. 4 Beck et al. (19507 Formalin, 2%, in Pyrausta. Sodium hydroxide, 2%. 10

c). Antiformin, 5%, in Begg and Sand, Formalin, 10%. 10 (1950), Mercuric chloride, 0.19 Drosophila. in hydrochloric acid, 0.5%. 20

d). Dilute soap solution. 5 Buddington, (1941), Hexyl resorcinol, 0.1%. 2 Culex.

The eggs were picked from the leaves on which they were laid and were put on to moist filter paper in a Petri dish. When a sufficient number had been obtained, they were all washed with distilled water into a 2"xl" specimen tube, after which the distilled water was pipetted off. The eggs were covered with the 0.1% solution of hexyl resorcinol, and were swirled around until all the eggs sank. The tube was now transferred to the sterile chamber, where the hexyl resorcinol was pipetted off the eggs. They were rinsed twice in sterile distilled water, and then covered with 70% alcohol. At the end of the allotted time, the alcohol was removed and the eggs were rinsed again in sterile distilled water, after which they were transferred to sterile -155— filter paper in sterilised Petri dishes. The eggs were incubated at 25°C. In the first trial, to test the efficacy of the treatment, the eggs were put in the hexyl resorcinol solution for 10 minutes and, after the rinses, in 70% alcohol for 45 minutes. This treatment was too drastic, and only 30% of the eggs hatched, compared with 80% for the unsterilised eggs. After some trials, the best times found were 2-3 minutes in the hexyl resorcinol, and 30 minutes in the alcohol. Immersion in the solutions for these times had no effect on the percentage hatch of the eggs° yet the times were adequate, because if newly hatched larvae from surface-sterilised eggs were allowed to crawl on a plate of potato-dextrose-agar, no growths appeared when the plate was incubated. d). The feeding chambers.

Very satisfactory chambers were made by drilling diameter holes to a depth of 3 16" in blocks of -I" thick 'Perspex'. These holes were grouped five to a block, so spaced that a i" diameter microscope cover slip could be placed over each. The blocks were kept in the chamber, in a covered dish containing 90% alcohol.

D. The preliminary experiment to test the growth of larvae on the dietsL with aseptic culture conditions.

A minimum number of 1200 larvae was obtained for these tests, from a batch of 1800 surface-sterilised eggs. A new series of diets was made up, identical with that described on pages 148-150, except that the agar concentration was reduced to 4%, and Nipagin M (methyl p- hydroxy benzoate) was added at a concentration of 0.2% to -156- inhibit possible mould growth. The agar concentration was reduced to 4% because newly hatched first instar larvae were used, instead of the second instar larvae which were used to find the optimum agar concentration. Each perspex block contained five holes; these were to give five replicates for each diet. The blocks were removed from the alcohol as required, and were dried with sterilised cotton wool. A diameter disc of filter paper was dropped in each hole; this paper was to absorb any moisture exuded from the food, as in the previous set of experiments a number of larvae had drowned in the liquid which had collected in the corners of the specimen tube. A small spatula was used to cut a piece of food of such size that when laid on the filter paper it just reached the top of the hole. Ten larvae were put on the food in each hole, and a 1" round microscope cover slip was dropped over each hole to form a small chamber. The slip was held down by a ring of Vaseline. At no point could a larva be more than 2mm. from the food. The larvae were transferred to the food with a camel- hair brush which had been sterilised by standing in 70% alcohol. It was necessary to rinse the brush thoroughly in distilled water, because any slight contamination of the food with alcohol made the larvae move away from it. The test blocks were stacked in a large covered dish containing a *" deep layer of water, and put in the C.T. room at 25°C. The light in the room was on for 16 hours in every 24 hours. The results were rather startling, because after 24 hours nearly all the larvae were dead° exceptions were on test diets 8 and 9, which had a higher concentration of sucrose present. All the larvae were dead after 48 hours. Mould could not be a contributory factor to this mortality, because the food was quite clean in every case, and smelt -157- quite normal. It was feared that the surface sterilisation of the eggs had destroyed essential gut symbionts. Another set of experiments was arranged, to check this possibility.

E. A comparison of the growth of larvae from surface sterilised eggs, and of larvae from untreated eggs.

This experiment was done to see if the rapid death of the larvae in the previous experiment was caused by the surface sterilisation of the eggs, either directly, or indirectly by the killing of gut symbionts on the egg surface. The basic diet alone was used for this experiment. A number of eggs, sufficient to give 100 larvae, was surface sterilised, then incubated at 25°C. Another lot of untreated eggs was incubated at the same time. Twenty small chambers were prepared, as before, each containing a disc of filter paper and a piece of the basic diet. Ten newly-hatched larvae from surface sterilised eggs were placed in each of 10 chambers, and 10 larvae from untreated eggs were put in each of the other 10. The blocks containing the chambers were stacked in a covered dish containing a shallow layer of water, and put in the C.T. room at 25°C. After 24 hours most of the larvae were dead in both sets of chambers, but about four in each hundred survived to the second day, after which all but one were dead. This one was from a surface sterilised egg, and it survived for a week, with a slight increase in size. The experiment gave several results of interest, and one observation gave the probable cause for the early death of the larvae. -158-

In the first place, none of the diets became mouldy in either of the two sets, although in one set the larvae were from eggs which were not surface sterilised. It was expected that these larvae would transfer mould spores from the egg-case to the diet. Possibly some spores were transferred, but the Nipagin M retarded their growth adequately. The result suggests, however, that for experiments where it is not important to account for the possible influence of gut symbionts, surface sterilisation may not be necessary. The main cause of contamination in the first experiment was the spores in the laboratory air: when transfer of the food and larvae was done in the sterile chamber, the contamination could not take place. Secondly, there was no difference between the results for the two sets of larvae. In both cases most were dead within 24 hours. The possibility that the larvae from surface sterilised eggs might have died quicker because of the loss of gut symbionts, or from a direct effect of the sterilising solutions, was eliminated. Of course, if growth had continued long enough, a long-term effect of the loss of symbionts might have become apparent, but at this stage the present deaths were caused by some other factor. The effect of surface sterilisation could be seen on the dead larvae: whereas these larvae retained their form and colour for several days in the sealed chambers, the dead larvae from untreated eggs became blackened and crumpled within a few hours. The third, and most important, observation was that the larvae had actually fed on the diet. The abdomen was distended, and the anal segments were elongated* examinat- ion showed that this was not a natural growth effect, but was caused by constipation. The agar, apparently, did not give enough roughage to allow the larvae to excrete it. From this it was concluded that the first thing -159- required was a satisfactory inert base for the diet, with sufficient roughage to enable the larvae to eliminate waste. This could be investigated using the basic diet until a base allowing optimum growth on the basal diet was found. Only then would it be worthwhile testing the effect of the different formulations. This was the last experiment which could be carried out, but suggestions for further work are given later.

---oOo--- -160-

VII. DISCUSSION.

The results obtained from the experiments are discussed under their respective headings, with the over-all result presented at the end.

A. The orientation of the larvae of Phaedon to the food. a). Odour.

It was clear from the preliminary experiments (p. 52) that the larvae were stimulated by an odour given off by the leaves of the natural food-plants, which are all members of the Cruciferae. It seemed probable that this odour was that of the mustard oil, allyl iso-thiocyanate, which Verschaeffelt (1910), and Thorsteinson (1953), had successfully used to bring about orientation to the source by larvae of Pieris and Plutella, which also feed on plants of the Cruciferae. The mustard oil is produced in the plant by the hydrolysis of the glucoside sinigrin (potassium myronate) by the enzyme rarrosin. Sinigrin is not the only glucoside found in the Cruciferae: sinalbin and gluco-cheirolin are two others, each of which hydrolyses to give a distinct mustard oil, but these were not tested in the present work. It should be pointed out here that all references to larval response to given concentrations of mustard oil refers to the concentrations in the solution at source, and not to the concentration in the air. The actual concentrations to which the larvae were responding must have been very much lower than those cited, but there was no method of determining concentrations in the air-stream. The accur- acy of methods for making such determinations in some complicated olfactometers is open to doubt. The main requirement here was to find the concentrations required at the source, either a leaf or a spot of mustard oil, to -161- bring about orientation to that source, rather than to determine the perceptive limits of the sensory cells. Various concentrations of the mustard oil, allyl iso- thiocyanate, in aqueous solution, stimulated the larvae when tested in a modified Varley and Edwards type olfactometer. The dilution of the odour by the air-stream in this olfactometer gave rather high figures for the attractive concentrations, and no repellent effect was noted because of this dilution. A closed chamber, in which the odour diffused out from the source, gave much lower figures for the attractive range. This range was from 0.5 to 250 p.p.m., but with no apparent optimum concentration within this range once the threshold value was passed. Concen- trations above 500p.p.m. were repellent when tested in a small closed chamber. Stimulated larvae showed a typical behaviour. They stopped when the odour reached them, and raised the anterior part of the body to wave the head from side to side, with the appendages extended. The head was then lowered and the larvae set off towards the odour source, stopping every so often to repeat the 'testing' procedure. There could be little doubt from the response and behaviour of the larvae that the mustard oil was the natural orientation stimulus. The behaviour changed if the odour built up to repellent concentrations of 500p.p.m. or more in this case the larvae either turned rapidly away, or else stopped, with the head down and the appendages retracted. That the repellent effect was not noted in the olfactometer was due entirely to the method of taking readings, (p. 74). The first movement of the larvae, whether into or out of the test air-stream, was recorded. Clearly, the dilution of the odour by the air-stream, and the need to draw off 'neutral' air from the test region immediately after the lid of the chamber had been sealed down, meant -162- that initially, relatively low concentrations of the mustard oil reached the larvae. Thereafter the concentration increased. It would have been better to have observed the time spent by the larvae in the test air-stream, and the way they moved. This was tried with a few larvae, and the response of one larva to the pure oil is shown in fig. 7. (p.80). The larva first moved into the test air-stream, but soon moved out again when the concentration built up to its full level, which was repellent. The simulation of the natural process of producing mustard oil, by mixing a solution of sinigrin with a solution of the enzyme myrosin, also attracted the larvae. The glucoside sinalbin, with myrosin, was also effective, but in this case the mustard oil produced was acrimyl iso- thiocyanate. (see Table 9, pp. 77-78). Evidently the mustard oils as a group are effective. Sinigrin, sinalbin, or myrosin alone had no effect on the larvae. At one stage (Table 9, pp. 77-78), it was observed that larvae seemed to be showing an increased response when the mustard oil was offered on crushed pea leaf than when it was offered alone. Pea leaf by itself was not attractive. This suggested that the odour of some substance in the leaf, though not attractive in itself, complemented the effect of the oil, but further experiments did not confirm this. (p. 82, and Table 10, p. 83). Some workers, such as Chin (1950), have claimed that orientation to odour must operate at very short distances, of not more than a few millimetres. But the experimental method, of holding bits of food near the larvae for a short time and observing the response, is open to criticism. No odour gradients are set up, because of the short time for which the food is offered, and under these circumstances the larvae would not detect any odour unless the food was very close. Admittedly, odour gradients are rarely set up -163- under natural conditions because of the turbulence of the air, but at least the source is stationary, and the odour is carried away from it over a period of time until it is too dilute for detection. One has only to consider the distance over which a field of slightly decayed cabbage can be detected by the human nose. Under natural conditions, with the lack of odour gradients to guide the insect, the medium-range effect is probably to excite the insect and elicit a searching response, until it is close enough for short-range orientation by an odour gradient. Some escaped adults of Phaedon were observed to come from a distance of 5-6ft. to the vicinity of a spot of spilt mustard oil. The larvae of Phaedon showed orientation over the maximum distance tested in the olfactometer and chamber tests, which was 4 inches. In the chamber the dispersal of the odour was by diffusion alone. In all these cases, unlike the tests carried out by Chin, the odour was tested over a period of 3-4 minutes. Two observations, incidental to the above work, were rather interesting. One was that batches of larvae differed in their behaviour to light on different days. One day a batch would be positively phototactic, and all move towards the light of a window, whereas the next day they would be negatively phototactic. For this reason the experiments were carried out in the darkroom, with a single diffused light immediately above the chamber. No definite explanation can be offered for this behaviour of the second instar larvae, but it seemed to occur when the larvae used were approaching the moult to the third instar. The other observation was that when the larvae were put into the olfactometer, the first move tended to be away from the side touched by the brush, despite the presence of the attractive odour. To counteract this, alternate pairs of larvae were transferred with the brush touching the opposite side -164- of the body. b). Colour.

Although the larvae would move towards the source of the odour, only about 20% of those moving nearer actually located the source, which was a solution of mustard oil soaked on white cotton wool. Clearly, something further was required for complete orientation: a visual response seemed to be the most likely, and this was confirmed experimentally. If the odour source was coloured, then up to 80% of the larvae moving towards the source were able to locate it, the actual percentage depending on the colour used. More detailed experiments were done with coloured paper cones (p. 86 et seq.), and it was clear from graphs 2 and 3A and 3B, that there was a variable response to colour, with the orange, yellow, and yellow-green attracting many more larvae than the blue or red regions. The orange gave a peak response. The colours attracted more larvae than greys or a white of equivalent, or higher, brightness. This response to colour could be caused in one of several ways° by the changed contrast of the source against its background; by a response to the brightness of the light reflected from the different coloured papers; or by a definite attraction by certain colours, which must pre- suppose the presence of colour vision. A response to changed contrast seems unlikely, because the response was not related to the brightness of the different colours, which was determined photographically (p. 89). A response to brightness could be on a grey scale, the insects being colour-blind, or to colour brightness, the insects then having colour vision. In either case, the variable response would be brought about by a colour -165- sensitivity curve modifying the apparent brightness of the colours. The presence of such a curve might explain why orange, which reflected less light than the yellow, attracted more larvae. If brightness was actually causing the attraction, the maximum response to a colour at the peak of the sensitivity curve, assuming 100% sensitivity at that point, would be equal to that obtained with a grey reflecting white light of equal brightness. This follows because the quantum of energy is expressed as the frequency of a wave-length multiplied by Planck's Constant, so that whether the physiological effect in the eye is to give a grey image or a coloured image, wave-length is still important, and the brightness of the image is the same as that given by white light of equal brightness reflected from a grey cone. But it can be seen from graph 2 that more larvae went to the colours than to the greys or white of equivalent or higher brightness, so wave-length must be more important than brightness for the orientation response. The response of the larvae must therefore have been caused by colour vision, with an associated attraction to certain colours, especially orange. The response could probably be modified by a colour sensitivity curve. If this increased response to colours over that for an equally bright grey had not been obtained, it might have been argued that the curve in graph 2 represented a colour sensitivity response curve alone. These preliminary experiments showed, therefore, that the response of the larvae to colours must be due to colour vision and attraction as a function of wave-length, and not to brightness as a function of wave-length and intensity. The results gained from the use of coloured papers, however, must be treated with reserve, because very often these papers reflect wave-lengths other than those which give the physiological impression of colour in the human eye. -166-

The insect sees, for example, light well into the ultraviolet, and papers which reflect these wave-lengths, although appearing perhaps green to the human eye, may appear 'ultraviolet', or an intermediate colour, because of these wave-lengths. The precise wave-lengths, and the brightness, to which the larvae are responding thus remains obscure. The ideal is to use monochromatic lights of equal energy, under controlled conditions, to test the larvae. As monochromatic light sources are not easy to obtain, colour filters which transmit narrow wave-bands of the spectrum can be used as a substitute. In these experiments with Phaedon larvae, Kodak 'Wratten' filter combinations were used under controlled conditions to check and confirm the results obtained from the use of coloured paper cones. Precautions were taken to cut out the infra-red rays, which some of the filters transmitted to a considerable extent, and also ultraviolet rays, of which there was only slight transmission by a few filters. Previous workers do not seem always to have taken these factors into account with their filters, so that the actual energy of a particular wave-length being tested could have been much lower than they thought. Full details of transmissions outside the normal colour range are not normally published, and their importance in this work was not realised until it was found that the very high energy readings for some colours, obtained with a thermopile, could not be replicated with a photo-electric cell. High readings were not obtained with the thermopile if a bath of 5% copper chloride solution was placed between the light source and the filters. Curves supplied by Kodak Ltd. gave the extent of the transmission. The first experiment, with the colours presented singly at equal energy (P. 107 et seq.), was to see if the over-all result with coloured lights was the same as that -167- obtained with coloured papers. A definite curve of larval response was obtained (graph 5, p. 112). This proved that the larvae had a differential response to colours, with a peak response in the orange, - yellow - yellow-green regions, and the maximum at yellow or yellow-green. These colours all gave a higher response than white, but the response to red, blue-green, and blue was only as good as, or inferior to, that obtained with white. For the reason expressed on page 165, response to colour brightness of the peak colour should not be greater than the response to a white of equal energy, it therefore follows that the response to orange, yellow, and yellow- green recorded in this experiment (Table 20) must be true colour responses to wave-length, because in these cases the response to the colours exceeded the response to the white. The colours where the response was no better than, or inferior to, that with the white light, could arise from the action of a colour sensitivity curve, or from a lack of attraction to these colours. It was interesting also from this first set of experiments to see that lowering of the overall intensity caused a drop in the number of larvae reaching the individual colours. This does not necessarily mean that brightness itself was the cause, because all the colours were affected to the same extent: the effect probably arose because fewer larvae were in a physiological state able to detect the light at each decreased energy level. Larvae that at one time did not seem to perceive the dim lights did so at other times. From graph 5 it is also interesting to note that as the over-all relative intensities were lowered, a slight shifting in the peak response to shorter, bluer wave-lengths was noted. Thus, at a relative intensity of 0.81 and 0.425 foot candles the peak was in the orange and yellow -168- region (570-640W, while at intensities of 0.065 foot candles and below the main peak was in the yellow-green region (530-560mp). At even lower intensities, a double response peak occurred, one to orange light (600-640mg), and the other to yellow-green light (530-560m0, with a dip in the yellow region (570-590m11). The meaning of these results is not clear. Weiss et al. (1941,1942) found that in adult insects the lowering of the introductory intensity of monochromatic lights caused a shift in the response towards the blue region (see p. 22), which seems to parallel the effect noted here. Fingerman (1952), and Fingerman and Brown (1952), showed that the compound eye of Drosophila showed a 'Purkinje shift', in which, as the intensity of the monochromatic light stimulus decreased, there was a gradual shift in the over-all sensitivity of the eye towards the shorter wave-lengths. This showed the presence of two receptive mechanisms, one operating in bright light, and the other in dim light (see literature, p. 23). How- ever, it would not be reasonable to apply these conclusions to the effect noted with the simple ocellus of the larvae of Phaedon, without further work. The double peak effect is interesting, but again the cause is not known. Bertholf (1932), and Sanders (1933), noted similar double peaks with the honey-bee and Drosophila. The low point found in the yellow region with Phaedon larvae may represent a low absorption of yellow by the pigment of the eye, so that the energy perceived fell below a threshold level, in which case the eye would possess a sensitivity curve; or the yellow may be selectively absorbed by the tissues of the ocellus, before it reaches the pigment, in which case the energy level would be reduced below that required for threshold stimulation. Another possible explanation is that a sensitivity curve for the ocellus is represented by one peak, and the other peak represents a colour attraction by that colour, superimposed on the -169- sensitivity curve. Such an effect would be expected to become apparent only at low light intensities. A colour response curve to light of equal energy had now been shown, and it seemed that this response must be a true response to wave-length. A sensitivity curve for colour might be operating, but the presence of such a curve was not proved. The second experiment was carried out to check and confirm these results. In this second experiment (p. 113 et seq.), a white reference light was presented at one end of the chamber, at an intensity equal to that of the colours presented at the other end. If now the larvae were responding to colour brightness, numbers would be more or less equal at each end of the chamber when the peak colour was presented against the white, because both must appear of eaual brightness if presented at equal energy. This is discussed more fully on page 165. For colours on each side of the peak, fewer larvae would go to the coloured end of the chamber than to the white, because the white would appear brighter. The results of the experiment are given in Table 219 p. 114.. It can be seen that the yellow-green and blue-green lights attracted more larvae than the reference white, so that there must be a definite colour attraction acting, and not a response to brightness. The other colours on each side of the attractive wave-lengths were of equal, or of less, attractiveness than the reference white. This could indicate a lack of response to the wave-length of these colours, although the larvae could see them as well as the other colours; or, on the other hand, it could represent the action of a colour sensitivity curve, with an attractive effect superimposed for the peak yellow-green and blue-green wave-lengths. Colour vision and attraction to certain colours must therefore be important mechanisms in the orientation of the larvae to the food. The presence of a sensitivity curve remained uncertain. -170-

It seemed desirable to compare the colours one against the other. It is not really satisfactory to offer the colours in turn to the larvae and then conclude that the difference in reaponse to each colour is real. But if a permutation is used in which each colour is tested against every other colour, then these results are reliable, and can check those obtained when the colours are tested singly. If the two methods gave results which tallied, then the first method, of using lights singly, could be used as a reliable method. The colours were compared one against the other in the third experiment, the results of which are given in Table 22, page 116. The same relative order of response to the lights was found, with yellow-green and blue-green at the peak, and a fall-off in response at each side; testing the colours singly was therefore a valid procedure. It was found that by this method the blue was more attractive than red or orange, a result that was not shown when the colours were tested singly. Although the work so far had indicated that the larvae had true colour vision, with an associated attraction to certain colours, this had to be proved. Proof was obtained by two methods, one method using varying light energy for the colours, and the other using constant energy levels. The two completely different methods gave similar results, and gave complete confirmation. The first experiment, using an 'energy curve' for the colours, used the principle of increasing the energy output of the colours which elicited the least larval response, and decreasing the energy of the most stimulating colours (page 117 et seq.). Properly carried out, such a curve should compensate for any colour sensitivity curve of the eye, so that if the larvae were responding to brightness alone the response should be the same to each colour. If, however, the larval response was to wave-length, then most larvae should still be attracted to the dim colours which had -171- previously been at the peak of the response curve° a low response would still be elicited by the colours now presented at very high energy levels. The use of the reference white at the end of the chamber opposite the colours was to confirm the colour attraction effect. The energy level of the white light was above that of the yellow-green light, but well below that of the red and blue. Besides an unequal response to the colours indicating colour vision, if more larvae went to the dim colours than to the brighter reference white, these colours must be exerting a definite colour attraction for the larvae. Similarly, a lack of response to the very bright red and blue regions, with a higher response to the white, would indicate that these wave-lengths were not attractive to the larvae, and brightness was not important. The results of the experiment are given in Table 23, p. 119. Clearly, from the above argument, these figures indicate a larval response to wave-length, because despite the energy curve more larvae went to the yellow and yellow- green than to the other colours, and there was a graded fall- off in response to colours on each side. In fact the curve, and even the actual figures obtained, are very close to - those presented in Table 21, page 114, for the light at equal energy levels. The larvae therefore have colour vision. The larvae are also definitely attracted by yellow, yellow-green, and blue-green light, because with these more larvae went to the colour than to the reference white light. The evidence here for a colour sensitivity curve is slight, because although the figures for the numbers reaching the different colours show a definite response curve, this seems to be caused by the larval preference for certain colours. Also, with a colour sensitivity curve, the percentage reaching red should be about the same as -172- that reaching white, because of the energy corrections which had been applied; but in fact 1 reached the red and 42 reached the white. This indicates that there is a negative response by the larvae to the red light, and this conclusion must apply to the earlier experiments where it was concluded that a sensitivity curve might be important. An entirely different approach was used to confirm the presence of colour vision in these larvae. The experiment is described on page 119 et seq. Each end of the chamber was illuminated by a w3 semi- circle of white light, of equal energy per unit area. A small mirror in one beam cast a shadow in the middle of the semi-circle at one end. This mirror was used to reflect the coloured light onto the end wall of the chamber, within the encompassing arc of white light. The coloured light was of equal energy per unit area as the white light, so that the semi-circles at each end presented exactly the same area, and energy per unit area, to the larvae. Now surfaces which transmit equal amounts of energy per unit area appear equally bright, no matter what their size. The only effect that size could have would be to change the angle subtended by the light at the larval eye. Because the semi-circles of white light were the same size, equal numbers of larvae should go to each end, regardless of the dark region at the middle of one white semi-circle. Edge effects between this dark region and the white had no effect on the equality of response to each end, nor did a range of energy levels of white light which were presented instead of the dark area within the outer white semi-circle. If now the coloured lights were presented in turn within the white semi-circle, the response should still be equal to each end of the chamber if they were responding to bright-- ness alone. But if the response was due to colour vision, then varying numbers would go to each end, depending on the colour. -173-

The results are given in Table 24, p. 124. It can be seen that varying numbers of larvae go to the end with the colour, the response depending on the particular colour. It follows from the argument above that the larvae have colour vision. Red had no effect on the larvae, but more went to the end where white light surrounded orange, yellow, yellow- green, blue-green, and blue, than to the opposite plain white. The peak response was to the yellow-green. The numbers reaching the colours were higher than those reaching the central test region when this was illuminated with white light of equal energy per unit area: this response was therefore caused by attraction to the colours. The evidence for a sensitivity curve is obscured by this colour attraction. With the peripheral white light switched off, but with the central white light on, more larvae moved to the reference end than to this white, although both regions were of the same energy output per unit area. This meant that the angle subtended at the larval eye was important. When this central region alone was used to test the response to the colours, it was found that only the yellow and yellow-green attracted many more larvae than the reference white at the opposite end, and the blue-green attracted almost as many, despite the small area presented to the larvae compared with that of the reference white. These colours must show a strong attraction for the larvae. The larvae were not affected by red light, and orange and blue were not very stimulating wave-lengths. It may be concluded, therefore, i). That the larvae have colour vision, and respond to wave-length. ii). That the yellow, yellow-green, and blue-green -174-

wave-lengths attract the larvae, the yellow-green very strongly so. iii). That the presence of a colour sensitivity curve, inferred from the results of earlier experiments, cannot be proved because of the attraction by certain colours. A differential response to wave- length is probably more important than the brightness changes effected by a colour sensitivity curve.

B. The biting response.

In the preliminary tests (p. 53), an experiment was carried out to see if a common odour of mustard oil would cause biting on normally unacceptable leaves. No nibbles could be found on these unacceptable leaves which might indicate that the olfactory stimulus also elicited the biting response. In work with the olfactory response to the mustard oil, the biting response was observed for the larvae which reached the cones (Table 13, p. 93 ). These results show that in the absence of the mustard oil, but with colour present, no biting took place. But with mustard oil present at a concentration of 10p.p.m., biting occurred, although this was for relatively short periods, and feeding did not continue. After a while, although the stimulus was still present, the larvae wandered away from the cones. The mustard oil, which had provided the olfactory stimulus for orientation, was therefore eliciting a biting response. This response was not an olfactory one, however, as the larvae under these conditions should have advanced with their mandibles biting, whereas in fact the biting response was not elicited until the larvae reached the mustard oil source. Contact chemo-receptors must be respipsible for biting. -175-

C. Continued feeding. a). Taste.

Once it has orientated to the food, and taken a bite, something further is required by the larva to induce continued feeding. This was clear from the results of the leaf juice brushing experiment described on page 56 et seq. From the work of Thorsteinson and Verschaeffelt with larvae which fed on Cruciferae, and because the larvae here responded to mustard oil, it seemed most likely that the glucoside sinigrin (potassium myronate) was the stimulus for continued feeding, and this proved to be so. Concentrations of from 5 to 40,000p.p.m. sinigrin were tested in a 5% agar gel. Concentrations of 5,000 and 10,000p.p.m. gave the maximum amount of feeding, as can be seen from Tables 25 and 27, and the photographs in figs. 12 and 13. Thorsteinson (1953), with Plutella maculipennis, found that sinigrin at 2,000p.p.m., the maximum level tested, gave the highest frass count. Probably Thorstein- son was still not using the optimum concentration, because the figures for Phaedon larvae are 3 - 4 times higher. It is interesting that for his sinigrin preparation Thorstein- son gave a threshold concentration of about 2p.p.m.: in these experiments with Phaedon larvae, a response was obtained to the lowest concentration tested, which was 5ppm. The higher concentrations of sinigrin were not satisfactory: although a large amount was eaten, almost as much in some casedatA the optimum level, this was made up of many smaller nibbles. This can be seen from the photographs in fig. 13, page 136, for concentrations of 20,000 and 40,000p.p.m. This result was probably caused by a repellent effect of the higher concentrations. In early experiments the effect at high concentrations arose because the gels were prepared in advance, and melted down as required: at this second melting, the gel did not form -176- properly, and the texture of the gel was altered. In some cases the gel solated completely. Because of the requirements in an artificial diet, the effect of the inclusion of the mustard oil in the agar was noted. From tables 25 and 27, and figs. 12 and 13, it can be seen that there was no difference between the results when water alone was present, and when mustard oil was included. This was attributed to the fact that the larvae were put on the food from the start, and so did not require an orientation stimulus. That the larvae should eat as much with water present as with mustard oil rather defeated the idea of a biting stimulus. Further experiments with mustard oil were done, because Thorsteinson (1953) stated that in some experiments Plutella maculipennis larvae produced more frass on a diet when mustard oil was also present. He suggested that such an olfactory stimulus might initiate feeding more promptly. Tests were made with Phaedon larvae, using a range of mustard oil concentrations in an agar gel containing 8,000p.p.m. sinigrin, to see if the presence of the oil improved results, either directly by initiating rapid biting, or indirectly by attracting larvae back to the food after they had wandered away from it. The tests are described on page 138 et seq. The range of concentrations of mustard oil used, 5 to 200p.p.m., did not significantly increase the amount of agar eaten over that eaten with sinigrin alone. (Table 29, p. 139). Possibly the high concentration of sinigrin supplied sufficient mustard oil by hydrolysis to meet the threshold requirements of the sensory cells. The conclusion was that mustard oil was not necessary for biting, because the biting response arises after a time from hunger. And it can be seen from the results in tables 25 and 27 that the hunger response with water alone present gave just as much eaten as when the mustard oil was present. If sinigrin is present, then feeding continues normally, and much larger -177-

9moullts are eaten, but these are not increased if mustard oil is added. During these feeding experiments, two similar series of tests were made, one series colourless and the other coloured green, to see if the addition of colour to the diet would increase the amount eaten. Results can be seen in Tables 27, 29, and 31. Comparison of the results for green coloured agar against the corresponding ones with colourless agar showed no significant differences: colour is therefore an orientation stimulus, with no influence on feeding. This again must have been because the larvae were put on the agar at the beginning. The colour was originally tested with the idea that it might enable some of the larvae which wandered away from the food to find their way back again, and so indirectly increase the amount eaten. Anything that might improve feeding on an artificial diet must be investigated and included in this diet, to enable the larvae to feed successfully and continuously. Although the larvae could be induced to eat an amount of agar gel equal to that eaten when the agar contained turnip leaf-juice, the medium was unsatisfactory. The larvae soon wandered off round the tube, although some must have returned during the 24 hour feeding period, but at the end of this time only two or three larvae remained on the agar. This might be the result of a lack of an adequate leaf-like taste to supplement the stimulatory effect of the sinigrin; but then, it occurred also when leaf tissue was used. More probably the agar had an unsatisfactory physical texture, and evidence that this was so is presented in the discussion about the nutritional work. b). Physical factors.

The work with the Williams type penetrometer showed that the leaf toughness was an important factor for feeding -178- by the larvae on leaf tissues (page 59 et seq.). The res'- ults are clearly expressed in Graph 1, p. 66. Relatively large amounts were eaten of less tough leaves, but at a point about toughness 4 the slope of the graph changes sharply, and a smaller amount was eaten at increasing toughness levels, the difference being slight over a range from T4 to T12. Probably at this point, T4, the mandibular power of the first instar larvae was nearly matched by the elasticity and plasticity of the leaf tissues. This would slow down the rate at which the larvae could feed. At the same time, as can be seen from Table 6, p. 64, the number of nibbles taken to eat a given area of leaf increased threefold, again substantiating the view that toughness was an important factor. The growth of the larvae was also slower on the tougher leaves, and the adults which emerged were lighter in weight. (Table 8, p. 67). With this evidence, it was clear that one of the most important things in the formulation of an artificial diet for the larvae of Phaedon must be the production of a suitable inert base which would give a suitable physical texture to the diet. As a standard, against which other bases could be assessed, the optimum concentration of an agar gel was determined for second instar larvae. The experiment is described on page 140 et seq. Results are given in Table 31. With the range of agar concentrations used, 1.5 to 6%, the maximum amount eaten was with 5 - 6% agar. Representative photographs of the results are shown in fig. 14. 5% was chosen as the standard concentration, because 6% proved too difficult to flow into an even film.

D. The main factors in orientation and feeding..

The larvae of Phaedon cochleariae F. show chemotactic -179- responses in their choice of host plant identical with those found by Verschaeffelt (1910) and Thorsteinson (1953), with other species which feed on Cruciferae. The mustard oil, allyl iso-thiocyanate, brings about orientation to the food, together with a colour attraction by yellow, yellow-green, and, to a lesser extent, blue-green colours. When the insect reaches the food, a biting response is released by the contact stimulus of the mustard oil, rather than by the olfactory stimulus. In the absence of the mustard oil, a delayed biting response takes place because of hunger, and if the necessary gustatory stimulus, the glucoside sinigrin, is present, feeding continues normally. The feeding response may, however, be modified by physical factors such as toughness, which may be an inherent property of the leaf or one arising from wilting, which increases the plasticity of the tissues.

E. The importance of the orientation and feeding stimuli in Nature.

Although the adults of Phaedon were not tested fully, a few experiments showed that their responses were similar to those of the larvae to chemical stimuli. Colour vision was not tested in the adults, but is most certainly present if one takes the work of Schlegtendal (1934), with adult Chrysomela, as a criterion. Phaedon adults rarely fly. Only twice during the rearing of many thousand beetles was a beetle observed to spread the elytra, and even then flight did not take place. Probably long range dispersion is by a very few beetles of a colony which fly off at random. If they come within a few yards of a plant emitting an odour of mustard oil, they are probably stimulated by the odour to search for the source. How far a concentration gradient for the odour might aid -180- this is not c1ear2 such gradients are probably not very marked, except at very close range, because of natural air turbulence. After the preliminary excitation to search, the beetle is probably attracted to the plant by a combination of visual response to colour, and the olfactory resp- onse2 close to the plant a concentration gradient of the odour would be more marked. If the insect reaches the right plant, the contact chemo-receptors are stimulated by the mustard oil, and this elicits a biting response. Cont- inued feeding then follows as a response to the taste stimulus provided by the sinigrin. With the larvae, the stimuli are probably not all equally important in Nature. The adult beetle lays the eggs on the appropriate food plant, and the larvae hatch on this. The contact chemo-receptors are immediately stimulated, and the insect bites, and continues feeding in response to the taste stimulus. The orientation to odour and colour are not of immediate importance, but if the larvae are dislodged from the plant, or if the existing food plant has been stripped, then these responses are necessary either to enable the larvae to get back to its food plant, or to find a new one. In these cases the orientation stimuli probably act in the same way as for the adult insect. The physical factors, such as toughness, may not be so important in nature, because the larvae can crawl from older, tougher, leaves to less tough young growing tissues. But laboratory reared larvae showed no inclination to move from any particular leaf, whatever its toughness, once feeding had commenced.

F. The formulation of the diet.

It can be very difficult to investigate the nutritional requirements of an insect if the larvae find the diet offer- -181-

-ed in any way unpalatable. This is particularly so for larvae which do not require specific stimuli for feeding. It means that smaller amounts are eaten, and growth must inevitably be inferior to that on the natural food. In many cases where insect nutrition has been investigated, this has not been appreciated, and the inferior growth has been attributed to a lack of some essential dietary substance. When the larvae have definite feeding stimuli, as do the larvae of Phaedon, then the inclusion of the stimuli should at least induce the larvae to eat the artificial diet, and make it easier to improve the palatability in other directions, such as physical texture, and taste. These factors cannot be investigated if the larvae will not eat the diet in the first place. The diet used in these experiments contained the optimum concentrations of the stimulating substances, and had an inert base of agar. The formulation was mostly original, although the vitamin mixture also took into account the results published by other workers. Almost all the dietary essentials demonstrated by previous workers were included. The method seemed by far the best for preliminary work. Such things as amino-acid balance were ignored for the time being, and the mixture contained the acids in equal amounts, so that there should be little chance of a shortage of ahy one. By doing this, the effects of physical factors, and of the proportions ofthe major groups of nutrients (salts, vitamins, etc.) could be investigated and improved first. Then, by varying the proportions of these various groups of nutrients (pp. 148.149), an optimum mixture could be obtained for further investigating the suitability of various inert bases, using 5% agar as the reference standard. In this way, a diet would be obtained in which an optimum balance of the major nutrients had been attained, together with the optimum inert base. With these requirements met, the effects of variation within the major groups -182- of nutrients could be studied, and the results would be more valid than if the effects arising from the unsuitable balance of the major groups, and from the use of an unsuitable inert base, were superimposed on them. It was soon found that aseptic conditions were required (p. 151 et seq.). Complete surface-sterilisation of the eggs may not be necessary during preliminary work, because in the experiments described on p. 157 et seq., the sterile diet did not become mouldy when larvae from untreated eggs were put on it. This would suggest that sterile techniques for the transfer of the diet and larvae are essential to eliminate contamination from the air, but surface sterilisation of the eggs may not be essential, because the larvae do not carry many spores on the body. Of course, mould spores may have been transferred, but the growth of these was effectively retarded by the Nipagin M which was present. For work in which the possible effect of gut symbionts must be known, then surface-sterilisation of the eggs would be necessary, and the procedure described on pages 153-155 would be adequate. Using aseptic procedures, most of the larvae died within 48 hours, which was much sooner than when sterile conditions were not maintained. This was not because essential gut symbionts were killed during the surface sterilisation of the eggs, because in the experiment described on page 157 et seq., there was no difference between the results with larvae from surface-sterilised eggs and those with larvae from untreated eggs. The Nipagin N, which was not used in the first experimentm may have proved toxic. Beck et al. (1949) used the same substance as a mould inhibitor in the diet of Pyrausta, and reported that it had a toxic effects Wressel (1955), however, found no deleterious effect, and a colleague working with Lucilia larvae used it up to a concentration of 0.5% in the diet quite successfully. It seems that this substance is unlikely to have a serious -183- effect, although the possibility merits further investigation. The death of the larvae must have been caused by the unsatisfactory base used for the diets. Although the larvae fed on the diets for a tine, they then stopped, and most wandered away round the chamber. This was at first attributed to a repellent taste effect caused by the pure substances used in the diet. Further investigation showed that the abdomen of the larvae was distended, and the anal segments were elongated° the larvae were apparently const- ipated, and could not eat more. Possibly the rapid death was caused by the rupture of internal organs. It seemed that more roughage, to give a firmer diet, was required. Cellulose powder, or cellulose fibre, might have been adequate, but time was too short to confirm this. These results demonstrated the importance of first finding a suitable inert base for the artificial diet: only when such a base, allowing optimum growth, is found will it be worthwhile investigating the nutritional requirements. Apart from the physiological effect of the agar in these experiments, the results also indicated that a mechanical stimulus might be important for continued feeding. Macer- ated leaf tissue in an agar gel is not eaten to any greater extent than agar gel containing the optimum concentration of sinigrin alone, and feeding ceased on both after a while, whereas larvae on leaves continued feeding. The idea that a mechanical stimulus was important in an artificial diet was also put forward by Ripley et al. (1939), with Argyro- ploce, which would not feed on an agar gel; by Stride (1953), with Carpophilus hemipterus; and by Noland et al. (1949), with Blatella germanica, in which case results were improved by the addition of 30% Cellu flour to the diet. The inert base must therefore not only enable the -184- larvae to void the faeces: it must also provide a suitable mechanical stimulus for biting and feeding. Although the use of the small chambers (page 155) greatly facilitated handling in the sterile chamber, the larvae still wandered from the food, as they had done in the 2"xl" tubes. Relatively few returned, despite the presence of the olfactory and visual stimuli. As stated above, this was probably caused by the unsatisfactory physical texture of the diet, coupled with the alien taste of the constituents. No conclusions were reached about the optimum balance of the major nutrient groups, because of the early death of the larvae.

G. Suggestions for further work on the nutrition of Phaedon larvae.

1). The inert fraction must be improved, to give the larvae a suitable mechanical stimulus for feeding, and also sufficient roughage to allow the free passage of food through the gut, and the elimination of waste. A number of types of inert base, for example, agar and cellulose powder; agar and 'Kleenex' tissue; Cellofas 'B' and 'Kleenex' tissue, should be tried with a single basal diet until the base giving optimum growth with this diet is found. Particular formulations can then be tried, and their full pot- ential should be realised. ii). The diet, with the satisfactory inert fraction, should be tested with the main classes of nutrients in different ratios in the mixture, as described on page 149. Such things as amino-acid balance can be ignored at this stage. iii). The mixture which has ratios of nutrients that give

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optimum growth can now be taken as a standard. Instead of now haphazardly testing the effects of different proportions of amino-acids, chromatograms of the blood and of the hydrolysed body tissues should be compared against chromatograms for larvae reared on turnip leaves. The relative size of the spots on the chromatograms should give some idea where alteration of the amount of any particular amino-acid is required. iv). Up till ii), all transfer operations must be done under aseptic conditions, although up to this stage it may not be necessary to surface-sterilise the eggs. For iii), and later stages, the eggs must be thus sterilised, otherwise slight fungal growth on the diet, and gut symbionts, may provide some of the dietary essentials. v). The optimum balance within each of the major nutrient groups should be found, after which any significant difference in the growth rate from that recorded on leaves may be attributed to extra dietary requirements.

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VIII SUDZIARY.

1). The factors causing orientation to the food, and feeding, were investigated with the larvae of the Mustard beetle, Phaedon cochleariae F. This insect feeds on various Cruciferae. 2). Preliminary experiments with a chamber olfactometer showed that the larvae were attracted by the odour of turnip leaf. Leaf choice experiments showed that this odour did not cause a biting response due to olfaction. Painting juice of attractive leaves on to unattractive leaves caused feeding on these leaves, and showed that a taste stimulus was required. 3). The mustard oil, allyl iso-thiocyanate, was tested in a Varley and Edwards type olfactometer, and in various small chambers, and it was found that the odour of the oil attracted the larvae. Concentrations of from 0.5 to 250p.p.m. were effective, but concentrations above 500p.p.m. were repellent. The glucosides sinigrin and sinalbin were not attractive, nor was the enzyme myrosin; but a mixture of either glucoside and myrosin solutions was attractive. The action of the enzyme is to hydrolyse the glucoside to mustard oil. 4). The mustard oil did not cause complete orientation to the source. Coloured paper cones were tested, and were found to attract many more larvae to the odour source. Orange, yellow, and yellow-green were particularly effective. 5). The effect of colour was investigated further. Colour filter combinations were used to give narrow wave-band transmissions, and the energy of the wave-bands was equalised. A response curve to the colours was obtained, with the peak at yellow-green (530-560W. A double peak effect was obtained at low light intensities, with -187- one peak at yellow-green (530-560mu), and the other at orange (600-640mu); between these two, there was a marked drop in the response to yellow light (570-590mu). There was also a shift in the peak response towards the shorter wave-lengths as the intensity decreased. 6). By offering a white reference light against the colours of equal energy, an attraction to certain colours was shown. Colour vision, as a response to wave-length, was proved by using an 'energy curve' for the lights. The colour attraction was to the range of wave-lengths from 520-580 mu (blue-green to yellow), with a peak attraction at about 530-560mu (yellow-green). These results were confirmed by an equal energy method, which was completely different. 7). After orientation by odour and colour, the mustard oil provided a contact stimulus which elicited biting. 8). Biting continued in the presence of the glucoside sinigrin, at optimum concentrations of 5,000 - 10,000 p.p.m. The sinigrin was tested in a film of 5% agar coated on small cover slips, and the area eaten was assessed by a photo-electric method. 9). Colour or mustard oil included in the agar did not significantly affect the amount of feeding over that obtained with sinigrin alone. This was probably because the larvae were put straight on to the agar, and orientation stimuli were not needed. The high concentration of sinigrin used, 8,000p.p.m., probably hydrolysed to give an adequate amount of free mustard oil. 10). Although feeding on the agar plus sinigrin took place to the same extent as on agar containing turnip leaf tissue, in neither case was as much eaten as on turnip leaf. 5-6% was the optimum concentration of agar. 11). An artificial diet was/formulated and tested on the -188-

larvae. Aseptic techniques were found to be essential, and a successful method for the surface-sterilisation of eggs is described. Ultraviolet radiation was necessary to get completely sterile conditions in the inoculation cabinet. 12). Although the nutrients in the diet were probably adequate, the diet failed because of the physical texture of the gel. The agar gave neither a suitable mechanical stimulus for biting, nor a medium suitable for passage, through the gut. 13). The usual response sequence for the larvae of Phaedon cochleariae F. during food finding would be: a), a fairly long-range excitation response to the odour of a mustard oil; b), a shorter range orientation response to odour and colour; c), a biting response elicited by the contact stimulus of the mustard oil, or, if no mustard oil is present, delayed biting occurs from hunger; d), continued feeding is stimulated by the taste of sinigrin, a mustard oil glucoside this stimulus is re-inforced by a mechanical stimulus provided by the leaf tissue. Continued feeding is then modified by such physical factors as leaf toughness and texture.

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Acknowledgements.

This work was done during the tenure of a Research Studentship from the Agricultural Research Council. I am very grateful to my supervisor, Professor O.W. Richards, F.R.S., for facilities to undertake this work at the Imperial College Field Station, and for his guidance and criticism. I also wish to thank Mrs. L.B. Newbold for checking the final draft of the thesis. Imperial Chemical Industries Ltd. very kindly supplied samples of Cellofas 'A' and 'B°, and Whiffen and Sons Ltd. provided a pure sample of allyl iso-thiocyanate. J. and J. Colman, Ltd. very generously undertook the preparation in their laboratories of batches of the glucoside sinalbin, and of the enzyme myrosin, for use in this work.

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