ABSTRACT
Samples of all stages of Phyllopertha hortícola L. have been analysed for fat, total nitrogen and glycogen.
Total nitrogen increases throughout the larval feeding period, while fat and glycogen are laid down mainly in the latter half.
From November, when the third-instar larva goes into hibernation, until June, when the eggs have been matured and laid, no food is taken.
Of the store of fat and glycogen in the hibernating larva at the beginning of the winter, half is used up by the time the adult emerges. The other half is used in the formation of eggs.
Adult feeding provides energy for the post-oviposition activity period.
INTRODUCTION
The garden chafer is a lamellicorn with an annual life cycle. The egg, larva and pupa occur in light, well-drained soils, usually under grass. The adult lives in and above the sward and on bracken, hedges or trees nearby. The flight season lasts 4-6 weeks in May-June. Eggs are laid about in. deep and hatch in July. From August to November or December the larvae feed on plant roots and moult twice. The fully fed third-instar larvae empty the gut and hibernate in the soil over the winter, pupating about the end of April. Adults emerge from the soil about the end of May or beginning of June, mate, lay eggs and die.
There are two main phases in this life cycle. The short larval feeding period is devoted to growth and the storage of reserves. During the rest of the year these reserves are used for living, for metamorphosis and for reproduction. This paper presents a picture of the two phases in terms of the amounts of fat, nitrogen and glycogen in the body of the individual chafer.
METHODS
All the material for analysis came from one infested field in the Lake District. Not all the samples were taken from the same generation of chafers. Most of the samples of adults were taken in the 1951 flight season, and most of the samples of larvae in the autumn of 1951 (i.e. the following generation).
All the pupae and some of the adults analysed were collected as hibernating third-instar larvae and kept in a damp atmosphere until development reached the appropriate stage. Some adults were collected from the field during the flight season. The first group of larvae to be analysed was reared from eggs under grass in flowerpots out of doors. All the larvae were of the same age. Later groups were collected from the field at intervals during the feeding period. The age given for each group is a mean value, the number of days between the mean hatching date for the year in question and the date of collection. Eggs hatched over a period of 17-28 days (mean 22 days) in the six years 1948-52 (see Milne, 1956a), so the age variation among the larvae in a field sample is about ±10 days.
The individuals in each sample were weighed and assembled into groups by the following procedure. The individuals were arranged in order of ascending weight and marked off in lots containing as many individuals as there would be groups. The individuals in each lot were then assigned, at random, one to each group. For example, if four groups were required, the four lightest individuals were allotted at random to the four groups; this allotment was repeated, always with the four lightest individuals of those remaining, until the whole sample had been divided into four groups with similar means and ranges of weight. The groups were analysed for fat, nitrogen or glycogen (usually one each for fat and nitrogen and two for glycogen). In 1951, in all but the two youngest larval samples, individuals were analysed separately. In previous years the groups were analysed as a whole.
A modified Soxhlet process was used for individual fat analysis. The chafers were dried at 90° C. for 24 hr. and stored at—3° C. Before extraction they were crushed and put into small extraction thimbles (10 × 20 mm.). The thimbles were plugged with cotton-wool and vacuum-dried to constant weight over activated alumina at room temperature. After 8 hr. extraction with petroleum ether the thimbles were again vacuum-dried to constant weight. The weight of ether-soluble material is given by subtraction and not, as in the normal Soxhlet method, by direct weighing of the extract. The direct method was used for the estimation of fat in whole groups of individuals.
Total nitrogen was estimated by the Kjeldahl method using a sodium sulphatecopper sulphate-selenium catalyst for the digestion and an ammonia microstill (Markham, 1943).
Glycogen, in groups of individuals, was extracted by Pflüger’s method (Cole, 1926), hydrolysed with hydrochloric acid and the glucose estimated by Bertrand’s method (Plimmer, 1920). For the analysis of single individuals a modified Pflüger’s method was used (Good, Kramer & Somogyi, 1933) and the glucose obtained by hydrolysis estimated by the Hagedorn and Jensen method as described by Hawk, Oser & Summerson (1947, p. 861).
A group of newly emerged male adults was used to test the glycogen method for individuals. Seventy-two males were divided into three groups of twenty-four. One group was analysed immediately and one after the beetles had been dried at 90o C. for 24 hr. and stored at—30 C. for 2 months. The third group was also dried and kept for 2 months ; it was then analysed for fat and the extracted residues analysed for glycogen. The amounts of glycogen in the three groups were 2·13, 1·65 and 1·68 mg. per beetle respectively (means). The highest value (given by the analysis of fresh material) is significantly greater than the other two (p=0·001). There is no significant difference between the latter (P=0·8).
Another test indicated that it may be safe to estimate glycogen on material pickled in Camoy’s fluid. Two groups of twelve and twenty-two feeding third-instar larvae were analysed, the former group when fresh, the latter after 24 hr. in Camoy’s fluid. The mean weights of glycogen per larva were 4·32 mg. (fresh group) and 3·76 mg. (pickled group). There is no significant difference between these two means (p=0·4). Thus there is no evidence to show that 24 hr. in Camoy’s fluid affects the glycogen content of the larva.
Two further tests indicated that fat extraction made no significant difference to the nitrogen figures. Two lots of six newly emerged male adults were analysed for total nitrogen. One lot was analysed immediately after killing and the other lot after drying and fat extraction. The mean weights of nitrogen per beetle were 2·00 and 1·92 mg. respectively. This difference was not significant (P = 06). Five groups of individuals were used for the other test : 40 male pupae, 40 female pupae and three groups of female adults (40, 60 and 220 beetles respectively). Each group was halved and the halves analysed for nitrogen only and for fat and nitrogen. The individuals were not analysed separately. The nitrogen figures for each half group were expressed as nitrogen per individual and the nitrogen figures for the ‘fat and nitrogen’ half groups subtracted from their respective ‘nitrogen only’ values. The five differences were then subjected to Fisher’s ‘Unique Sample’ test. This showed that there was no significant difference between the two methods in this instance (P=o-3).
STORAGE OF RESERVES
In the population sampled, newly hatched larvae weighed about 3 mg. before they had begun to feed. Their subsequent increase in live weight with age is shown by the sample figures in Table 1. Larvae weighed about 20 mg. at the first moult and 70 mg. at the second ; after reaching a maximum of about 200 mg. in the third instar, the larvae weighed about 140 mg. just after going into hibernation. This decrease in weight is due chiefly to the emptying of the gut.
The water content falls during the feeding period. Water content of second-instar larvae is in the region of 90% ; the two samples of second-instar larvae gave mean values of 88-o% (25-day larvae) and 87-9% (34-day larvae). The samples of third-instar larvae gave mean values of 84-2, 80·2, 80·8 and 79·1% (mean ages: 72, 94, 109 and 128 days respectively—the last sample composed of hibernating larvae). The water content of individual third-instar larvae varies little. Ranges for the four samples are: 80·9−88·0, 77·4·85·0, 80·3−81·5 and 72·7·82·7 respectively.
In the early second-instar larvae (25 days old) there was apparently little more nitrogen than in the egg (see Table 2). This result is open to criticism. There was a shortage of larvae of this age and only twenty-two were available. The larvae were dried, extracted with petroleum ether and the extracted residues analysed for nitrogen. The tests described above indicated that the double analysis could be safely carried out on pupae and adults, but this safety cannot be assumed for the young larvae where the level of nitrogen is so much lower.
By the end of the second instar (34-day sample) the level of nitrogen has risen to an average of o-8i mg. per larva and by the middle of the feeding third instar (72-day sample) to 2-37 mg. There is little further increase and the hibernating larva contains 2-41 mg. of nitrogen (128-day sample). Nitrogen as a percentage of live weight increases steadily in the samples analysed. The five samples between 34 and 128 days give mean percentages of 1-21, 1-17, 1-49, 1-57 and 1-75 respectively. Variation of individual percentages within the samples is small (coefficients of variation, 5-7%).
There is very little fat in the young larva. The amount in the body does not really begin to rise until the beginning of the third instar. From 0-85 mg. per larva at the end of the second instar, the fat content rises to 3-8 mg. at 72 days and 5-0 mg. in the hibernating larva. Variation in individual fat content among the larvae of one sample is much greater than variation in live weight, water or nitrogen. Coefficients of variation lie between 38 and 48% in the samples analysed individually for fat. Coefficients of variation for live weight, water and nitrogen lie between 15 and 25%.
There is little glycogen in the 34-day larva. By 72 days (the middle of the feeding third instar) the level has risen to 3-5 mg. This level is maintained through the rest of the third instar. Individual glycogen content figures vary widely, giving coefficients of variation of 40-75 % for the third-instar samples.
From live-weight figures and from observation of the growing larva it has been suggested (Laughlin, 1956) that there are two ends to be attained by the feeding larva—growth in body size and deposition of reserve materials—and that the emphasis shifts from the former process to the latter as the feeding period progresses. Body growth takes place in the first, second and beginning of the third instars and the development of the fat body mainly in the third. The analysis data agree with this idea (see Fig. 1). Broadly speaking the nitrogen level reflects the amount of living tissue and hence the body size. The levels for fat and glycogen reflect the growth of the fat body and the amount of reserve material in the body. Nitrogen increases throughout the feeding period, while fat and glycogen are laid down mainly in the third instar (see also Chin, 1950).
THE HIBERNATING LARVA AND THE PUPA
The feeding period results in an average larva of 140 mg. (in this instance) which contains III mg. of water, 2·4 mg. of nitrogen, 5 mg. of fat and 4 mg. of glycogen. This material must last through the winter, provide energy and materials for metamorphosis and for the production of eggs by the female and spermatophores by the male.
Figures for three other samples of hibernating larvae and for one sample of pupae are available and are shown in Table 1. The samples were taken from the same field as the rest but were collected in different years. The analysis figures confirm the general picture given by the 1951 results and emphasize the fact that there is a considerable variation in live weight and fat content between hibernating larvae of different generations. The mean live weights of the samples cannot be compared closely because in October, November and December the mean weight of a population falls as larvae empty the gut and enter hibernation. Even after the gut is rid of all solid material a good deal of clear liquid is excreted and for the first few weeks of hibernation the weight of the hibernating larva continues to drop. However, it seems fairly certain that the samples taken in 1948 and 1951 were from populations (generations) of smaller larvae than those of 1949.
The thirty-two hibernating larvae of the 128-day sample of 1951 which were analysed for fat show a strong correlation between live weight and fat content (r=0·8215, P= <0·001). The mean fat content of the samples also varies with the mean weight of the larvae to a certain extent. The 5 mg. of the 1951 larvae and the 7·8 mg. of the 1949 larvae indicates that, as would be expected, large larvae contain more fat than small larvae. On the other hand, the 1948 larvae were little if any bigger than the 1951 larvae and yet contained 6·7 mg. of fat. This suggests that conditions during the feeding period can affect body size and reserve stores more or less independently of each other.
Over the winter of 1949-50, fat content dropped from nearly 8 mg. in the hibernating larva to about 5 mg. in the pupa. Nearly half the store of fat is used up over the winter. There is little fall in nitrogen content but this means no more than that little nitrogen was lost from the body.
THE EVENTS OF THE FLIGHT SEASON
The brief and generalized summary below is taken from two papers: Milne & Laughlin (1956) and Milne (1956a). There is much individual variation in the timing of development and of the different behaviour patterns.
In nature the pupa lies in a cell about 2 in. deep in the soil. When the adult emerges from the pupal skin, the elytra and parts of the abdominal cuticle are still unpigmented. During the next 7-8 days the cuticle hardens and darkens and the beetle moves from the cell to the surface of the sward. The rest of the beetle’s life (about 14 more days) is spent partly above ground. The males run and fly over the grass and over the bracken, trees or hedges surrounding the field, resting down among the grass stems, on bracken fronds or in the trees. The females come out on to the grass, mate and return to the soil to lay their eggs. With egg laying largely completed, they too spend some time flying and resting, mostly in the bracken or trees.
At ecdysis the ovaries contain no fully developed eggs. At most one oocyte is visible as a small swelling at the base of each ovariole. The abdomen is full of fat body. Ten to fifteen days later, at 15° C., the mature female has no fat body left and the ovaries and oviducts are packed with fully developed eggs with perhaps one or two immature oocytes left in the ovarioles. In nature, egg development may be somewhat faster. Females emerging from the soil (i.e. about a week after ecdysis) have most of their eggs mature and very little fat body left (Milne, 1956b).
Females do not begin to lay eggs until all are mature and the fat body used up. They do not begin to feed until all or most of the eggs have been laid. The life of the adult female falls into two parts : an egg maturation and opposition period and a period of feeding and activity after the bulk of the eggs have been laid.
UTILIZATION OF RESERVES
Two groups of female adults, collected as hibernating larvae, were analysed, the first group just after emergence from the pupa (12-36 hr.) and the second group 15-20 days later when egg development was complete. For the 15-20 days the females were kept at 15o C. in sifted soil and darkness.
Field samples of females which had laid most of their eggs were also analysed: one sample of females which had only just begun to feed and one sample off the bracken towards the end of the flight season.
The males from the collection of hibernating larvae were divided at random into five groups and killed 12-36 hr. after ecdysis. These groups were used to test combinations of analysis methods (see p. 567). A small field sample of males off the bracken at the end of the flight season was also analysed.
The collection of hibernating larvae produced 192 male and 150 female pupae. The mean weight of the female pupae was 131-4 mg. (standard error 1-724). The mean weight of forty-six newly emerged female adults was 90-1 mg. (standard error 1-835) and °f twenty-two mature female adults, 15-20 days after emergence, was 54-4 mg. (standard error 2-209). Both these groups of adults were random samples of the 150 female pupae. The difference of 80 mg. between the pupa and the mature female adult is accounted for mainly in two ways: at emergence a good deal of colourless fluid is left in the cast pupal cuticle; at emergence the gut is full of accumulated excretory products which are passed out of the body in the first few days of adult life.
Analysis results are given in Table 2. All females except those in the ‘newly emerged ‘sample were dissected before analysis. Mature eggs were dissected out and those taken from the ‘mature female’ sample were analysed separately. The ‘grass female* sample was collected on 18 June 1951, i.e. towards the end of ‘phase I activity’ (see Milne, 19566). Females were collected off the grass and dissected. Only females containing less than two mature eggs, no fat body and little or no food in the gut were analysed. Most of such females have just finished oviposition and are about to fly off to the bracken fringing the field (see Milne, 19566). The ‘bracken female’ sample was collected on 30 June 1951, i.e. towards the end of ‘phase 2 activity’ and of the flight season.
The mean live-weight figures for mature females given in Table 2 include the eggs the females were carrying. The body weight of these females (live weight minus weight of eggs) was calculated (mean 45-8 mg., standard error 1-55) and the figures put into an analysis of variance with the live weights of grass females and bracken females (F= 5-201, P=0·01). There is no significant difference between mature females and grass females but bracken females are significantly heavier than both the former samples (P= <0-05). The difference (about 7 mg.) is probably due to the large amounts of food in the guts of the bracken females.
There is no significant difference between the amounts of fat or glycogen in the last three (female) samples of Table 2. Analyses of variance gave variance ratios (F) of 1-456 (P=0·2) and 2-947 (P=0·1) for the fat and glycogen data respectively. The level of both substances is very low (only about half a milligram per female).
The eggs dissected from the mature female sample were analysed and the figures shown in Table 2 are the quantities per egg. This sample produced a mean number of 7-6 eggs per female. The difference in mean fat and glycogen content between newly emerged females and the bodies of mature females is by no means accounted for by the total fat and glycogen in the eggs produced (see Fig. 2). Evidently a considerable amount of energy is used up in the metabolic processes involved in egg manufacture. In the field the female will also use energy digging in the soil. The ‘mature females ‘were kept in loose soil, however, and will have used a minimum of energy for this purpose.
The water content of the egg is low—about 50%. The egg swells during the embryonic period, taking up about twice its own weight of water from the surroundings.
DISCUSSION
It has been shown (Raw, 1951 ; Milne & Laughlin, 1956) that adult feeding makes no difference to the number of eggs produced. Egg production is closely correlated with the weight of the pupa. Indeed, feeding only starts after all the eggs are mature and usually after all or most have been laid. Thus the reserves of the feeding larva form the main if not the only source of material for the production of eggs.
About half the fat and glycogen contained in the hibernating grub at the beginning of the winter is consumed before pupation. Almost all the rest is used up in the development of the adult and in maturation of the eggs. Fig. 2 shows how the still considerable fat and glycogen stores of the newly emerged adult (column A) are almost entirely dissipated by the time the eggs are mature (column B). Nor do field samples of females which have laid all or most of their eggs (columns C and D) show any recovery from this low level. Once egg development is completed, the adult female spends a very active life, burrowing in the ground to lay eggs, flying and walking about the bracken and, in some cases, taking off on long distance flights to colonize new areas (see Milne, 1956ft). There is no store of fat and glycogen left to provide all this energy (the half milligram of fat and glycogen that is left could probably not support flight for more than about 20 min.,* even if both sources of energy were used). Thus feeding by the adult female is essential for continued activity in the second half of adult life.
ACKNOWLEDGEMENTS
I am very grateful to Prof. V. B. Wigglesworth and to Dr. A. Milne for many helpful discussions. I would also like to thank Mr. A. Thompson, of the Department of Agricultural Chemistry in this college, for discussions on the methods used and for the loan of apparatus and other facilities.
REFERENCES
This figure was calculated knowing that (a) a bee of about too mg. uses about 10 mg. of sugar per hour in flight (Wigglesworth, 1950, p.392) and (6) the calorific value of fat is a-2 times that of carbohydrate; and assuming that (a) a garden chafer adult of about 50 mg. uses about half the sugar used by a bee of 100 mg. and (i) both fat and glycogen are available for use in flight.