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Glucose levels in Vespa orientalis: The effect of starvation
Comp. Biochem. Physiol.. 1975. Vol. 52A. pp. 91 to 96. Pergamon Press. Printed in Great Britain
GLUCOSE LEVELS IN VESPA ORIENTALIS: THE EFFECT OF STARVATION JACOB ISHAY Department of Physiology and Pharmacology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel
(Received 20 June 1974)
Abstract--I. Distinct differences in glucose level have been found in the various tissue fluids of the larvae as well as between the different age groups and castes. The glucose level gradient order in the larvae is: saliva (2000-5000 rag%)--* haemolymph (100-200rag%)---. midgut (zero or near zero mg%). 2. During starvation the larval midgut is utilized to a greater extent than other tissues. 3. The glucose level in larval haemolymph may be altered by feeding the larvae on substances such as dextrose, distilled water, alcohol or alloxan. 4. The larvae saliva contains a factor (or factors) which produces hyperglycemia in larval haemolymph in vitro. Addition of mammalian insulin partially depresses the resultant hyperglycemia.
INTRODUCTION
colony were sorted according to age (e.g. larvae of instars 1-3 or 4-5) and sex, and their body fluids and tissues separated as rapidly as possible into test-tubes chilled at 0°C. In view of the finding that in haemolymph standing at room temperature, the glucose level tends to rise spontaneously (Fischl & Ishay, 1971), most of the determinations were made very close to the time of body-fluids withdrawal from the hornets, or in cases where frozen material was tested--immediately upon its thawing. Larval saliva was collected by gentle tickling of the oral region, larval haemolymph--by puncture and aspiration, larval midgut fluid by squashing of dissected midguts, and larval fat-body by dissection. The gastric juices of adults were expressed by pressure on the abdomen. Tissue fluid samples were taken both immediately as well as following starvation for several days. The various determinations were carried out on either fresh samples or on ones that had undergone prolonged freezing at -20°C. In every instance the test material was first centrifuged at x 2000 g for 20min and the supernate was used for the various determinations. Glucose determination, as based on glucose oxidase, was done according to the method of Kingsly & Getchell (1960), usually at 27°C and at pH 7. Dialysis of haemolymph or larval saliva was carried out against distilled water at 0°C for 24hr. The insulin was protamine Zn insulin 80 U/ml (Rafa Co., Israel). Hydrolysis was performed by boiling the samples for 3hr in a I N HCI solution in a water bath.
THE PHENOMENON of food-exchange between the various colony members of social insects has been called trophallaxis by Wheeler (1928) a n d the term is used now by most students of social insects to imply mainly the transfer of alimentary contents in liquid or solid state, either mutually or unilaterally. A m o n g social wasps it has been proven that in the course of food exchange between the adults a n d the larvae, the adults offer the larvae bits of meat a n d receive, in return, droplets of larval saliva which are extremely rich in sugar (Ishay & Ikan, 1968). In the case of Vespa orientalis, for instance, larval saliva contains 4 0 0 0 - 5 0 0 0 m g % glucose whereas the glucose content of larval h a e m o l y m p h ranges only between 10 a n d 200 rag% (Fischl & Ishay, 1971). This finding raises many interesting questions. Is there, for instance, a difference in the glucose level of larvae at different stages of development or belonging to different castes? Does starvation affect the glucose level? W h a t could be the reason for such a high glucose concentration in larval saliva? Are any hyperglycemic factors involved? The present study was instigated in an attempt to answer some of these questions t h r o u g h systematic determination of the glucose level in the various tissues of the different castes of the Oriental hornet.
RESULTS MATERIALS AND METHODS
Glucose levels in various tissues of Vespa orientalis
Body fluids of numerous hornets, all of the same species, age, sex and caste, were pooled. In nature, individuals of all the various castes occur concurrently only during a certain period of the year, and it was hence necessary to carry out the determinations on fresh material during August-October, when the colony comprises thousands of individuals at all stages of development. Such complete colonies were sought in the field and upon discovery, were brought in toto (under aether anaesthesia) to the laboratory during the early morning hours, when the workers are still in the nest. In the laboratory, the members of each
Figure 1 summarises in histogram form the data o n different glucose levels in members of a single hornet colony. Each d a t u m is a mean of three determinations carried o u t o n fresh material immediately after removal of the nest from its natural habitat. The data have been arranged in accordance with the different functional groups or castes. As can be seen in Fig. l, there are marked differences in glucose levels: (a) between various tissues of the same hornet; (b) between the different age groups within the same 91
92
JACOB ISHAY iooo I--
caste of hornets; and (c) between hornets of the same age group (stage) but of different castes. On the other hand, certain tissues of all members of the colony show a uniform or almost uniform glucose level. As for the differences in glucose level, the following conclusions may be drawn:
Males
6 0 0 I--
9 n
4001--
2 0 0 I--,..L, I I ~ Workers
4000~
rml
1. For the larva, there is invariably a glucose level gradient of the various tissues (or fluids) whose order is saliva (2000-5000 mg%)--* haemolymph (100200 rag%)--, midgut (zero or near zero mg%). 2. The glucose levels in larvae of instars 4-5 are invariably higher than in larvae of instars 1-3. 3. There is also a glucose level gradient for the larvae of the different castes, whose order is queen larva --~ worker larva---, male larva.
3000
2000 0
Q .£
I000
9 I-I . . . .
200 ~
Queens
5000
I Eggs 2 Saliva larvae :5 Hemolymph 4 Midgut 5 Saliva larvae 6 Hemolymph
8 4000
7 Midgut
I-3 I-3 I-3 4-5 4-5 4-5
8 Hemolymph prepupae 9 Hemolymph pupae IOHemolymph imagines I Gastric juices
3000
2000
I000 800 600 400 200
9 i-] Fig. 1. Glucose levels in various tissue fluids of Vespa orientalis. All data was transformed to logarithms (natural) and the analysis performed (L = line, R = repeat, T = time). The differences between the glucose level in the tissue fluid larvae (saliva, haemolymph, midgut) is significant (P < 0.0005). The differences between the glucose level in saliva of l-3rd instar and that of 4--5th instar is significant (P < 0.0005). Same degree of significance between glucose levels saliva of male, worker and queen larvae.
The highest glucose levels are recorded in larval saliva. For male larvae of instars 1-3, for instance, the glucose level in the saliva is about three times that in the haemolymph, while for queen larvae of instars 4-5, this ratio rises to 100: 1. There are, however, also instances where the glucose levels are similar. Thus there is the same glucose level (very low and approaching 0 mg%) in the midgut of all stages from all castes, while the glucose levels in the haemolymph of prepupae and pupae are quite similar. For comparison purposes, glucose level determinations were carried out in five additional colonies. Despite some variability in the results for the different colonies, the general picture was essentially the same and the level ratios were quite similar. Glucose levels in starvation
Figure 2 summarises in histogram form the data on the glucose levels in saliva and haemolymph of 5th instar larvae starved for up to 9 days. From the data, several findings may be noted: I. During starvation, the glucose level in the saliva drops with time but is always higher than in haemolymph.
V. orientalis- Worker larvae 6000
Starvation
5600
5200
Glucose level in saliva (top)and in hemolymph(bottom)
o_
4400
.c
4000!
E
- -_i
-
30 oVo d e x t r o s e - a d / / b d u r n
Starvation
4800 0
Queen larvae
-
!
Distilled w o t e r - a d lib#urn
' 5600 I"
Saliva
5200 -
_~
2800 .-
_.m
2400
o
2000 t600 1200
~
80O 4O0
II
0
.>,IB114
.~ 1614
6 t6
017
1~161gC
204 II *2 .=0~04~
I
day
2 5 4 5 6 7 6 9
doy
254567
9
doy
125456
69
doy
125456
8 9
Fig. 2. Glucose level in larvae haemolymph and saliva during 9 consecutive days of starvation. The differences between the glucose level in haemolymph and saliva are significant in every experiment and in every day (P < 0.0005). The increase in glucose level in haemolymph after feeding the larvae with 30% dextrose solution is significant (P < 0.05). The decrease in glucose level upon starvation is significant by worker larvae starting day 5 and by queen larvae starting day 6 (P < 0.005).
Glucose levels in Vespa orientalis Table 1. Weight loss following starvation of 5th-instar larvae
93
Table 2. Effect ofstarvation on midgut weightin 5th-instar larvae
Hours of starvation
Mean larval body weight (g)
S.D.
Hours of starvation
Mean weight of larval midgut (g)
S.D.
48 72 96
1.60400 1.53185 1'37030
0.095502 0.089454 0'104878
48 72 96
0.2232000 0.1940741 0-1733333
0.0665 0.0693 0'0723
2. The glucose level in queen larval saliva is more "stable" than in worker larval saliva. 3. The imbibation of distilled water produces a stabilisation of glucose level in saliva but an instability of the glucose level in haemolymph, whereas upon feeding on dextrose, there is gradual increase in the glucose levels of both saliva and haemolymph. The tall columns in the histogram represent the glucose level in saliva. The numbers within the columns represent the glucose levels in haemolymph. In all the experiments, unfed larvae started dying after 9 days of starvation, and the experiments were terminated at this stage. It should be noted, however, that individual larvae, whether totally starved or offered water or dextrose, survived up to 16 days but rarely attained the cocoon-spinning stage.
Determination of weight loss following starvation For this purpose, a comb containing some 300 queen larvae at 5th instar was separated from center to periphery into three equal segments and the larvae from each were removed for weighing on three consecutive days. Results were as in Table 1. deviation The coefficient of deviation V = standard mean was 0.15854 after 48hr, 0.12515 after 72hr and 0'28066 after 96 hr. Thus there was at first a slight drop in the value of this coefficient, which was followed by a sharp increase. As for the rate of weight loss: The average weight of larvae on the 3rd day (72hr) was 95.502~ that on the 2nd (48 hr), while that of larvae on the 4th day was 89'454~ that on the third day. The midgut from larvae starved under the same conditions was weighed separately, with results as in Table 2. The coefficient of deviation was 0.08354 at 48 hr, 0'12723 at 72 hr and 0-21953 at 96 hr. There was thus marked increase in the value of this coefficient after every additional day of starvation. As for the rate of weight loss: On day 3, the average weight was 86"95170 that on day 2, while on day 4, the average weight was 89"313~o that on day 3. From these results, it is clear that from day 2 (48 hr) to day 3 (72hr), there is greater weight loss of the midgut than of the total body. The midgut weight drops by about 13~ whereas the total body weight drops by about 4.5~. Weight of the midgut dropped, on average, by 29-1 mg whereas that of the body dropped by 72'1 mg. Thus, weight loss of the midgut constituted about 40.6~ of the total weight loss. From day 3 to day 4, weight of the midgut dropped approximately of the same order as the total body weight (by about 1170). Weight of the midgut dropped an average of 20.7mg. Total body weight dropped an average of 161'6rag. Hence the drop in midgut weight constituted only 12.970 of the total weight loss.
Since larval saliva was found to contain such a high glucose level, it was deemed interesting to ascertain the yield of glucose per larva per single daily milking following various treatments. All larvae were of the same colony and at the 4th-5th instar. They were placed in small combs of 25-30 larvae each, which were maintained in the shade at 27°C. Feeding, when carried out, was by pipette, ad libitum, twice daily. The larvae were milked once daily in the morning in accordance with the feeding procedure adopted. Results are summarised in Table 3. As expected, there was in the course of starvation, a decrease in the amount of glucose secreted, but surprisingly there was no drop in the amount of liquid secreted in the saliva. When distilled water was offered, there was increase both in the amount of fluid secreted as well as in the amount of glucose. Dextrose feeding produced increase both in total fluid and in glucose (dextrose) secretion, which was reflected in the marked increase in glucose levels. Alloxan feeding resulted in a sharp drop (to about 5070) in the total amount of fluid secreted and also marked decrease in the glucose per larva. The final result, however, was a marked increase in the glucose level! On day 4, all the larvae which had received alloxan died. Alcohol feeding produced a gradual but marked drop in the amount of fluid secreted and a parallel drop in the amount of secreted glucose, but the final result was only a slight drop in the glucose level. On day 4, there was no secretion of saliva! Otherwise, however, the larvae behaved normally, retaining their mobility and continuing to produce hunger signals (Schaudinischky & Ishay, 1968) as usual. From all the determinations carried out thus far, it is clear that the glucose level in larval saliva is markedly higher than in larval haemolymph, and this both in well-fed, satiated larvae as well as in larvae under prolonged starvation. The high glucose level in larval saliva could be due to various reasons, like the following two, for instance: 1. The possibility that the glucose in the saliva is derived from internal tissues and that the cells lining the salivary ducts polarise and filter it out to the lumen of the ducts, whence it is secreted in concentrated form. 2. The possibility that the fluid secreted into the lumen of the salivary ducts contains factors which produce hyperglycemia in saliva only, whether by the absorption of glucose from the haemolymph via the cells lining the ducts or by the absorption of glucose precursors from the haemolymph. To resolve this problem, larval saliva was incubated with larval haemolymph. As pointed out earlier, larval saliva shows a relatively very high glucose level and contains both di- and polysaccharides (Maschwitz, 1966; Ishay & Ikan, 1968), so that direct incuba-
JACOB ISHAY
94
Table 3. Glucose yield per larva under various dietary conditions
Treatment
Saliva amount per l daily milking (cm 3) S.D.
Trial day
Total starvation Total starvation Total starvation Total starvation Offered distilled water only water only water only water only Offered 30~ Dextrose Sol. Dextrose Sol. Dextrose Sol. Dextrose Sol. Dextrose Sol. Dextrose Sol. Offered alloxan I0 mg/ml in distilled water distilled water distilled water Offered a 10~ solution of ethyl alcohol ethyl alcohol ethyl alcohol
0.0110 0.0110 0.0200 0.0125
0.0036 0.0037 0.0029 0.0027
3090 2844 2964 2022
0.34000 0.31284 0.54280 0.25270
I 2 3 4
0'0110 0.0100 0.0250 0.0250
0.0036 0.0030 0.0070 0.0065
3090 3258 3418 3372
0.34000 0.32580 0.85450 0.84300
1 2 3 4 5 6
0.0110 0.0250 0.0750 0.0142 0.0200 0.0200
0.0036 0.0040 0.0021 0.0034 0.0051 0.0031
3090 3420 4758 4260 3630 4176
0.34000 0.85500 0-35680 0.60500 0.72600 0'83520
1 2 3
0.0110 0.0050 0.0058
0.0036 0.0021 0.0038
3090 3540 4584
0.34000 0.17700 0.26587
1 2 3
0.0110 0.0090 0.0058
0.0036 0.0024 0.0031
3090 2970 2760
0.34000 0.26730 0.16008
L a r v a l hemolymph Vespa oriental/l~
6500 -
Hemolyrnph Hemolymph + 12U insUlin/ml Hernalymph + larval saliva (dialysed) I:1 Hernolymph larval • s a l i v a ( d i a l y s e d ) ÷ 12U insulin
6000
3 5500 5000E o
(2_
4500
4000
3500
4
30OO _D 2 5 0 0 '..9 2000
1500 I000
I
500
30
I
I
60
90
I 120
I -"T~-~r 150
Glucose yield/larva single daily milking (mg)
I 2 3 4
tion of larval saliva with haemolymph could only yield misleading results. Hence, larval saliva was first dialysed in the cold (at 0°C) for 24 hr against distilled water, in order to remove all materials of low molecular weight, and only the dialysate was incubated with haemolymph. A typical result of such incubation is given in Fi~. 3. In connection with Fig. 3, it should be pointed out that similar results were obtained also by incubat-
I 2 3 4
Glucose level in larval saliva (in mg%)
~80
2 210
240
rain
Fig. 3. Incubation together of larval haemolymph and dialysed larval saliva, with and without insulin. Line 2 is less than line 1 at all times (P < 0.0005). Line 1 is less than lines 3 and 4 at all times (P < 0.0005). Line 4 is less than line 3 at all times (P < 0.0005).
ing saliva from Paravespula germanica larvae with haemolymph from Vespa orientalis larvae, and vice versa, which means that the phenomenon is not speciesspecific. Neither is the phenomenon stage-specific, inasmuch as similar results were obtained also by the incubation of larval saliva with prepupal or pupal haemolymph. F r o m the results shown in Fig. 3, the following merit attention: (a) Incubation of haemolymph with larval saliva that had undergone dialysis and whose glucose level is 0 mgVo, causes a sharp rise in the glucose level of such incubated haemolymph, relative to the glucose level in unincubated haemolymph. This rise in glucose level continued in the experiment for ~ hr, following which there was a slight decline. (b) The incubation with insulin of equivalent amounts of haemolymph, resulted in a drop in the glucose level. The hypoglycemia was slight in incubations of insulin and normal haemolymph, but became very pronounced in incubations of insulin with haemolymph that had undergone prior incubation with dialysed larval saliva. (c) After several hours of incubation of haemolymph with larval saliva, the glucose level approximated or even exceeded the ordinary glucose level of larval saliva. Boiling of the larval saliva prior to its incubation with haemolymph, brought to a stop its hyperglycemic activity. This finding, coupled to the fact that the active factor in saliva is non-dialysable, leads to the conclusion that we are dealing here with the activity of a protein or protein-bound substance, whose mol. wt is greater than 10,000. DISCUSSION
Till now, all tests to determine the glucose level in various body fluids of wasps or hornets have been
Glucose levels in Vespa orientalis run on randomly selected insects from various colonies. In the present study, individuals from the same colony have been used, the various determinations being carried out immediately upon collection of the body fluids. The importance of this has been established in a previous study (Fischl & Ishay, 1971), which showed that the glucose level in the body fluids of wasps rises during the first hours after their extraction. Some primary findings of the present study merit mention: 1. The occurrence of a distinct and constant gradient of the glucose level between the various tissues of the same individual insect, between the various age groups of the same caste and between individuals of the same age groups but of different castes. 2. Starvation or the various treatments, do alter the glucose level in the body fluids of hornets, but there is nevertheless a marked difference in glucose level between larval saliva, whose glucose content is always very high, and larval haemolymph. 3. During the first 2 days of starvation, the midgut contracts and dwindles more than the other body tissues. It is reasonable to assume that weight loss of the midgut is due to the fact that the food contents of the midgut are being used up, but it may also be that stored food reserves in it are being utilised. In this connection, it should be mentioned that in a previous study (Ishay et al., 1971), it was found that larval midgut contains 11.4-18"6~ lipids as dry weight. Insofar as adult hornets are essentially predatory insects, one can reasonably assume that they forage for and feed the larvae primarily on animal proteins (e.g. other insects or bits of flesh from the carcasses of vertebrates). This strengthens the impression that the lipids found in such high concentration in larval midgut actually serve as a food reserve and are the first to be utilised upon starvation of the larva. 4. Larval saliva contains factors which cause in vitro hyperglycemia of larval haemolymph. It is not clear yet whether one or several mechanisms are involved in this process, i.e. whether the rise in glucose level is due to the degradation of haemolymph polysaccharides or rather due to the formation of glucose de novo by way of gluconeogenesis. The problem requires further investigation. Hyperglycemic factors have been detected also in larval midgut (Ishay et al., 1973), but in this case it has been clearly demonstrated that the process is in part due to gluconeogenesis, inasmuch as incubation of midgut with haemolymph and C14-glutamic acid has shown incorporation of the labeled substance into C14-gtucose. However, there is this difference between midgut and larval saliva, in that the former is an internal, normally irremovable organ while the latter is regularly being passed on to the adult hornets in the nest. Does this transfer of hyperglycemic factors from larvae to the adults suggest that the adults lack such factors? It can be assumed that the larval transfer of saliva to the adults in the course of the process named trophallaxis, may serve two purposes: (a) to provide the adults with a food rich in carbohydrates so as to enable them to maintain the necessary nest functions between foraging trips to the field, which could explain the high glucose level of larval saliva; and
95
(b) that both the glucose and hyperglycemic factors in the saliva can participate in the process of detoxication in which substances like phenols and their derivatives are converted into the corresponding /3-Dglucosides. Such reactions normally occur in the insect fat-body (Hines & Smith, 1963). It seems that the acceleration of glucose formation by factors in larval saliva, larval midgut, larval fat-body or other tissues, may serve the latter purpose, that is to say, if more glucose is available there is greater potential for conjugation of phenol compounds which implies enhanced potential for detoxication. In the conjugated form, the phenols are not subjected to oxidation by phenolase (Brunet, 1965). It is reasonable to expect such accelerated ability of detoxication to be found in the digestive tract of the larvae: as is known, in the larvae of almost all the Apocrita, the midgut is a blind sac and does not communicate with the hindgut until the final instar, the faecal contents only being evacuated at the conclusion of the larval stage (Imms, 1960). It is similarly reasonable to expect to find a capacity for detoxication in the larval saliva which is offered to the adults. As already mentioned, the adult hornets, when foraging in the field, collect food from various sources, but chiefly flesh from carcasses or other organic matter in different stages of putrefaction, so that it is quite likely that the adult hornets utilise materials in larval saliva to form conjugates for the purpose of detoxication. In a previous study (Saslavsky et al., 1973), it was found that toxic materials have less effect on adult hornets in a natural, larva-containing nest than on hornets in a larva-less nest, which strongly suggests that the larvae play a r61e in detoxication processes. A similar finding was reported by Derevici & Nitu (1971) who observed that DDT was less toxic to bees in larva-populated hives (in the spring) than in larva-free hives (in the winter). 5. Bovine insulin counteracts hyperglycemia h7 vitro. Incubation of haemolymph together with insulin retards the rise in glucose level if the starting level is low, or causes a sharp drop in the glucose level if the starting level is high (Fig. 3). It should be noted in this connection that the induction of clear-cut hypoglycemia required the use of a relatively high concentration of insulin (12 units insulin/ml haemolymph). At the same time, the hypoglycemia obtained in incubation with insulin, amounts to several hundreds of m g ~ below that in incubations without insulin. The finding that insulin exerts its effect also in vitro is rather perplexing and poses the question as to the fate of the glucose which disappears or is removed from the haemolymph. There are no membranes involved, neither are there any cells to which the glucose might be transported. Possibly there is condensation or polymerisation of glucose to polysaccharide, along with a depression of the accelerated formation of glucose by the microsomes which may be present and active even in previously frozen haemolymph. On the other hand, the marked differences in glucose level between the various tissues of the larvae point to mechanisms which act to permanently maintain these differences. In a review article by Falkmer et al. (1973), evidence is offered both to the presence of insulin in various invertebrates as well as to the effect of the insulin from various vertebrates on the glucose level in inverte-
96
JACOB ISHAY
brates. In general lines, the influence of insulin in invertebrates is similar to that in vertebrates, namely, it produces hypoglycemia in vivo. However, there are no reports in the literature on the in vitro effect of insulin, as described in the present paper. 6. The occurrence of differences in glucose level in the saliva of larval males, workers and queens suggests the possibility that these differences are somehow genetically associated with the different castes and that they may be of a communicative value, i.e. enabling the nursing workers to distinguish between the larvae of the different castes and directing them to differential-preferential feeding of the various caste larvae. This does not rule out the possibility that apart from differences in glucose level of the larval saliva, there are also differences of no less importance in the amount of saliva secreted by the various larval castes or in the amount of other materials present in the saliva.
REFERENCES
BRUNET P. C. J. (1965)The metabolism of aromatic compounds. In Aspects of Insect Biochemistry (Edited by GOODWXN T. W.), pp. 49-77. Academic Press, London. DEREVJC1A. & NITtJ V. (1971) Recherches concernant l'action du D.D.T. sur les abeilles--1. D6termination de rac6tylcholinest6rase chez les abeilles normales et les abeilles intoxiqu6es dans des conditions vari6es. Bull. Apic. 14, 31-47. FALK~R S., EMDXNS., HAVU N., LUNDGREt~G., MARQUES M., OSTBERGY., STEXNERD. F. & THOMASN. W. (1973)
Insulin in invertebrates and cyclostomes. Am. Zoologist, 13, 625-638. FXSCHLJ. & ISHAVJ. (1971) The glucose levels and carbohydrate autolysis in Vespa orien~alis, lnsectes Soc. 18, 203-214. HINES W. J. W. & SMITH M. J. H. (1963) Some aspects of intermediary metabolism in the desert locust (Schistocerca greoaria ). J. Insect. Physiol. 9, 463-468. IMMS A. D. (1960) A General Textbook of Entomology. Methuen and Co., Ltd, London. ISHAVJ. & IKAN R. (1968) Food exchange between adults and larvae in Vespa orientalis F. Anita. Behav. 16, 298303. ISnAV J., GOLDBERG S. & IKAN R. (1971) The lipids in Vespa orientalis larvae. Lipids 6, 850-851. ISHAY J., GITTER S. & FISCHL J. (1973) Gluconeogenesis in vitro in Vespa orientalis hemolymph. Proc. VIPh Int. Congr. Soc. Insects, London, 165-175. KINGSLV G. R. & GETCHELL G. (1960) Glucose oxidase method for determination of glucose in blood and other biological fluids. Clin. Chem. 6, 466-475. MASCHWITZ U. (1965) Larven als Nahrungsspeicher in Vespenvolk (Ein Beitrag zum Trophallaxieproblem). Verh. dr. :ool. Ges. 50, 530-534. SASLAVSKY H., ISHAY J. & IKAN R. 0973) Alarm substances as toxicants in the Oriental hornet colony. Life Sci. 12, 135-144. SCHAUDINISCHKYL. & ISHAY J. (1968) On the nature of the sounds produced within the nest of the Oriental hornet Vespa orientalis F. (Hymenoptera). J. Acoust. Soc. Am. 44, 1290-1301. WHEELER W. M. (1928) The Social Insects. Kegan Paul, Trench, Trubner, New York. Key Word Index--Glucose; Vespa orientalis; starvation; alloxan; hyperglycemia.
GLUCOSE LEVELS IN VESPA ORIENTALIS: THE EFFECT OF STARVATION JACOB ISHAY Department of Physiology and Pharmacology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel
(Received 20 June 1974)
Abstract--I. Distinct differences in glucose level have been found in the various tissue fluids of the larvae as well as between the different age groups and castes. The glucose level gradient order in the larvae is: saliva (2000-5000 rag%)--* haemolymph (100-200rag%)---. midgut (zero or near zero mg%). 2. During starvation the larval midgut is utilized to a greater extent than other tissues. 3. The glucose level in larval haemolymph may be altered by feeding the larvae on substances such as dextrose, distilled water, alcohol or alloxan. 4. The larvae saliva contains a factor (or factors) which produces hyperglycemia in larval haemolymph in vitro. Addition of mammalian insulin partially depresses the resultant hyperglycemia.
INTRODUCTION
colony were sorted according to age (e.g. larvae of instars 1-3 or 4-5) and sex, and their body fluids and tissues separated as rapidly as possible into test-tubes chilled at 0°C. In view of the finding that in haemolymph standing at room temperature, the glucose level tends to rise spontaneously (Fischl & Ishay, 1971), most of the determinations were made very close to the time of body-fluids withdrawal from the hornets, or in cases where frozen material was tested--immediately upon its thawing. Larval saliva was collected by gentle tickling of the oral region, larval haemolymph--by puncture and aspiration, larval midgut fluid by squashing of dissected midguts, and larval fat-body by dissection. The gastric juices of adults were expressed by pressure on the abdomen. Tissue fluid samples were taken both immediately as well as following starvation for several days. The various determinations were carried out on either fresh samples or on ones that had undergone prolonged freezing at -20°C. In every instance the test material was first centrifuged at x 2000 g for 20min and the supernate was used for the various determinations. Glucose determination, as based on glucose oxidase, was done according to the method of Kingsly & Getchell (1960), usually at 27°C and at pH 7. Dialysis of haemolymph or larval saliva was carried out against distilled water at 0°C for 24hr. The insulin was protamine Zn insulin 80 U/ml (Rafa Co., Israel). Hydrolysis was performed by boiling the samples for 3hr in a I N HCI solution in a water bath.
THE PHENOMENON of food-exchange between the various colony members of social insects has been called trophallaxis by Wheeler (1928) a n d the term is used now by most students of social insects to imply mainly the transfer of alimentary contents in liquid or solid state, either mutually or unilaterally. A m o n g social wasps it has been proven that in the course of food exchange between the adults a n d the larvae, the adults offer the larvae bits of meat a n d receive, in return, droplets of larval saliva which are extremely rich in sugar (Ishay & Ikan, 1968). In the case of Vespa orientalis, for instance, larval saliva contains 4 0 0 0 - 5 0 0 0 m g % glucose whereas the glucose content of larval h a e m o l y m p h ranges only between 10 a n d 200 rag% (Fischl & Ishay, 1971). This finding raises many interesting questions. Is there, for instance, a difference in the glucose level of larvae at different stages of development or belonging to different castes? Does starvation affect the glucose level? W h a t could be the reason for such a high glucose concentration in larval saliva? Are any hyperglycemic factors involved? The present study was instigated in an attempt to answer some of these questions t h r o u g h systematic determination of the glucose level in the various tissues of the different castes of the Oriental hornet.
RESULTS MATERIALS AND METHODS
Glucose levels in various tissues of Vespa orientalis
Body fluids of numerous hornets, all of the same species, age, sex and caste, were pooled. In nature, individuals of all the various castes occur concurrently only during a certain period of the year, and it was hence necessary to carry out the determinations on fresh material during August-October, when the colony comprises thousands of individuals at all stages of development. Such complete colonies were sought in the field and upon discovery, were brought in toto (under aether anaesthesia) to the laboratory during the early morning hours, when the workers are still in the nest. In the laboratory, the members of each
Figure 1 summarises in histogram form the data o n different glucose levels in members of a single hornet colony. Each d a t u m is a mean of three determinations carried o u t o n fresh material immediately after removal of the nest from its natural habitat. The data have been arranged in accordance with the different functional groups or castes. As can be seen in Fig. l, there are marked differences in glucose levels: (a) between various tissues of the same hornet; (b) between the different age groups within the same 91
92
JACOB ISHAY iooo I--
caste of hornets; and (c) between hornets of the same age group (stage) but of different castes. On the other hand, certain tissues of all members of the colony show a uniform or almost uniform glucose level. As for the differences in glucose level, the following conclusions may be drawn:
Males
6 0 0 I--
9 n
4001--
2 0 0 I--,..L, I I ~ Workers
4000~
rml
1. For the larva, there is invariably a glucose level gradient of the various tissues (or fluids) whose order is saliva (2000-5000 mg%)--* haemolymph (100200 rag%)--, midgut (zero or near zero mg%). 2. The glucose levels in larvae of instars 4-5 are invariably higher than in larvae of instars 1-3. 3. There is also a glucose level gradient for the larvae of the different castes, whose order is queen larva --~ worker larva---, male larva.
3000
2000 0
Q .£
I000
9 I-I . . . .
200 ~
Queens
5000
I Eggs 2 Saliva larvae :5 Hemolymph 4 Midgut 5 Saliva larvae 6 Hemolymph
8 4000
7 Midgut
I-3 I-3 I-3 4-5 4-5 4-5
8 Hemolymph prepupae 9 Hemolymph pupae IOHemolymph imagines I Gastric juices
3000
2000
I000 800 600 400 200
9 i-] Fig. 1. Glucose levels in various tissue fluids of Vespa orientalis. All data was transformed to logarithms (natural) and the analysis performed (L = line, R = repeat, T = time). The differences between the glucose level in the tissue fluid larvae (saliva, haemolymph, midgut) is significant (P < 0.0005). The differences between the glucose level in saliva of l-3rd instar and that of 4--5th instar is significant (P < 0.0005). Same degree of significance between glucose levels saliva of male, worker and queen larvae.
The highest glucose levels are recorded in larval saliva. For male larvae of instars 1-3, for instance, the glucose level in the saliva is about three times that in the haemolymph, while for queen larvae of instars 4-5, this ratio rises to 100: 1. There are, however, also instances where the glucose levels are similar. Thus there is the same glucose level (very low and approaching 0 mg%) in the midgut of all stages from all castes, while the glucose levels in the haemolymph of prepupae and pupae are quite similar. For comparison purposes, glucose level determinations were carried out in five additional colonies. Despite some variability in the results for the different colonies, the general picture was essentially the same and the level ratios were quite similar. Glucose levels in starvation
Figure 2 summarises in histogram form the data on the glucose levels in saliva and haemolymph of 5th instar larvae starved for up to 9 days. From the data, several findings may be noted: I. During starvation, the glucose level in the saliva drops with time but is always higher than in haemolymph.
V. orientalis- Worker larvae 6000
Starvation
5600
5200
Glucose level in saliva (top)and in hemolymph(bottom)
o_
4400
.c
4000!
E
- -_i
-
30 oVo d e x t r o s e - a d / / b d u r n
Starvation
4800 0
Queen larvae
-
!
Distilled w o t e r - a d lib#urn
' 5600 I"
Saliva
5200 -
_~
2800 .-
_.m
2400
o
2000 t600 1200
~
80O 4O0
II
0
.>,IB114
.~ 1614
6 t6
017
1~161gC
204 II *2 .=0~04~
I
day
2 5 4 5 6 7 6 9
doy
254567
9
doy
125456
69
doy
125456
8 9
Fig. 2. Glucose level in larvae haemolymph and saliva during 9 consecutive days of starvation. The differences between the glucose level in haemolymph and saliva are significant in every experiment and in every day (P < 0.0005). The increase in glucose level in haemolymph after feeding the larvae with 30% dextrose solution is significant (P < 0.05). The decrease in glucose level upon starvation is significant by worker larvae starting day 5 and by queen larvae starting day 6 (P < 0.005).
Glucose levels in Vespa orientalis Table 1. Weight loss following starvation of 5th-instar larvae
93
Table 2. Effect ofstarvation on midgut weightin 5th-instar larvae
Hours of starvation
Mean larval body weight (g)
S.D.
Hours of starvation
Mean weight of larval midgut (g)
S.D.
48 72 96
1.60400 1.53185 1'37030
0.095502 0.089454 0'104878
48 72 96
0.2232000 0.1940741 0-1733333
0.0665 0.0693 0'0723
2. The glucose level in queen larval saliva is more "stable" than in worker larval saliva. 3. The imbibation of distilled water produces a stabilisation of glucose level in saliva but an instability of the glucose level in haemolymph, whereas upon feeding on dextrose, there is gradual increase in the glucose levels of both saliva and haemolymph. The tall columns in the histogram represent the glucose level in saliva. The numbers within the columns represent the glucose levels in haemolymph. In all the experiments, unfed larvae started dying after 9 days of starvation, and the experiments were terminated at this stage. It should be noted, however, that individual larvae, whether totally starved or offered water or dextrose, survived up to 16 days but rarely attained the cocoon-spinning stage.
Determination of weight loss following starvation For this purpose, a comb containing some 300 queen larvae at 5th instar was separated from center to periphery into three equal segments and the larvae from each were removed for weighing on three consecutive days. Results were as in Table 1. deviation The coefficient of deviation V = standard mean was 0.15854 after 48hr, 0.12515 after 72hr and 0'28066 after 96 hr. Thus there was at first a slight drop in the value of this coefficient, which was followed by a sharp increase. As for the rate of weight loss: The average weight of larvae on the 3rd day (72hr) was 95.502~ that on the 2nd (48 hr), while that of larvae on the 4th day was 89'454~ that on the third day. The midgut from larvae starved under the same conditions was weighed separately, with results as in Table 2. The coefficient of deviation was 0.08354 at 48 hr, 0'12723 at 72 hr and 0-21953 at 96 hr. There was thus marked increase in the value of this coefficient after every additional day of starvation. As for the rate of weight loss: On day 3, the average weight was 86"95170 that on day 2, while on day 4, the average weight was 89"313~o that on day 3. From these results, it is clear that from day 2 (48 hr) to day 3 (72hr), there is greater weight loss of the midgut than of the total body. The midgut weight drops by about 13~ whereas the total body weight drops by about 4.5~. Weight of the midgut dropped, on average, by 29-1 mg whereas that of the body dropped by 72'1 mg. Thus, weight loss of the midgut constituted about 40.6~ of the total weight loss. From day 3 to day 4, weight of the midgut dropped approximately of the same order as the total body weight (by about 1170). Weight of the midgut dropped an average of 20.7mg. Total body weight dropped an average of 161'6rag. Hence the drop in midgut weight constituted only 12.970 of the total weight loss.
Since larval saliva was found to contain such a high glucose level, it was deemed interesting to ascertain the yield of glucose per larva per single daily milking following various treatments. All larvae were of the same colony and at the 4th-5th instar. They were placed in small combs of 25-30 larvae each, which were maintained in the shade at 27°C. Feeding, when carried out, was by pipette, ad libitum, twice daily. The larvae were milked once daily in the morning in accordance with the feeding procedure adopted. Results are summarised in Table 3. As expected, there was in the course of starvation, a decrease in the amount of glucose secreted, but surprisingly there was no drop in the amount of liquid secreted in the saliva. When distilled water was offered, there was increase both in the amount of fluid secreted as well as in the amount of glucose. Dextrose feeding produced increase both in total fluid and in glucose (dextrose) secretion, which was reflected in the marked increase in glucose levels. Alloxan feeding resulted in a sharp drop (to about 5070) in the total amount of fluid secreted and also marked decrease in the glucose per larva. The final result, however, was a marked increase in the glucose level! On day 4, all the larvae which had received alloxan died. Alcohol feeding produced a gradual but marked drop in the amount of fluid secreted and a parallel drop in the amount of secreted glucose, but the final result was only a slight drop in the glucose level. On day 4, there was no secretion of saliva! Otherwise, however, the larvae behaved normally, retaining their mobility and continuing to produce hunger signals (Schaudinischky & Ishay, 1968) as usual. From all the determinations carried out thus far, it is clear that the glucose level in larval saliva is markedly higher than in larval haemolymph, and this both in well-fed, satiated larvae as well as in larvae under prolonged starvation. The high glucose level in larval saliva could be due to various reasons, like the following two, for instance: 1. The possibility that the glucose in the saliva is derived from internal tissues and that the cells lining the salivary ducts polarise and filter it out to the lumen of the ducts, whence it is secreted in concentrated form. 2. The possibility that the fluid secreted into the lumen of the salivary ducts contains factors which produce hyperglycemia in saliva only, whether by the absorption of glucose from the haemolymph via the cells lining the ducts or by the absorption of glucose precursors from the haemolymph. To resolve this problem, larval saliva was incubated with larval haemolymph. As pointed out earlier, larval saliva shows a relatively very high glucose level and contains both di- and polysaccharides (Maschwitz, 1966; Ishay & Ikan, 1968), so that direct incuba-
JACOB ISHAY
94
Table 3. Glucose yield per larva under various dietary conditions
Treatment
Saliva amount per l daily milking (cm 3) S.D.
Trial day
Total starvation Total starvation Total starvation Total starvation Offered distilled water only water only water only water only Offered 30~ Dextrose Sol. Dextrose Sol. Dextrose Sol. Dextrose Sol. Dextrose Sol. Dextrose Sol. Offered alloxan I0 mg/ml in distilled water distilled water distilled water Offered a 10~ solution of ethyl alcohol ethyl alcohol ethyl alcohol
0.0110 0.0110 0.0200 0.0125
0.0036 0.0037 0.0029 0.0027
3090 2844 2964 2022
0.34000 0.31284 0.54280 0.25270
I 2 3 4
0'0110 0.0100 0.0250 0.0250
0.0036 0.0030 0.0070 0.0065
3090 3258 3418 3372
0.34000 0.32580 0.85450 0.84300
1 2 3 4 5 6
0.0110 0.0250 0.0750 0.0142 0.0200 0.0200
0.0036 0.0040 0.0021 0.0034 0.0051 0.0031
3090 3420 4758 4260 3630 4176
0.34000 0.85500 0-35680 0.60500 0.72600 0'83520
1 2 3
0.0110 0.0050 0.0058
0.0036 0.0021 0.0038
3090 3540 4584
0.34000 0.17700 0.26587
1 2 3
0.0110 0.0090 0.0058
0.0036 0.0024 0.0031
3090 2970 2760
0.34000 0.26730 0.16008
L a r v a l hemolymph Vespa oriental/l~
6500 -
Hemolyrnph Hemolymph + 12U insUlin/ml Hernalymph + larval saliva (dialysed) I:1 Hernolymph larval • s a l i v a ( d i a l y s e d ) ÷ 12U insulin
6000
3 5500 5000E o
(2_
4500
4000
3500
4
30OO _D 2 5 0 0 '..9 2000
1500 I000
I
500
30
I
I
60
90
I 120
I -"T~-~r 150
Glucose yield/larva single daily milking (mg)
I 2 3 4
tion of larval saliva with haemolymph could only yield misleading results. Hence, larval saliva was first dialysed in the cold (at 0°C) for 24 hr against distilled water, in order to remove all materials of low molecular weight, and only the dialysate was incubated with haemolymph. A typical result of such incubation is given in Fi~. 3. In connection with Fig. 3, it should be pointed out that similar results were obtained also by incubat-
I 2 3 4
Glucose level in larval saliva (in mg%)
~80
2 210
240
rain
Fig. 3. Incubation together of larval haemolymph and dialysed larval saliva, with and without insulin. Line 2 is less than line 1 at all times (P < 0.0005). Line 1 is less than lines 3 and 4 at all times (P < 0.0005). Line 4 is less than line 3 at all times (P < 0.0005).
ing saliva from Paravespula germanica larvae with haemolymph from Vespa orientalis larvae, and vice versa, which means that the phenomenon is not speciesspecific. Neither is the phenomenon stage-specific, inasmuch as similar results were obtained also by the incubation of larval saliva with prepupal or pupal haemolymph. F r o m the results shown in Fig. 3, the following merit attention: (a) Incubation of haemolymph with larval saliva that had undergone dialysis and whose glucose level is 0 mgVo, causes a sharp rise in the glucose level of such incubated haemolymph, relative to the glucose level in unincubated haemolymph. This rise in glucose level continued in the experiment for ~ hr, following which there was a slight decline. (b) The incubation with insulin of equivalent amounts of haemolymph, resulted in a drop in the glucose level. The hypoglycemia was slight in incubations of insulin and normal haemolymph, but became very pronounced in incubations of insulin with haemolymph that had undergone prior incubation with dialysed larval saliva. (c) After several hours of incubation of haemolymph with larval saliva, the glucose level approximated or even exceeded the ordinary glucose level of larval saliva. Boiling of the larval saliva prior to its incubation with haemolymph, brought to a stop its hyperglycemic activity. This finding, coupled to the fact that the active factor in saliva is non-dialysable, leads to the conclusion that we are dealing here with the activity of a protein or protein-bound substance, whose mol. wt is greater than 10,000. DISCUSSION
Till now, all tests to determine the glucose level in various body fluids of wasps or hornets have been
Glucose levels in Vespa orientalis run on randomly selected insects from various colonies. In the present study, individuals from the same colony have been used, the various determinations being carried out immediately upon collection of the body fluids. The importance of this has been established in a previous study (Fischl & Ishay, 1971), which showed that the glucose level in the body fluids of wasps rises during the first hours after their extraction. Some primary findings of the present study merit mention: 1. The occurrence of a distinct and constant gradient of the glucose level between the various tissues of the same individual insect, between the various age groups of the same caste and between individuals of the same age groups but of different castes. 2. Starvation or the various treatments, do alter the glucose level in the body fluids of hornets, but there is nevertheless a marked difference in glucose level between larval saliva, whose glucose content is always very high, and larval haemolymph. 3. During the first 2 days of starvation, the midgut contracts and dwindles more than the other body tissues. It is reasonable to assume that weight loss of the midgut is due to the fact that the food contents of the midgut are being used up, but it may also be that stored food reserves in it are being utilised. In this connection, it should be mentioned that in a previous study (Ishay et al., 1971), it was found that larval midgut contains 11.4-18"6~ lipids as dry weight. Insofar as adult hornets are essentially predatory insects, one can reasonably assume that they forage for and feed the larvae primarily on animal proteins (e.g. other insects or bits of flesh from the carcasses of vertebrates). This strengthens the impression that the lipids found in such high concentration in larval midgut actually serve as a food reserve and are the first to be utilised upon starvation of the larva. 4. Larval saliva contains factors which cause in vitro hyperglycemia of larval haemolymph. It is not clear yet whether one or several mechanisms are involved in this process, i.e. whether the rise in glucose level is due to the degradation of haemolymph polysaccharides or rather due to the formation of glucose de novo by way of gluconeogenesis. The problem requires further investigation. Hyperglycemic factors have been detected also in larval midgut (Ishay et al., 1973), but in this case it has been clearly demonstrated that the process is in part due to gluconeogenesis, inasmuch as incubation of midgut with haemolymph and C14-glutamic acid has shown incorporation of the labeled substance into C14-gtucose. However, there is this difference between midgut and larval saliva, in that the former is an internal, normally irremovable organ while the latter is regularly being passed on to the adult hornets in the nest. Does this transfer of hyperglycemic factors from larvae to the adults suggest that the adults lack such factors? It can be assumed that the larval transfer of saliva to the adults in the course of the process named trophallaxis, may serve two purposes: (a) to provide the adults with a food rich in carbohydrates so as to enable them to maintain the necessary nest functions between foraging trips to the field, which could explain the high glucose level of larval saliva; and
95
(b) that both the glucose and hyperglycemic factors in the saliva can participate in the process of detoxication in which substances like phenols and their derivatives are converted into the corresponding /3-Dglucosides. Such reactions normally occur in the insect fat-body (Hines & Smith, 1963). It seems that the acceleration of glucose formation by factors in larval saliva, larval midgut, larval fat-body or other tissues, may serve the latter purpose, that is to say, if more glucose is available there is greater potential for conjugation of phenol compounds which implies enhanced potential for detoxication. In the conjugated form, the phenols are not subjected to oxidation by phenolase (Brunet, 1965). It is reasonable to expect such accelerated ability of detoxication to be found in the digestive tract of the larvae: as is known, in the larvae of almost all the Apocrita, the midgut is a blind sac and does not communicate with the hindgut until the final instar, the faecal contents only being evacuated at the conclusion of the larval stage (Imms, 1960). It is similarly reasonable to expect to find a capacity for detoxication in the larval saliva which is offered to the adults. As already mentioned, the adult hornets, when foraging in the field, collect food from various sources, but chiefly flesh from carcasses or other organic matter in different stages of putrefaction, so that it is quite likely that the adult hornets utilise materials in larval saliva to form conjugates for the purpose of detoxication. In a previous study (Saslavsky et al., 1973), it was found that toxic materials have less effect on adult hornets in a natural, larva-containing nest than on hornets in a larva-less nest, which strongly suggests that the larvae play a r61e in detoxication processes. A similar finding was reported by Derevici & Nitu (1971) who observed that DDT was less toxic to bees in larva-populated hives (in the spring) than in larva-free hives (in the winter). 5. Bovine insulin counteracts hyperglycemia h7 vitro. Incubation of haemolymph together with insulin retards the rise in glucose level if the starting level is low, or causes a sharp drop in the glucose level if the starting level is high (Fig. 3). It should be noted in this connection that the induction of clear-cut hypoglycemia required the use of a relatively high concentration of insulin (12 units insulin/ml haemolymph). At the same time, the hypoglycemia obtained in incubation with insulin, amounts to several hundreds of m g ~ below that in incubations without insulin. The finding that insulin exerts its effect also in vitro is rather perplexing and poses the question as to the fate of the glucose which disappears or is removed from the haemolymph. There are no membranes involved, neither are there any cells to which the glucose might be transported. Possibly there is condensation or polymerisation of glucose to polysaccharide, along with a depression of the accelerated formation of glucose by the microsomes which may be present and active even in previously frozen haemolymph. On the other hand, the marked differences in glucose level between the various tissues of the larvae point to mechanisms which act to permanently maintain these differences. In a review article by Falkmer et al. (1973), evidence is offered both to the presence of insulin in various invertebrates as well as to the effect of the insulin from various vertebrates on the glucose level in inverte-
96
JACOB ISHAY
brates. In general lines, the influence of insulin in invertebrates is similar to that in vertebrates, namely, it produces hypoglycemia in vivo. However, there are no reports in the literature on the in vitro effect of insulin, as described in the present paper. 6. The occurrence of differences in glucose level in the saliva of larval males, workers and queens suggests the possibility that these differences are somehow genetically associated with the different castes and that they may be of a communicative value, i.e. enabling the nursing workers to distinguish between the larvae of the different castes and directing them to differential-preferential feeding of the various caste larvae. This does not rule out the possibility that apart from differences in glucose level of the larval saliva, there are also differences of no less importance in the amount of saliva secreted by the various larval castes or in the amount of other materials present in the saliva.
REFERENCES
BRUNET P. C. J. (1965)The metabolism of aromatic compounds. In Aspects of Insect Biochemistry (Edited by GOODWXN T. W.), pp. 49-77. Academic Press, London. DEREVJC1A. & NITtJ V. (1971) Recherches concernant l'action du D.D.T. sur les abeilles--1. D6termination de rac6tylcholinest6rase chez les abeilles normales et les abeilles intoxiqu6es dans des conditions vari6es. Bull. Apic. 14, 31-47. FALK~R S., EMDXNS., HAVU N., LUNDGREt~G., MARQUES M., OSTBERGY., STEXNERD. F. & THOMASN. W. (1973)
Insulin in invertebrates and cyclostomes. Am. Zoologist, 13, 625-638. FXSCHLJ. & ISHAVJ. (1971) The glucose levels and carbohydrate autolysis in Vespa orien~alis, lnsectes Soc. 18, 203-214. HINES W. J. W. & SMITH M. J. H. (1963) Some aspects of intermediary metabolism in the desert locust (Schistocerca greoaria ). J. Insect. Physiol. 9, 463-468. IMMS A. D. (1960) A General Textbook of Entomology. Methuen and Co., Ltd, London. ISHAVJ. & IKAN R. (1968) Food exchange between adults and larvae in Vespa orientalis F. Anita. Behav. 16, 298303. ISnAV J., GOLDBERG S. & IKAN R. (1971) The lipids in Vespa orientalis larvae. Lipids 6, 850-851. ISHAY J., GITTER S. & FISCHL J. (1973) Gluconeogenesis in vitro in Vespa orientalis hemolymph. Proc. VIPh Int. Congr. Soc. Insects, London, 165-175. KINGSLV G. R. & GETCHELL G. (1960) Glucose oxidase method for determination of glucose in blood and other biological fluids. Clin. Chem. 6, 466-475. MASCHWITZ U. (1965) Larven als Nahrungsspeicher in Vespenvolk (Ein Beitrag zum Trophallaxieproblem). Verh. dr. :ool. Ges. 50, 530-534. SASLAVSKY H., ISHAY J. & IKAN R. 0973) Alarm substances as toxicants in the Oriental hornet colony. Life Sci. 12, 135-144. SCHAUDINISCHKYL. & ISHAY J. (1968) On the nature of the sounds produced within the nest of the Oriental hornet Vespa orientalis F. (Hymenoptera). J. Acoust. Soc. Am. 44, 1290-1301. WHEELER W. M. (1928) The Social Insects. Kegan Paul, Trench, Trubner, New York. Key Word Index--Glucose; Vespa orientalis; starvation; alloxan; hyperglycemia.