Ent. exp. & appl. 31 (1982) 147--153. Ned. Entomol. Ver. Amsterdam

WATER INTAKE BY THE TERMITE MACROTERMES MICHAELSENI

ROBERT SIEBER and ELIZABETH D. KOKWARO

The International Centre for Insect Physiology and Ecology, P.O. Box 30772, Nairobi, Kenya

Workers and reproductives of the fungus-growing termite Macrotermes michaelseni (SjOstedt) protrude the hypopharynx during water intake. Scanning electron microscopic and histological investigations showed that the front part of the hypopharynx, which is pressed onto the wet surface, is covered by a dense system of unidirectionally arranged trichomes. It is assumed that water intake is accomplished by capillary forces along these trichomes. Soldiers were never observed protruding their hypopharynx which is distinctly smaller and without any trichomes on the surface.

Major workers, deprived of water for 15 hr, protruded the hypopharynx as soon as the water was supplied and maintained this drinking position for approximately 2 rain. The amount of water imbibed by thirsty workers was 15.5 + 5.9% of their fresh weight before drinking. No correlation between the length of time the hypopharynx remained extended and the amount of water taken in could be found. Imbibed water was initially stored in the foregut and from there transferred to the water sacs. This continuous process was nearly completed 4 hr after water intake. Although transfer of imbibed water to the water sacs was observed only in reproductives and workers, soldiers and larvae also possess these liquid reservoirs.

KEy WOgDS: Macrotermes michaelseni, Isopte- ra, Macrotermitinae, water intake, hypopha- rynx

Water demand in fungus-growing termites seems to be met either by producing metabolic water or by imbibing it from a water source. J. P. Watson (1972) could not find radioactive material in nests of Macrotermes natalensis (Haviland) after he had labelled water from the adjacent water table. From these results he concluded that water must be provided by met- abolic water produced within the mound. On the other hand, Hewitt et al. (1971) described newly emerged alates of Hodotermes mossam- bicus (Hagen), confined in pairs, drinking half of their weight in water which suggests the di- rect intake from some water source. Further on, J. A. L. Watson et al. (1971) described wa- ter being transferred into special structures, usually described as salivary gland reservoirs (Noirot, 1969) which Hewitt et al. (1971) called "water sacs". Drinking by Macrotermes mi- chaelseni reproductives was also observed from pairing to the development of the first workers as well as by workers at places of water supply outside the nest and from the fungus comb af- ter deposition of the semiliquid faecal pellets (Sieber & Leuthold, 1981). In this species, be-

havioural observations showed that the hypo- pharynx was always protruded and pressed on- to the wet surface during active water intake.

This paper describes the structure of the hy- popharynx of M. michaelseni and provides in- formation about its function in water intake. In addition, the morphology of the salivary reser- voirs (here also called water sacs) is detailed and compared among the different castes. The path of water in major workers was deter- mined.

MATERIALS AND METHODS

Termites: Macrotermes michaelseni is a fungus- growing termite that builds mounds in semiarid grasslands in Africa (Ruelle, 1970; 1977). A mature field colony consists of the royal cou-. pie, major and minor workers, major and mi- nor soldiers as well as larvae and eggs. In addi- tion, nymphs can be found after the long rains in March--May and the alates fly during the short rains in November and December. Alates were collected during their flight in Ka- jiado, 80 km south of Nairobi and subsequently reared as described (Sieber & Leuthold, 1981). In addition, field mounds were opened and ter- mites were collected.

Structure o f the hypopharynx and the labium: For scanning electron microscopy (SEM), hy-

148 Robert Sieber and Elizabeth D. Kokwaro

popharynx and labium were dissected and fixed in 70% ethanol for 3---5 days and subse- quently dehydrated through ascending grades of alcohol into absolute alcohol. In order to re- move surface debris, the specimens were cleaned in an ultrasonic cleaner for 3-4 min. The wax coating of the cuticle was extracted for 4 hr with ethyl acetate in a soxhlet appara- tus. Clean specimens were then double-coated with carbon and gold in a vacuum evaporator and observed in a Jeot SEM. For histological studies, the hypopharynx was dissected and fixed in Bouin's fluid, embedded in Paraplast, sectioned, and stained with haematoxylin- eosin.

Behavioural observations: The imbibing of water by workers was observed either in the nest of incipient colonies, through a watchglass (Sieber & Leuthold, 1981) or by depriving field-collected workers of water for 15 hr and subsequently adding a small cube of wet, sponge-like material or moistened filter paper into their petri dish.

To determine the relationship between the duration of drinking and the amount of water consumed, field-collected workers were de- prived of water for 15 hr, marked with a quick drying colour spot on the abdomen and weighed. Together with other workers, they were then placed on moistened filter paper and the time during which the hypopharynx was protruded in each termite, recorded. Each ter- mite was weighed again after it had stopped drinking.

Path of imbibed water: Three groups of 20 major workers from 3 different field colonies, deprived of water for 15 hr, were transferred to filter paper which was moistened with either tap water or methylene blue-coloured water. Immediately after the termites had stopped drinking they were kept on dry filter paper. At intervals of 30 rain the animals were then im- mobilized either by CO 2 or in a freezer. From these termites the volumes of the foregut and the water sacs were determined: the abdomen was opened dorsally with forceps and the length and width of the foregut measured with an ocular micrometer on a dissecting micro- scope. The volume was determined using the formula for the volume of a sphere if the fore- gut contained water, or of a cylinder, if it was empty. The volumes of the water sacs were de- termined to be empty; l/, filled (liquid recog- nizable in them); t/2 filled (distinctly extend- ed); 3/a filled (filling the forepart of the abdo-

men); and full (the water sacs being strongly swollen, the left one extending (~ the length of the abdomen).

RESULTS

Morphology of the hypopharynx: The structure of the hypopharynx is the same in reproduc- fives and both worker castes. The anterior sur- face, the part which is pressed onto the wet substrate during water intake, is completely covered with unidirectionaUy oriented, hairlike structures (Figs 1 & 2). Histological sections through the hypopharynx of workers or repro- ductives revealed that these structures are sep- arated from the epidermis by the endocuticle (Fig. 3) and are therefore acellular "tricho- mes". These sections also confirmed our dis- secting observations that the hypopharynx is a hollow structure, connected with the head cap- sule.

The inner surface of the labium, on which the hypopharynx lies in its normal position (Fig. 4), was investigated by means of SEM. Fig. 5 shows that the surface of the glossae is covered by a system of stubby structures.

Unlike the worker's hypopharynx, that of the soldier is a significantly smaller, structure- less tongue and without any trichomes on the surface. The larval hypopharynx has the same structure as that of the workers, but without any trichomes on its surface.

Layout of the water sacs: In the final stages of all castes as well as in larvae, a pair of water sacs was found as described in the reproduc- tires of Hodotermes mossambicus (Watson et ad., 1971). Variably extended water sacs were observed in workers before or after imbibing water. When empty, the left water sac lies more dorsally than the right. In the case of a full foregut and after imbibing water, the left water sac lies under the foregut and the right one directly on it. When the water sacs are full, the left one parallels the line of the pleura, reaching the last third of the abdomen, where- as the right sac bends in front of the midgut. Full water sacs do not narrow progressively to- wards the head as described for the reproduc- tives of H. mossambicus (Watson et al., 1971) but decrease rather abruptly in diam just be- fore opening into the ducts. The ducts of the water sacs run along the salivary ducts and the longitudinal connectives of the ventral nerve cord. The ducts of the former are of uniform diam throughout and about twice the diam of the salivary ducts. In workers, reproductives

Water intake by M a c r o t e r m e s 149

Figs 1--3. Hypopharynx of minor worker. I - - Side view. Anter ior and dorsal surface partly visible. Scanning electron micrograph (350 ×). 2 - - Parallel a r rangements of tr ichomes on anterior surface (4000 ×). 3 - - Horizontal longitudinal section (I0 p.m) through front part. T = tr ichomes, E = endocuticle, Ep = epider- mis, H = haemocoel .

150 Robert Sieber and Elizabeth D. Kokwaro

4

o,

• t ~ *'

' "* O ~ 4 ' ' 11) 11

/ ' 7 I

rigs o,--/. 4 - - Normal position of hypopharynx (H) in mouth cavity of workers and reproductives. Side view of reproductives head after removing left maxilla. 5 - - Inner surface of labium of major worker (120 x). 6 - - Position of head during protrusion of hypopharynx by major worker. 7 - - Protruded hypopharynx of major worker. View through observation glass.

and larvae, the ducts of the salivary glands dis- charge into the ducts of the water sacs before opening at the base of the hypopharynx. Un- like the reproductives, where the ducts run within the head capsule, in workers and sol- diers these ducts leave the head capsule ven-

trally and run to the external opening through a tube formed between the floor of the head capsule and the postmentum (particularly elongated in minor and major soldiers).

Water intake: When thirsty workers were of- fered a moistened medium they might walk on

Water intake by Macrotermes 151

it for several min, touching the wet surface with antennae and palpae. Subsequently they stopped moving, approached the moistened surface with the head and protruded the hypo- pharynx (Fig. 6). In this position they re- mained immobile, moving the antennae slight- ly and were not disturbed by other termites touching them. During this time a pulsating muscle movement could be observed within the frontal area of the head capsule. The pro- truded hypopharynx pressed onto the surface of a watchglass could be observed in the obser- vation nests. Fig. 7 shows the close contact be- tween the glass and the hypopharynx as it is protruded between the glossae. After the hy- popharynx was retracted into the mouth cavi- ty, it was moved several times against the labium.

Duration of water intake: The total duration of hypopharynx protrusion was monitored in 50 major workers, originating from 5 different field colonies. It was protruded once only (82%), twice (16%) or thrice (2%) at the same place with an average total duration of 140.4 + 59.0 sec (SD). Taking into consider- ation the different weights of the workers, the amount of water drunk was calculated as a % of the weight of thirsty workers and found to be 15.5 + 5.9%. However, there was no cor- relation between the length of time the hypo- pharynx was extended and the amount of wa- ter imbibed. In all the observed cases, the ter- mites imbibed about twice the amount of water they had lost during the 15 hr of desiccation.

Deposition of water after drinking: The dis- section of major workers before and at differ- ent times after imbibing water showed that the average volume of the foregut in the three groups of animals increased by a factor of 13.1, 32.0 and 40.9, respectively (Fig. 8). The in- crease in volume was in fact slightly greater than this, because both methods of immobiliza- tion (CO,. or chilling) induced workers to re- lease a small droplet of water whenever water was present in the foregut. Fig. 8 also shows that the decrease in volume of the foregut and, as a consequence, the increase in volume of the water sacs is a more or less continuous pro- cess. This water translocation was generally completed 4 hr after water intake; however, the large standard deviations observed after 5 and 6 hr show that in a few termites the foregut still contained water and that water transfer to the water sacs had not yet been completed in these individuals.

To find out whether workers drink water again after the foregut has become empty, thirsty workers were first offered plain tap wa- ter and then, when the foregut was empty, methylene blue-coloured water. In no case could a worker be found with full water sacs and with blue water in the gut during the fol- lowing 24 hr, although no difference could be found in water intake between tap water and stained water.

To determine from which part of the gut wa- ter was absorbed, thirsty workers were offered blue-coloured water. Since no blue material

J l

wa~ ,make

x

IV.

/ . / 1 "

HOURS AFTER WATER INTAKE

Full

! z

V4

Fig. 8. Volumes _+ SD of foreguts (open signs) and water sacs (filled signs) before and at different times after imbibing water. Each point represents 20 major workers from same field colony.

152 Robert Sieber and Elizabeth D. Kokwaro

could be found in the midgut by the time it had appeared in the water sacs it was concluded that water was absorbed through the walls of the foregut.

DISCUSSION

A first possible function of the hypopharynx in termites was described by Grass6 (1978). He described that in Macrotermes muelleri (Sjfstedt) fresh faecal pellets deposited on the fungus comb were molded into balls by the hy- popharynx. Sieber & Leuthold (1981) also ob- served the hypopharynx being protruded in M. michaelseni, but this behavioural element was strictly correlated with water intake.

Similar to the fibrils on the bladder-like structures on the hypopharynx of Arenivaga in- vestigata (O'Donel l , 1976), our SEM observa- tions on the worker 's and reproductive's hypo- pharynx revealed a dense trichome system on that part which touches the surface during wa- ter intake. Since active movement of trichomes is not possible, it must be assumed that water is transferred from the source into the mouth cavity by means of capillary forces. This hy- pothesis is strengthened by the fact that these trichomes lie in parallel courses. During a drinking bout this orderly arrangement may be upset. The intense in-and-out movement of the hypopharynx against the opposing projections on the inner face of the glossae may serve to rearrange or "comb out" the trichomes. In ad- dition, the rhythmic muscular contractions ob- served within the head capsule suggest that ad- ditional force or suction may be provided through pumping movements of the oesopha- gus or by altering the shape of the hypopha- rynx itself.

The details of structure reported support previous behavioural observations by Sieber & Leuthold (1981): trichomes are found only on the hypopharynx of workers and reproduc- tives, the only two castes which have been ob- served to drink by protruding the hypopha- rynx.

Our investigations also confirm the findings of Watson et al. (1971) that imbibed water reaches the foregut and is transferred from there to the reservoirs. This fact has been con- firmed in major and minor workers as well as in reproductives before the appearance of their adult offspring. From these findings, the term water sac seems to be more appropriate than salivary gland reservoir. However, additional chemical investigations need to be undertaken

to find out whether saliva is also stored in these reservoirs. In addition, the composition of the liquid in the reservoirs of the soldiers and lar- vae should be elucidated, especially because these animals have never been observed imbib- ing free water.

This project was supported by the Swiss Direc- torate for Development Co-operation and Humanitarian Aid. We thank Prof. W. L. Nut- ting and Dr. R. H. Leuthoid for criticism: Mr. M. O. Kotengo for technical assistance, Mr. Ch. Bwire for drawing Fig. 4 and Mr. P. Lisa- mula for processing the photographs.

ZUSAMMENFASSUNG

Wasseraufnahme bei der Termite Macrotermes michaelseni

Arbeiter und Geschlechtstiere der pilzziichtenden Termite Macrotermes michaelseni (Sjrstedt) stiJlpen zur Wasseraufnahme den Hypopharynx aus. Scan- ning elektronenmikroskopische sowie histologische Untersuchungen zeigten, dass der vorderste Teil des Hypopharynx. der auf die feuchte Oberfl~iche ge- presst wird, yon einem dichten System einheitlieh angeordneter Trichome bedeckt ist. Es wird ange- nommen, dass die Wasseraufnahme mittels Kapil- larkrafte entlang der Trichome erfolgt. Soldaten konnten nie beim Ausstiilpen ihres Hypopharynx beobachtet werden. Dieser ist zudem deutlich klein- er und ohne Trichome auf der Oberfl~iche.

Grosse Arbeiter. die w~ihrend 15 h auf trockenem Filterpapier gehalten wurden, stiilpten bei Wasser- zugabe den Hypopharynx racist nur einmal aus und verblieben in dieser Stellung w~ihrend ca 2 Min. Die Menge aufgenommenen Wassers betrug in durstigen Arbeitern 15.5 + 5.9% des Frischgewichtes vor der Wasseraufnahme. Zwischen der Dauer, w~ihrend der der Hypopharynx ausgestiilpt blieb und dem aufge- nommenen Wasser konnte keine Beziehung gefun- den werden. Aufgenommenem Wasser wird zuerst im Vorderarm gelagert und yon dort in die Was- sers~ieke transferiert. Dieser kontinuierliche Vor- gang war 4 h nach der Wasseraufnahme in den meis- ten Termiten abgeschlossen. Obwohl ein Transport von aufgenommenem Wasser in die Wassersacke nur bei Arbeitern und Gesehlechtstieren beobachtet werden konnte, besitzen auch Soldaten und Larven Fliissigkeitsrese rvoire.

REFERENCES

Hewitt, P. H., Nel, J. J. C. & Schoeman~ I. (1971). Influence of group size on water imbibition by Hodotermes mossarnbicus alate termites. J. In- sect Physiol. 17 : 587---600.

Noirot. Ch. (1969). Glands and secretions, pp. 89--- 123. In: K. Krishna & F. Weesner (Eds): Biology

Water intake by M a c r o t e r m e s 153

o f termites, Vol. 1. New York, London, Academ- ic Press.

GrassY, P. P. (1978). Sur la v~ritable nature et le r61e des meules ~ champignons construites par les termites Macrotermitinae (Isoptera, Termiti- dae). Paris. C. R. Acad Sc. 287 (D) 1223---1226.

O'Donell, M. J. (1976). The site of water vapour ab- sorption in Arenivaga investigata, pp. 115--121. In: K. Schmidt-Nielsen, L. Bol~s & S. R. H. Maddrell (Eds): Comparative Physiology - - Wa- ter, Ions and Fluid Mechanics. Cambridge Univ. Press, London.

Ruelle, J. E. (1970). A revision of the termites of the genus Macrotermes from the Ethiopian Region (Isoptera: Termitidae). Bull. Br. Mus. nat. Hist. 24 : 365-----444.

Ruelle, J. E. (1977). Macrotermes michaelseni (Sj6stedt) a new name for Macrotermes mossam- bicus (Hagen) (lsoptera: Termitidae). J. ent. Soc. S. Afr. 40 : 119.

Sieber, R. & Leuthold, R. H. (1981). Behavioural elements and their meaning in incipient colonies of the fungus-growing termite Macrotermes mi- chaelseni (Isoptera: Macrotermitinae). lnsectes Sociaux, in press.

Watson, J. A. L., Hewitt, P. H. & Nel, J. J. C. (1971). The watersacs of Hodotermes mossambi- cus. J. Insect Physiol. 17 : 1705--1709.

Watson, J. P. (1972). Some observations on the wa- ter relations of mounds of Macrotermes natalensis (Haviland) Fuller. Insectes Sociaux 19 : 87--93.

Accepted: July 12, 1981