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 investig- ated, 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 sol- utions 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 resp- onse. 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 contain- ing 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 ---o0o--- -7- I. INTRODUCTION. So far, relatively little work has been done towards elucidating the many factors that must influence the feed- ing 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 resp- onse 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 import- ance 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 formulat- ion 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 simil- ar 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 syn- thetic 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 attract- ants 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.