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Food exchange between adults and larvae in Vespa orientalis F
Anim. Behav., 1968,16,298-303
FOOD EXCHANGE BETWEEN ADULTS AND LARVAE IN VESPA
ORIENTALIS F BY
J . ISHAY
Department of Physiology and Pharmacology, Tel-Aviv University Medical School AND
R . IKAN
Department of Organic Chemistry, The Hebrew University, Jerusalem
A constant exchange of food between adults and larvae has been observed among social insects such as termites, ants and wasps . In the course of such food exchange, the larvae release droplets of secretion and this secretory process has been termed 'trophallaxis' . Scientific opinion was divided until recently as to the significance of trophallaxis . Janet (1895) and Roubaud (1916) believed the oral secretion of the larvae to be highly attractive to the adults . Weyrauch (1936) suggested that this secretion serves to regulate the temperature and humidity in the nest . Brian & Brian (1952) concluded that trophallaxis among wasp larvae is merely an oral method of excretion insofar as the larvae lack any other means of excreting waste products . Montagner (1963), Montagner & Courtois (1963) and Montagner (1964) have studied the food habits of a number of European species of social hornets by means of radioactive gold (Au 198) . They concluded that the males are incapable of feeding themselves and subsist solely on food provided by the workers or by the larvae . Montagner (1963) reported in detail on the characteristic behaviour of adult hornets while visiting the larvae, a behaviour which caused the larvae to release droplets of saliva . He also noted that the old females `demand' food from younger females as well as from larvae . A similar pattern of behaviour has been observed by Ishay (1962) in Vespa orientalis and by Pardi (1950) in wasps of the genus Polistes. Morimoto (1960) fed Polistes larvae on radioactive phosphorus (P 32 ) and was then able to detect radioactivity in the saliva 3 hr after the experimental meal . Maschwitz (1965) reported that the saliva of hornet larvae is highly nutritious, containing sugars and amino acids . All these recent observations confirm Wheeler's (1928) opinion as to the importance of trophallaxis in forming a close bond between the various members of insect families, particularly between the larvae and the nursing workers . The present paper reports on trophallaxis in the Oriental hornet, Vespa orientalis F.
Methods Colonies of Vespa orientalis comprising eggs, larvae of various instars, pupae, workers and a queen, were collected in the field and brought to the laboratory nursery where they were placed in special nests built to simulate the natural habitat (Ishay, 1964) . Members of the colonies were observed while in the nests, where they behaved naturally . Paint markings on the thorax were used to designate either individual hornets or entire colonies, according to the nature of the observation . The food provided consisted of honey, summer fruit, bees and chopped meat . In order to determine the role of the larvae in preserving the social structure of the colony, four experiments were carried out . In the first, two colonies were brought from the field to the laboratory, and all the larvae in one of these colonies were removed from their cells . Both colonies were then maintained under identical conditions and 48 hr later the hornets were allowed to enter and leave the nest freely as well as to fly about within a nest enclosure . In the second experiment, three colonies were raised in the laboratory, but the workers were allowed to forage in the field ; the larvae in all three colonies were removed from their cells . The third experiment involved three colonies in which the combs of the nest were covered with a thin, transparent nylon sheet, separating the larvae from the adults . In the fourth experiment, the adults of three different nests were separated from the larvae by a mosquito net which was placed on the combs . All these experiments were carried out during the months of June, July and August. In order to determine the nature of the larval secretion, another series of experiments was carried out as follows : C14-labelled protein* *Prepared from disrupted Chlorella vulgaris cells which had been grown in a C1402 environment . The labelled protein was freed from lipids by solvent extraction, and from water-soluble compounds by dialysis . 298
ISHAY & IKAN : FOOD EXCHANGE BETWEEN ADULTS AND LARVAE IN INSECTS was fed to larvae and workers, and the saliva composition determined after various time intervals . The labelled protein was obtained from Radiochemical Centre, Amersham, England . The radioactivity was measured in a Nuclear Chicago detector, in a Radiochromatogram Scanner, Packard Model 7200, and in a Vanguard Autoscanner 880 . Larval saliva was collected with a micropipette by tickling the mandibular region of the larvae, thus simulating the method employed by the adult hornets . The saliva of the adults was obtained by applying gentle pressure to the abdominal region of the workers . Larval haemolymph was collected by slightly wounding the skin of the larva with a hypodermic needle . The carbohydrates and amino acids of saliva and haemolymph were separated on ionexchange columns ; carbohydrates were determined by a modified anthrone method (Mokrasch, 1952) and amino acids by micro-Kjeldahl . The proteins in the saliva and haemolymph were separated by Schaefer's (1964) method. The presence of proteases in saliva was determined with gelatine (Northrop, 1939) . Results Direct Observations Larval care . Each worker in the nest spends most of its time caring for the larvae . It pays frequent visits to the `infant pool', probing inside with its maxillae in order to examine the contents. If a larva is detected in the cell, the worker supplies it with meat particles or droplets of food . The larva, in turn, releases droplets of saliva . This behaviour occurs as long as the temperature reading at the nest entry is above 26°C . When the temperature drops below this value, the workers become less active, gathering about the `infant pool' and covering with their bodies all cells which contain larvae . Larval care is for the workers an essential `transition stage' in passing from one duty to another (e .g . from building or repairing cells to foraging for food or guard duty etc .) . It should be emphasized that never have we observed a worker to pass from one assignment to another without first caring for larvae. The workers visit the larvae regularly during all hours of the day and while the temperature is above minimum . Each visit lasts from 2 to 13 sec, depending on the extent of the larva's hunger. Small larvae of the first and second instar are paid an average of fifty-three visits an hour ; medium larvae of instars 3 and 4, are visited
299
seventy-four times an hour ; and larvae of the 5th instar-about ninety-eight times an hour. The big larvae are visited also by the queen, but she comes to receive rather than to give food . This is evident from the fact that occasionally, when the larva is unwilling to provide the queen with saliva, it retracts towards the bottom of the cell as the queen approaches . A hungry queen will plunge her head into the cell until she reaches the mandible of the larva and forces it to release a drop of saliva . The queen's visit usually lasts from 80 sec to 3 min . Sometimes the larvae receive unchewed particles of meat which they are then able to chew by themselves . A morsel of meat about 1 . 5 to 2 mm in diameter is chewed up by big larvae within 3 to 4 min . Once they have chewed and swallowed the food offering, the bigger larvae (5th instar) produce specific sounds by rubbing their mandibles against the cell wall . These sounds are clearly audible to human ears and serve as a signal to the workers to bring more food . The small larvae are incapable of producing these sounds because they are unable to reach the cell wall with their mandibles . The feeding problem here is resolved by special workers who pay regular visits to the cells and upon detecting a hungry young larva, tap the tip of their abdomen against the cell walls . The tapping noises produced by such workers serve as a signal to other workers to come quickly and feed the larvae (Schaudinischcky & Ishay, in prep .) . Adult-larva interdependence . In the first experiment, in which the larvae were removed from one of two colonies, only the colony retaining its larvae continued life as usual ; the other colony dwindled gradually despite renewed oviposition by the queen, because the workers failed to care for the new-born larvae . Six days later, the nest was abandoned . This experiment was repeated twice more with identical results . In the second experiment, in which larvae were removed from three colonies whose workers were free to fly to the field, all three nests were abandoned within 11 days, the queens having died in the nests . A few of the hornets succeeded in joining nearby colonies which did carry larvae. In the third experiment, in which the combs of three nests were covered by thin, transparent nylon sheets, separating larvae from the adults, the adults left the nest within 4 days, and the queens were found dead on the floor of the nests. In the fourth experiment, in which the combs of three colonies were covered by mosquito
3 00
ANIMAL BEHAVIOUR, 16, 2-3
nets, the adults remained in the nest and on the first day made futile attempts to tear the nets apart . In the meantime, adult hornets newly hatched from the net-covered combs, commenced mediating between the hornets on the outside and the larvae within, passing particles of meat from the adults to the larvae, and droplets of saliva from the larvae to the adults . On the second day, the workers started building new combs outside the net-covered areas ; the queens immediately oviposited in the new cells, and the colonies continued life as usual . Experimental Data Larval saliva. In Table I are given the quantities of saliva formed by the different larval instars which can be transferred to the adults by means of trophallaxis . Table I. Maximum Quantity of Saliva Released by Larvae Isolated from the Adults for Twelve Hours (Data Based on Sixty Larvae from Each Instar)
Instar
Average weight of larva (in mg)
Average volume of saliva (in ml)
Average volume of saliva per body weight (in ml/mg)
1
4.4
0 . 0004
9 . 1 x 10 - 5
2
33 . 4
0 . 0018
5 .4 x 10- 5
3
178 .0
0 .0025
1 .4 x 10- 5
4
292 . 6
0 .0051
1 . 7 x 10 - 5
5
919 . 3
0 . 0024
2 . 6 x 10 - 6
It is interesting to note that except in one instance (instar 4), the saliva volume to body weight ratio decreases with increasing weight of the larva . In Table II are given the carbohydrate and protein contents of the crude saliva and the haemolymph of 5th instar larvae . Table II . Percentage of Carbohydrate and Protein in the Crude Saliva and the Haemolymph of Fifth Instar Larvae (Average of Twenty Determinations)
Fluid
Total Total carbohydrates nitrogen
Nitrogen of free amino acids
Saliva
5 . 50
0 . 21
0 . 13
Haemolymph
2 .05
1 . 37
0 . 24 •
T his value approaches the maximum recorded so far in insects (Duchateau & Florkin, 1958) .
As is clear from Table II, sugar concentration is considerably higher in larval saliva than in haemolymph . On the other hand, there are fewer nitrogenous substances in the saliva than in haemolymph . Table III. Determination of Carbohydrate Concentration in Larval Haemolymph at Different Hours of the Dayt Time
Total carbohydrates in haemolymph (in %)
0400
2 . 15
1000
1 .73
1800
2 .40 tAverage of sixty determinations .
The data in Table III suggest that the carbohydrate concentration in the larval haemolymph is lowest during the time of maximal worker activity. Composition of carbohydrates in the saliva and haemolymph of larvae and workers . The chroma-
tography was carried out with butanol-acetic acid-ethyl ether-water (9 :6 :3 :1) as mobile phase and various mono-, di- and oligo-saccharides as markers . The radioactivity was measured in a Packard Model 7200 Radiochromatogram Scanner. The findings were as follows . (a) In larval saliva : glucose, fructose, sucrose, maltose, trehalose, 4G-a-glycosyl-sucrose and oligosaccharides . (b) In larval haemolymph : glucose, fructose, disaccharide (unidentified), trisaccharide (unidentified) and tetrasaccharide (unidentified). (c) In worker saliva : glucose only. (d) In worker haemolymph : glucose, fructose, maltose, trehalose, 4G-a-glycosyl-sucrose and oligosaccharides (unidentified) . Feeding of 5th-instar larvae with C 14-protein . Fifth-instar hornet larvae were given C14labelled protein by mouth . Equal samples of saliva were withdrawn at regular intervals and stored at - 30 ° C . Minute samples of saliva were placed on 5 cm x 20 cm glass plates (0 . 25 mm thick) . The chromatography was carried out as before . As can be seen from Table IV, glucose formation reaches a peak 3 hr after the labelled protein meal and then declines . When labelled protein was fed to workers which had been kept away from the larvae from the time of eclosion, no degradation of the protein occurred, that is, no sugars were found . Furthermore, no proteases were detected by
ISHAY & IKAN : FOOD EXCHANGE BETWEEN ADULTS AND LARVAE IN INSECTS
301
Table IV . Saliva Composition of Fifth-instar Hornet Larvae at Various Time intervals after Feeding on C 1 4-labelled Protein' Hours after labelled protein meal' •
Material in larval saliva
3
4
5
20
fr
2
Protein C 14
11
8.7
1
1
1
1
Dissaccharide C 14
-
-
3.4
1 .6
2.1
3 .6
5 .8
82
Glucose C 14
1
1
14
25 .4
'Each datum is an average of ten samples and is expressed in relative area units . 'A different group of ten larvae each was `milked' at each of the designated time intervals . No incubation of saliva was carried out, as it was assumed that such occurred in vivo, body temperature of the larva being approximately 30°C . means of gelatine in the saliva of adults who had been separated from the larvae for a period of one week. In vitro incubation of crude 5th-instar larval saliva with C 14-protein. Five ml of crude 5thinstar larval saliva was incubated at 30 ° C with samples of C 1 4-protein . Measurements were made with the use of a Vanguard Autoscanner 880 . The results of this incubation are presented in Table V. It should be pointed out that the saliva used in the above experiment was crude, i .e . it had not undergone centrifugation . We cannot be certain, therefore, that the observed protein degradation is attributable to the fluid part of the saliva and not to its cellular components . Incubation of hornet homogenate with C 14labelled protein . The homogenates were prepared by crushing larvae, pupae or workers with ultra-turrax in water . C 14-labelled protein was then added and the suspension was kept at 30°C for 20 hours, then chromatographed on activated charcoal and celite (1 :1) column, and developed with distilled water followed by aqueous ethanol up to 50 per cent . The fractions obtained were concentrated in vacuo and fractionated on silica gel chromato plates as described . The radioactivity of glucose and protein only was measured, although other carbohydrates
and free amino acids may also have been present . The results shown in Table VI suggest that the ability to degrade proteins is lost immediately after pupation . Indeed tests for protease activity, using gelatine, failed to show protease activity in the saliva of workers which had been removed from the larvae immediately upon eclosion . In contrast, larval saliva showed clear-cut protease activity . Discussion The strong attraction of the adult hornets to the secretion of the larvae has already been described earlier . This larval secretion has been shown to contain 5 . 5 per cent sugars (mono-, diand oligo-saccharides) and 1 . 3 per cent protein and free amino acids . Despite the low concentration of these compounds in the saliva, it can still be regarded as nutritious . It is interesting to compare the amount of sugars and nitrogenous compounds in the saliva of hornet larvae with that in Royal Jelly of bees or in mammalian milk . Such a comparison is made in Table VII . The concentration of sugars in Vespa orientalis larval saliva is low compared with that found by Maschwitz (1965) in the larval saliva of European wasps, but the concentration of nitrogenous compounds is very much the same . Indeed, the protein concentration in the larval saliva is
Table V . In Vitro Incubation of Crude Fifth-instar Larval Saliva with C t 4-labelled Protein (Average Ratios from Six Samples) Material detected
Hours of incubation 1
Glucose C t 4/Protein C
14
6 .6/1
3 11 . 3/1
5 33 . 6/1
6 17 . 7/1
3 02
ANIMAL BEHAVIOUR, 16, 2-3 Table VI. Labelled Sugars in Homogenates of Various Hornet Stages after Twenty Hours of Incubation With C14-labelled Protein Test homogenate*
Labelled sugars
Labelled proteinglucose ratio
Fat body of 5th-instar larvae
present
1 :5 . 2
5th-instar larvae just before pupation
present
1 :3 . 3
Pupae before metamorphosis
absent
Pupae after metamorphosis (bright)
absent
Workers, 5 hr after eclosion**
absent
Workers, 2 days after eclosion**
absent
Workers, a week after eclosion**
absent
*In each case six samples were taken from the 5 g homogenate . **From eclosion till examination time, the workers were kept isolated from the larvae. Table VII. Percentage of Sugar and Protein in Hornet Larval Saliva, Royal Jelly of Bees and Mammalian Milk
Materials
Larval saliva of Vespa orientalis ph 4 . 5
Larval saliva of Vespa vulgaris, V. crabro and Vespula germanica (after Maschwitz, 1965)
Royal Jelly from the cells of bee larvae 3 to 4 days old ; ph 4 to 4 .5 (after Decourt, 1956)
Mammalian milk (after West & Todd, 1959) human
bovine
Sugars
5 .5
8 .9
12. 5
7 .2
4.9
Proteinst
1 .3
1 .8
12.3
1 .5
3.5
tThe percentage of proteins is calculated by multiplying the total nitrogen (N) by the factor 6 .25
close to that in human milk . The concentration of sugars and nitrogenous compounds in Royal Jelly is quite high, compared with that in vespan saliva . It should be remembered, however, that the feeding of apian larvae is totally different from that of vespan larvae in that Royal Jelly, like mammalian milk, is an adult secretion intended as food for the larvae, whereas the larval saliva of hornets is intended as food for the adults and is given in return for meat particles provided by the adults . Continuous observations (Ishay, 1964) have shown that the queen of the Oriental hornet will not leave the nest as long as there are a number of workers in it. During this period, which lasts from June to December, the queen feeds solely on the larval secretion, and when the larvae are removed from the nest, it dies of starvation . The average weight of the nest-founding queen is 746 mg. It has been stated by Janet (1895) and by Rivnay & Bytinsky-Salz (1949) that the queen lays about 1500 eggs per season, but
according to Ishay & Ikan (in preparation) the annual number of eggs is over 4000. Each egg weighs about 2 mg, which means that the total weight of the eggs is 3000 to 8000 mg, or four to eleven times the queen's weight in the spring . The weight of the queen's ovarioles in the spring, before the start of oviposition, is 9 to 10 . 1 mg, whereas their weight during the height of oviposition (in September) is 153 -1 mg, or more than fifteen times their previous weight . Imagine, then, the amount of larval secretion necessary to account for this remarkable increase in weight, for it is the larvae who provide the queen with the raw material needed for developing its load of eggs . Equally benefited are the workers who `milk' the larval secretion during breaks between foraging trips, and the males who collect larval saliva droplets whenever and for as long as they come in contact with the larvae. The findings of the present paper clearly indicate that the colony cannot survive without
ISHAY & IKAN : FOOD EXCHANGE BETWEEN ADULTS AND LARVAE IN INSECTS
larvae, for the adults are soon forced to leave the nest or die of starvation . Only the larvae are able to digest proteins and degrade them into amino acids and sugars . A similar situation exists also among butterflies (Lepidoptera), where only the larvae feed on proteins, while the adults feed merely on the carbohydrates present in nectar . So far as is known, the only digestive enzyme present in adult Lepidoptera capable of feeding is invertase ; starch, fat or protein mixed in the food remain unchanged in the stomach (Snodgrass, 1961, as cited by House, 1965) . Moreover, the adults of many Diptera, Hymenoptera, Lepidoptera and other insect orders, are known to subsist quite adequately on sugar solution alone (House, 1965) . That only larvae digest proteins is a common enough phenomenon among insects, but in the case of wasps this has especial significance, for the degradation products of proteins are transferred from the larvae to the adults and serve as raw materials for nitrogen metabolism, which is especially important for the queen, who produces the eggs . Consequently it is clear that social interrelations are a sine qua non for Oriental hornet populations, and that the phenomenon of trophallaxis helps to create an organic bond between the various members of the colony . Summary Colonies of Vespa orientalis cannot exist without the larvae or, more specifically, without larval salivary secretion, which is the only source of raw materials for nitrogen metabolism and egg production available, to the workers and the queen, who do not themselves possess the proteases necessary for protein degradation . This finding suggests that social life among wasps is probably a symbiosis between the adults and larvae . Acknowledgments This study was partly supported by a grant from the Israel Foundation Trustees for Research and Education . It is part of a Ph .D . thesis carried out by J . Ishay at the Hebrew University of Jerusalem . The authors are indebted to Mrs Leslie Mandl, of the Israel Pharmacological Trust, and to Dr J. Lengy of the Tel-Aviv University, for critical review of the manuscript . REFERENCES' Brian, M . V. & Brian, A. D . (1952) . The wasp Vespula sylvestris Scopoli : Feeding, foraging and colony development . Trans. R . Soc. Lond., 103, 1-26.
303
Decourt, P . (1956) . La gel6e royale d'Apis meiifica et son activit6 chez les vert6br6s sup6rieurs . Revue Path. gin . Physiol. clin ., 682, 1641-1663 . Duchateau, G . & Florkin, M . (1958). A survey of aminoacideias with special reference to the high concentration of free amino acids in insects' haemolymph . Archs. int. Physiol. Biochim ., 66, 573-591 . House, H . L . (1965) . Chapter on Digestion. In The Physiology of Insects ( ed . b y M . Rockstein) . Vol. 2, pp . 815-853 . Ishay, J. (1962) . Observations on the biology of the oriental wasp Vespa orientalis F . in Israel . M .Sc. Thesis, Tel-Aviv University (in Hebrew) . Ishay, J. (1964) . Observations sur la biologie de la guepe orientate Vespa orientalis F . Insectes roc., 11, 193-206. Janet, Ch. (1895) . Etudes sur les fourmis, les guepes et les abeilles . Mem. Soc . Zool. Fr., 8, 1-140. Maschwitz, U . (1965) . Larven als Nahrungsspeicher im Wespenvolk (Ein Beitrag zum Throphallaxieproblem) . Verh . dt . zool. Ges., 50, 530-534 . Mokrasch, L. C . (1952) . Analysis of hexose phosphates and sugar mixture with the anthrone reagent . J. Biol. Chem ., 208, 55-59. Montagner, H . (1963). Etude preliminaire des relation entre les adultes et le couvain chez les guepess sociales du genre Vespa au moyen d'un radioisotope. Insecles roc. 10, 153-165 . Montagner, H . (1964) . Etudes du comportement alimentaire et des relations trophallactiques du mile au sein de la soci6t6 de guepes . Insectes soc., 11, 301-316 . Montagner, H. & Courtois, G . (1963) . Donn6es nouvelles sur le comportement alimentaire et les 6changes trophallactiques chez les guepes sociales . C .R . Acad. Sci. Paris, 256, 4092-4094 . Morimoto, R. (1960) . Experimental study on the trophallactic behaviour in Polistes (Hymenoptera, Vespinae) . Actu. hym . Fukuoka, 1, 99-103 . Northrop, J. H . (1939) . Crystalline Enzymes. New York : Columbia University Press . Pardi, L. (1950) . Dominazione e gararchia in alcuni invertebrati. Colloq . int . Cent. Nat . Rech . Sci. Paris, 34, 183-197. Rivnay, E . & Bytinsky-Salz, H . (1949) . The Oriental hornet (Vespa orientalis F .) : Its biology in Israel . Agr . Res . Sta . Rehovot, 52,1-32 . Roubaud, E . (1916) . Recherches biologiques sur les guepes solitaires et sociales d'Afrique . La g6n6se de la vie sociale at l'6volution de l'instinct maternel chez les Vespides. Annis. Sci. nat ., 10, 1-160 . Schaefer, C . H . (1964) . Free amino acids in the Virginia Pine Sawfly, Neodiprion prati Dyar . : Their chromatographic determination and biosynthesis . J. Insect. Physiol., 9, 363-369. West, E . S . & Todd, W . R . (1959) . Textbook of Biochemistry. New York : Macmillan . Weyrauch, W. (1936) . Wie entsteht ein Wespennest? Z. Morph. Okol. Tiere, 31, 64-105 . Wheeler, W . M . (1928) . The Social Insects . New York : Kegan Paul, Trench, Triibner . (Received 12 March 1967 ; revised 12 September 1967 : Ms . number : 737)
FOOD EXCHANGE BETWEEN ADULTS AND LARVAE IN VESPA
ORIENTALIS F BY
J . ISHAY
Department of Physiology and Pharmacology, Tel-Aviv University Medical School AND
R . IKAN
Department of Organic Chemistry, The Hebrew University, Jerusalem
A constant exchange of food between adults and larvae has been observed among social insects such as termites, ants and wasps . In the course of such food exchange, the larvae release droplets of secretion and this secretory process has been termed 'trophallaxis' . Scientific opinion was divided until recently as to the significance of trophallaxis . Janet (1895) and Roubaud (1916) believed the oral secretion of the larvae to be highly attractive to the adults . Weyrauch (1936) suggested that this secretion serves to regulate the temperature and humidity in the nest . Brian & Brian (1952) concluded that trophallaxis among wasp larvae is merely an oral method of excretion insofar as the larvae lack any other means of excreting waste products . Montagner (1963), Montagner & Courtois (1963) and Montagner (1964) have studied the food habits of a number of European species of social hornets by means of radioactive gold (Au 198) . They concluded that the males are incapable of feeding themselves and subsist solely on food provided by the workers or by the larvae . Montagner (1963) reported in detail on the characteristic behaviour of adult hornets while visiting the larvae, a behaviour which caused the larvae to release droplets of saliva . He also noted that the old females `demand' food from younger females as well as from larvae . A similar pattern of behaviour has been observed by Ishay (1962) in Vespa orientalis and by Pardi (1950) in wasps of the genus Polistes. Morimoto (1960) fed Polistes larvae on radioactive phosphorus (P 32 ) and was then able to detect radioactivity in the saliva 3 hr after the experimental meal . Maschwitz (1965) reported that the saliva of hornet larvae is highly nutritious, containing sugars and amino acids . All these recent observations confirm Wheeler's (1928) opinion as to the importance of trophallaxis in forming a close bond between the various members of insect families, particularly between the larvae and the nursing workers . The present paper reports on trophallaxis in the Oriental hornet, Vespa orientalis F.
Methods Colonies of Vespa orientalis comprising eggs, larvae of various instars, pupae, workers and a queen, were collected in the field and brought to the laboratory nursery where they were placed in special nests built to simulate the natural habitat (Ishay, 1964) . Members of the colonies were observed while in the nests, where they behaved naturally . Paint markings on the thorax were used to designate either individual hornets or entire colonies, according to the nature of the observation . The food provided consisted of honey, summer fruit, bees and chopped meat . In order to determine the role of the larvae in preserving the social structure of the colony, four experiments were carried out . In the first, two colonies were brought from the field to the laboratory, and all the larvae in one of these colonies were removed from their cells . Both colonies were then maintained under identical conditions and 48 hr later the hornets were allowed to enter and leave the nest freely as well as to fly about within a nest enclosure . In the second experiment, three colonies were raised in the laboratory, but the workers were allowed to forage in the field ; the larvae in all three colonies were removed from their cells . The third experiment involved three colonies in which the combs of the nest were covered with a thin, transparent nylon sheet, separating the larvae from the adults . In the fourth experiment, the adults of three different nests were separated from the larvae by a mosquito net which was placed on the combs . All these experiments were carried out during the months of June, July and August. In order to determine the nature of the larval secretion, another series of experiments was carried out as follows : C14-labelled protein* *Prepared from disrupted Chlorella vulgaris cells which had been grown in a C1402 environment . The labelled protein was freed from lipids by solvent extraction, and from water-soluble compounds by dialysis . 298
ISHAY & IKAN : FOOD EXCHANGE BETWEEN ADULTS AND LARVAE IN INSECTS was fed to larvae and workers, and the saliva composition determined after various time intervals . The labelled protein was obtained from Radiochemical Centre, Amersham, England . The radioactivity was measured in a Nuclear Chicago detector, in a Radiochromatogram Scanner, Packard Model 7200, and in a Vanguard Autoscanner 880 . Larval saliva was collected with a micropipette by tickling the mandibular region of the larvae, thus simulating the method employed by the adult hornets . The saliva of the adults was obtained by applying gentle pressure to the abdominal region of the workers . Larval haemolymph was collected by slightly wounding the skin of the larva with a hypodermic needle . The carbohydrates and amino acids of saliva and haemolymph were separated on ionexchange columns ; carbohydrates were determined by a modified anthrone method (Mokrasch, 1952) and amino acids by micro-Kjeldahl . The proteins in the saliva and haemolymph were separated by Schaefer's (1964) method. The presence of proteases in saliva was determined with gelatine (Northrop, 1939) . Results Direct Observations Larval care . Each worker in the nest spends most of its time caring for the larvae . It pays frequent visits to the `infant pool', probing inside with its maxillae in order to examine the contents. If a larva is detected in the cell, the worker supplies it with meat particles or droplets of food . The larva, in turn, releases droplets of saliva . This behaviour occurs as long as the temperature reading at the nest entry is above 26°C . When the temperature drops below this value, the workers become less active, gathering about the `infant pool' and covering with their bodies all cells which contain larvae . Larval care is for the workers an essential `transition stage' in passing from one duty to another (e .g . from building or repairing cells to foraging for food or guard duty etc .) . It should be emphasized that never have we observed a worker to pass from one assignment to another without first caring for larvae. The workers visit the larvae regularly during all hours of the day and while the temperature is above minimum . Each visit lasts from 2 to 13 sec, depending on the extent of the larva's hunger. Small larvae of the first and second instar are paid an average of fifty-three visits an hour ; medium larvae of instars 3 and 4, are visited
299
seventy-four times an hour ; and larvae of the 5th instar-about ninety-eight times an hour. The big larvae are visited also by the queen, but she comes to receive rather than to give food . This is evident from the fact that occasionally, when the larva is unwilling to provide the queen with saliva, it retracts towards the bottom of the cell as the queen approaches . A hungry queen will plunge her head into the cell until she reaches the mandible of the larva and forces it to release a drop of saliva . The queen's visit usually lasts from 80 sec to 3 min . Sometimes the larvae receive unchewed particles of meat which they are then able to chew by themselves . A morsel of meat about 1 . 5 to 2 mm in diameter is chewed up by big larvae within 3 to 4 min . Once they have chewed and swallowed the food offering, the bigger larvae (5th instar) produce specific sounds by rubbing their mandibles against the cell wall . These sounds are clearly audible to human ears and serve as a signal to the workers to bring more food . The small larvae are incapable of producing these sounds because they are unable to reach the cell wall with their mandibles . The feeding problem here is resolved by special workers who pay regular visits to the cells and upon detecting a hungry young larva, tap the tip of their abdomen against the cell walls . The tapping noises produced by such workers serve as a signal to other workers to come quickly and feed the larvae (Schaudinischcky & Ishay, in prep .) . Adult-larva interdependence . In the first experiment, in which the larvae were removed from one of two colonies, only the colony retaining its larvae continued life as usual ; the other colony dwindled gradually despite renewed oviposition by the queen, because the workers failed to care for the new-born larvae . Six days later, the nest was abandoned . This experiment was repeated twice more with identical results . In the second experiment, in which larvae were removed from three colonies whose workers were free to fly to the field, all three nests were abandoned within 11 days, the queens having died in the nests . A few of the hornets succeeded in joining nearby colonies which did carry larvae. In the third experiment, in which the combs of three nests were covered by thin, transparent nylon sheets, separating larvae from the adults, the adults left the nest within 4 days, and the queens were found dead on the floor of the nests. In the fourth experiment, in which the combs of three colonies were covered by mosquito
3 00
ANIMAL BEHAVIOUR, 16, 2-3
nets, the adults remained in the nest and on the first day made futile attempts to tear the nets apart . In the meantime, adult hornets newly hatched from the net-covered combs, commenced mediating between the hornets on the outside and the larvae within, passing particles of meat from the adults to the larvae, and droplets of saliva from the larvae to the adults . On the second day, the workers started building new combs outside the net-covered areas ; the queens immediately oviposited in the new cells, and the colonies continued life as usual . Experimental Data Larval saliva. In Table I are given the quantities of saliva formed by the different larval instars which can be transferred to the adults by means of trophallaxis . Table I. Maximum Quantity of Saliva Released by Larvae Isolated from the Adults for Twelve Hours (Data Based on Sixty Larvae from Each Instar)
Instar
Average weight of larva (in mg)
Average volume of saliva (in ml)
Average volume of saliva per body weight (in ml/mg)
1
4.4
0 . 0004
9 . 1 x 10 - 5
2
33 . 4
0 . 0018
5 .4 x 10- 5
3
178 .0
0 .0025
1 .4 x 10- 5
4
292 . 6
0 .0051
1 . 7 x 10 - 5
5
919 . 3
0 . 0024
2 . 6 x 10 - 6
It is interesting to note that except in one instance (instar 4), the saliva volume to body weight ratio decreases with increasing weight of the larva . In Table II are given the carbohydrate and protein contents of the crude saliva and the haemolymph of 5th instar larvae . Table II . Percentage of Carbohydrate and Protein in the Crude Saliva and the Haemolymph of Fifth Instar Larvae (Average of Twenty Determinations)
Fluid
Total Total carbohydrates nitrogen
Nitrogen of free amino acids
Saliva
5 . 50
0 . 21
0 . 13
Haemolymph
2 .05
1 . 37
0 . 24 •
T his value approaches the maximum recorded so far in insects (Duchateau & Florkin, 1958) .
As is clear from Table II, sugar concentration is considerably higher in larval saliva than in haemolymph . On the other hand, there are fewer nitrogenous substances in the saliva than in haemolymph . Table III. Determination of Carbohydrate Concentration in Larval Haemolymph at Different Hours of the Dayt Time
Total carbohydrates in haemolymph (in %)
0400
2 . 15
1000
1 .73
1800
2 .40 tAverage of sixty determinations .
The data in Table III suggest that the carbohydrate concentration in the larval haemolymph is lowest during the time of maximal worker activity. Composition of carbohydrates in the saliva and haemolymph of larvae and workers . The chroma-
tography was carried out with butanol-acetic acid-ethyl ether-water (9 :6 :3 :1) as mobile phase and various mono-, di- and oligo-saccharides as markers . The radioactivity was measured in a Packard Model 7200 Radiochromatogram Scanner. The findings were as follows . (a) In larval saliva : glucose, fructose, sucrose, maltose, trehalose, 4G-a-glycosyl-sucrose and oligosaccharides . (b) In larval haemolymph : glucose, fructose, disaccharide (unidentified), trisaccharide (unidentified) and tetrasaccharide (unidentified). (c) In worker saliva : glucose only. (d) In worker haemolymph : glucose, fructose, maltose, trehalose, 4G-a-glycosyl-sucrose and oligosaccharides (unidentified) . Feeding of 5th-instar larvae with C 14-protein . Fifth-instar hornet larvae were given C14labelled protein by mouth . Equal samples of saliva were withdrawn at regular intervals and stored at - 30 ° C . Minute samples of saliva were placed on 5 cm x 20 cm glass plates (0 . 25 mm thick) . The chromatography was carried out as before . As can be seen from Table IV, glucose formation reaches a peak 3 hr after the labelled protein meal and then declines . When labelled protein was fed to workers which had been kept away from the larvae from the time of eclosion, no degradation of the protein occurred, that is, no sugars were found . Furthermore, no proteases were detected by
ISHAY & IKAN : FOOD EXCHANGE BETWEEN ADULTS AND LARVAE IN INSECTS
301
Table IV . Saliva Composition of Fifth-instar Hornet Larvae at Various Time intervals after Feeding on C 1 4-labelled Protein' Hours after labelled protein meal' •
Material in larval saliva
3
4
5
20
fr
2
Protein C 14
11
8.7
1
1
1
1
Dissaccharide C 14
-
-
3.4
1 .6
2.1
3 .6
5 .8
82
Glucose C 14
1
1
14
25 .4
'Each datum is an average of ten samples and is expressed in relative area units . 'A different group of ten larvae each was `milked' at each of the designated time intervals . No incubation of saliva was carried out, as it was assumed that such occurred in vivo, body temperature of the larva being approximately 30°C . means of gelatine in the saliva of adults who had been separated from the larvae for a period of one week. In vitro incubation of crude 5th-instar larval saliva with C 14-protein. Five ml of crude 5thinstar larval saliva was incubated at 30 ° C with samples of C 1 4-protein . Measurements were made with the use of a Vanguard Autoscanner 880 . The results of this incubation are presented in Table V. It should be pointed out that the saliva used in the above experiment was crude, i .e . it had not undergone centrifugation . We cannot be certain, therefore, that the observed protein degradation is attributable to the fluid part of the saliva and not to its cellular components . Incubation of hornet homogenate with C 14labelled protein . The homogenates were prepared by crushing larvae, pupae or workers with ultra-turrax in water . C 14-labelled protein was then added and the suspension was kept at 30°C for 20 hours, then chromatographed on activated charcoal and celite (1 :1) column, and developed with distilled water followed by aqueous ethanol up to 50 per cent . The fractions obtained were concentrated in vacuo and fractionated on silica gel chromato plates as described . The radioactivity of glucose and protein only was measured, although other carbohydrates
and free amino acids may also have been present . The results shown in Table VI suggest that the ability to degrade proteins is lost immediately after pupation . Indeed tests for protease activity, using gelatine, failed to show protease activity in the saliva of workers which had been removed from the larvae immediately upon eclosion . In contrast, larval saliva showed clear-cut protease activity . Discussion The strong attraction of the adult hornets to the secretion of the larvae has already been described earlier . This larval secretion has been shown to contain 5 . 5 per cent sugars (mono-, diand oligo-saccharides) and 1 . 3 per cent protein and free amino acids . Despite the low concentration of these compounds in the saliva, it can still be regarded as nutritious . It is interesting to compare the amount of sugars and nitrogenous compounds in the saliva of hornet larvae with that in Royal Jelly of bees or in mammalian milk . Such a comparison is made in Table VII . The concentration of sugars in Vespa orientalis larval saliva is low compared with that found by Maschwitz (1965) in the larval saliva of European wasps, but the concentration of nitrogenous compounds is very much the same . Indeed, the protein concentration in the larval saliva is
Table V . In Vitro Incubation of Crude Fifth-instar Larval Saliva with C t 4-labelled Protein (Average Ratios from Six Samples) Material detected
Hours of incubation 1
Glucose C t 4/Protein C
14
6 .6/1
3 11 . 3/1
5 33 . 6/1
6 17 . 7/1
3 02
ANIMAL BEHAVIOUR, 16, 2-3 Table VI. Labelled Sugars in Homogenates of Various Hornet Stages after Twenty Hours of Incubation With C14-labelled Protein Test homogenate*
Labelled sugars
Labelled proteinglucose ratio
Fat body of 5th-instar larvae
present
1 :5 . 2
5th-instar larvae just before pupation
present
1 :3 . 3
Pupae before metamorphosis
absent
Pupae after metamorphosis (bright)
absent
Workers, 5 hr after eclosion**
absent
Workers, 2 days after eclosion**
absent
Workers, a week after eclosion**
absent
*In each case six samples were taken from the 5 g homogenate . **From eclosion till examination time, the workers were kept isolated from the larvae. Table VII. Percentage of Sugar and Protein in Hornet Larval Saliva, Royal Jelly of Bees and Mammalian Milk
Materials
Larval saliva of Vespa orientalis ph 4 . 5
Larval saliva of Vespa vulgaris, V. crabro and Vespula germanica (after Maschwitz, 1965)
Royal Jelly from the cells of bee larvae 3 to 4 days old ; ph 4 to 4 .5 (after Decourt, 1956)
Mammalian milk (after West & Todd, 1959) human
bovine
Sugars
5 .5
8 .9
12. 5
7 .2
4.9
Proteinst
1 .3
1 .8
12.3
1 .5
3.5
tThe percentage of proteins is calculated by multiplying the total nitrogen (N) by the factor 6 .25
close to that in human milk . The concentration of sugars and nitrogenous compounds in Royal Jelly is quite high, compared with that in vespan saliva . It should be remembered, however, that the feeding of apian larvae is totally different from that of vespan larvae in that Royal Jelly, like mammalian milk, is an adult secretion intended as food for the larvae, whereas the larval saliva of hornets is intended as food for the adults and is given in return for meat particles provided by the adults . Continuous observations (Ishay, 1964) have shown that the queen of the Oriental hornet will not leave the nest as long as there are a number of workers in it. During this period, which lasts from June to December, the queen feeds solely on the larval secretion, and when the larvae are removed from the nest, it dies of starvation . The average weight of the nest-founding queen is 746 mg. It has been stated by Janet (1895) and by Rivnay & Bytinsky-Salz (1949) that the queen lays about 1500 eggs per season, but
according to Ishay & Ikan (in preparation) the annual number of eggs is over 4000. Each egg weighs about 2 mg, which means that the total weight of the eggs is 3000 to 8000 mg, or four to eleven times the queen's weight in the spring . The weight of the queen's ovarioles in the spring, before the start of oviposition, is 9 to 10 . 1 mg, whereas their weight during the height of oviposition (in September) is 153 -1 mg, or more than fifteen times their previous weight . Imagine, then, the amount of larval secretion necessary to account for this remarkable increase in weight, for it is the larvae who provide the queen with the raw material needed for developing its load of eggs . Equally benefited are the workers who `milk' the larval secretion during breaks between foraging trips, and the males who collect larval saliva droplets whenever and for as long as they come in contact with the larvae. The findings of the present paper clearly indicate that the colony cannot survive without
ISHAY & IKAN : FOOD EXCHANGE BETWEEN ADULTS AND LARVAE IN INSECTS
larvae, for the adults are soon forced to leave the nest or die of starvation . Only the larvae are able to digest proteins and degrade them into amino acids and sugars . A similar situation exists also among butterflies (Lepidoptera), where only the larvae feed on proteins, while the adults feed merely on the carbohydrates present in nectar . So far as is known, the only digestive enzyme present in adult Lepidoptera capable of feeding is invertase ; starch, fat or protein mixed in the food remain unchanged in the stomach (Snodgrass, 1961, as cited by House, 1965) . Moreover, the adults of many Diptera, Hymenoptera, Lepidoptera and other insect orders, are known to subsist quite adequately on sugar solution alone (House, 1965) . That only larvae digest proteins is a common enough phenomenon among insects, but in the case of wasps this has especial significance, for the degradation products of proteins are transferred from the larvae to the adults and serve as raw materials for nitrogen metabolism, which is especially important for the queen, who produces the eggs . Consequently it is clear that social interrelations are a sine qua non for Oriental hornet populations, and that the phenomenon of trophallaxis helps to create an organic bond between the various members of the colony . Summary Colonies of Vespa orientalis cannot exist without the larvae or, more specifically, without larval salivary secretion, which is the only source of raw materials for nitrogen metabolism and egg production available, to the workers and the queen, who do not themselves possess the proteases necessary for protein degradation . This finding suggests that social life among wasps is probably a symbiosis between the adults and larvae . Acknowledgments This study was partly supported by a grant from the Israel Foundation Trustees for Research and Education . It is part of a Ph .D . thesis carried out by J . Ishay at the Hebrew University of Jerusalem . The authors are indebted to Mrs Leslie Mandl, of the Israel Pharmacological Trust, and to Dr J. Lengy of the Tel-Aviv University, for critical review of the manuscript . REFERENCES' Brian, M . V. & Brian, A. D . (1952) . The wasp Vespula sylvestris Scopoli : Feeding, foraging and colony development . Trans. R . Soc. Lond., 103, 1-26.
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Decourt, P . (1956) . La gel6e royale d'Apis meiifica et son activit6 chez les vert6br6s sup6rieurs . Revue Path. gin . Physiol. clin ., 682, 1641-1663 . Duchateau, G . & Florkin, M . (1958). A survey of aminoacideias with special reference to the high concentration of free amino acids in insects' haemolymph . Archs. int. Physiol. Biochim ., 66, 573-591 . House, H . L . (1965) . Chapter on Digestion. In The Physiology of Insects ( ed . b y M . Rockstein) . Vol. 2, pp . 815-853 . Ishay, J. (1962) . Observations on the biology of the oriental wasp Vespa orientalis F . in Israel . M .Sc. Thesis, Tel-Aviv University (in Hebrew) . Ishay, J. (1964) . Observations sur la biologie de la guepe orientate Vespa orientalis F . Insectes roc., 11, 193-206. Janet, Ch. (1895) . Etudes sur les fourmis, les guepes et les abeilles . Mem. Soc . Zool. Fr., 8, 1-140. Maschwitz, U . (1965) . Larven als Nahrungsspeicher im Wespenvolk (Ein Beitrag zum Throphallaxieproblem) . Verh . dt . zool. Ges., 50, 530-534 . Mokrasch, L. C . (1952) . Analysis of hexose phosphates and sugar mixture with the anthrone reagent . J. Biol. Chem ., 208, 55-59. Montagner, H . (1963). Etude preliminaire des relation entre les adultes et le couvain chez les guepess sociales du genre Vespa au moyen d'un radioisotope. Insecles roc. 10, 153-165 . Montagner, H . (1964) . Etudes du comportement alimentaire et des relations trophallactiques du mile au sein de la soci6t6 de guepes . Insectes soc., 11, 301-316 . Montagner, H. & Courtois, G . (1963) . Donn6es nouvelles sur le comportement alimentaire et les 6changes trophallactiques chez les guepes sociales . C .R . Acad. Sci. Paris, 256, 4092-4094 . Morimoto, R. (1960) . Experimental study on the trophallactic behaviour in Polistes (Hymenoptera, Vespinae) . Actu. hym . Fukuoka, 1, 99-103 . Northrop, J. H . (1939) . Crystalline Enzymes. New York : Columbia University Press . Pardi, L. (1950) . Dominazione e gararchia in alcuni invertebrati. Colloq . int . Cent. Nat . Rech . Sci. Paris, 34, 183-197. Rivnay, E . & Bytinsky-Salz, H . (1949) . The Oriental hornet (Vespa orientalis F .) : Its biology in Israel . Agr . Res . Sta . Rehovot, 52,1-32 . Roubaud, E . (1916) . Recherches biologiques sur les guepes solitaires et sociales d'Afrique . La g6n6se de la vie sociale at l'6volution de l'instinct maternel chez les Vespides. Annis. Sci. nat ., 10, 1-160 . Schaefer, C . H . (1964) . Free amino acids in the Virginia Pine Sawfly, Neodiprion prati Dyar . : Their chromatographic determination and biosynthesis . J. Insect. Physiol., 9, 363-369. West, E . S . & Todd, W . R . (1959) . Textbook of Biochemistry. New York : Macmillan . Weyrauch, W. (1936) . Wie entsteht ein Wespennest? Z. Morph. Okol. Tiere, 31, 64-105 . Wheeler, W . M . (1928) . The Social Insects . New York : Kegan Paul, Trench, Triibner . (Received 12 March 1967 ; revised 12 September 1967 : Ms . number : 737)