Insectes Sociattx, Paris 1983, V o l u m e 30, n ~ 2, pp . 204-209

�9 M a s s o n , Pa r i s , 1983

ESTABLISHMENT OF FUNGUS COMB IN LABORATORY

COLONIES OF MACROTERMES 3IICHAELSENI AND ODONTOTERMES AIONTANUS (ISOPTERA, MACROTERMITINAE)

Robert SIEBER * The International Centre of Insect Physiology and Ecology, P,O. Box 30772, Nairobi, Kenya Re~u le 20 f6vrier 1982. Accept6 le 30 ju in 1982.

S U M M A R Y

Workers in incipient l abora tory colonies of Macrotermes michaelseni and Odonto- termes montanus bui ld feecal combs bu t are unab le to inoculate t h e s e combs ,with fungus. These c o m b s are eaten up and the colonies die unless fungus comb f rom a field colony is in t roduced 'when the f i rs t ,wor, kers s ta r t foraging. This s tudy invest igated w h e t h e r ,workers of the two species could produce fungus comb w h e n fed o n basidio- spores of the appropr ia te Termitomyces species. When the f i rs t ,workers ,,vere offered basidiospores on fil ter paper, 92.5 '% of M. michaetseni Colonies and 85 '% of O. montanus colonies produced Eungus combs. When bas id iospores vcere offered to O. montanus in a wa te r suspension on the inner surface of the nest , 82 .5% of colonies p roduced fungus combs.

Z U S A M M E N F A S S UNG

H|zgarten-lnduktion in Laborkolonlen yon Macrotermes rnichael~3ni und Odontotermes rnontanua {Isoptera : Macrotermitinae}

Arbei ter in j u n g e n Labonkolonien yon Macrotermes michaelseni und Odontotermes montanus produzieren F'~Lka!ienh~iufchen, ,k/Jnnen diese aber n icht mi t Pilz inoku l i e ren . Diese Hgufchen 'werden wieder aufgefressen und die Kolonien s terben, w e n n zum Zeit- purAct des Fouragierbeginns d e r e r s ten A r b e i t e r k e i n Pilzgarten von e iner Feld~kotonie zugegeben wird. In dieser Arbeit ~vurde un t e r such t , ob Arbei ter dieser be iden Ar ten Pi lzgarten produzieren, ~ e r m sie mi t Bas id iosporen de r e n t s p r e c h e n d e n Termitomyces Art gef~t ter t wurden. , 'Wurde d e n e r s t en Arbe i t e rn Basidiosporen auf F ihe rpap ie r a n g e - boten , so produzier ten 92.5 '% der M. michaetseni :Kolonien und 85 % der O. montanus Kolonien Pilzgarten. Wurden Bas id iosporen in e iner Wassersuspension auf die innere Nes tober fNche yon O. montanus Kolonien verabre ich t , b i ldeten 82-5 % der Kolonien Pilzgarten.

* Present address : Division of Animal Physiolo~cy, Zoological Ins t i tu te , Univers i ty of Bern , Er lachs t rasse 9 a, CH-3012 Bern, Switzerland.

FUNGUS COMB I N M. MICHAELSENI 205

I N T R O D U C T I O N

All termite species of the subfamily Macrotermitin~e build fmcal combs, upon which, in mos t if not all cases, a fungus of the basidiomycete genus T~rmitornyces is grown to produce a fungus comb. The only possible exceptions are Sphgerotermes spha~rothorax (SjiJstedt) with sterile combs ,(GRASS~ and NOIROT, 1949) and Madagascar species of Micro termes (SANDS, 1969), which are said to have no fungus, but require fur ther investigation. When laboratory colonies are started with dealate reproduct ives some macro- termitines inoculate their fmcal combs with the fungus, while others are unable to do so. Young laboratory colonies of Pseudacanthotermes spiniger Sj6stedt are unable to start fungus combs, and die after 1.5 - 2 months (LiiscHER, 1951). Colonies of Ancis t ro termes guineensis (Silvestri), Macro- termes michaetseni- (Sj6stedt) and Odonto termes montanus Harris are simi- lar, but are able to inoculate their combs and to survive if a piece of fungus comb f rom the field is offered to the workers (SANDS, 1960; SIEBER and LEUTHO~, 1981; unpublished data). Conversely, it has been found that MacrOterraes belIicosus (Smeathman) (as Bett icosi termes natalensis), Micro- termes unicoIor Snyder, Microtermes sp, hr. usambaricus, and two additional Micro termes species are able to inoculate their combs spontaneously (GRAss~ and NOIROT, 1955 ; AKrITaR, 1978 ; JOr~NSOt~, 1981). It has been assumed that spontaneous fungal growth could only occur if the alates carr ied conidioo spores f rom the fungus combs of their parental nest (Liiscr~R, 1951 ; SAsos, 1960) and J o ~ s o g (198I) has recently found conidiospores in the gut of swarming reproducfives. The offspring workers were able to use released conidiospores f rom the reproductives to inoculate their comb.

To explain development of fungus combs in those species where the reproduct ives do not carry conidiospores, SANDS (1960) suggested that foraging workers might carry basidiospores into the nest during foraging. The aim of the present s tudy was to investigate whe ther fungus comb establishment could be induced by feeding the first foraging workers of M. michaelseni and O. montanus with basidiospores collected f rom mushrooms ' of the cor- responding Termi tomyees sp.

M A T E R I A L AND M E T H O D S

Termites : MacroIermes michaeIseni Mates avere collected from fight traps at Kajiado, 80 km south of Nairobi during swarming in November and December 1980. They ~vere established in pairs and reared in II cm plastic Petri dishes in moistened soil at a constant temperature of 29 ~ C (S~E~R and LEUTnOLO, 1981). To permit observation of eota~/5~ development and the establishment of the fungus comb, a smMl piece of the soil ceiling of the nest was removed.

Odontotermes montanus Mates were collected from Muguga, 20 km west of Nairobi where they s~armed on November 14th t980. Pairs were again established on moistened

206 R. S I E B E R

soil, but in 6.5 cm diameter Petri dishes. The two reproductives immediately dug to the bottom of the Petri dish, where they removed all soil and started to raise the incipient colony. This allowed a continuous insight into the colony through the bottom of the dish. Since the mortality rate was very high at a constant temperature of 29 ~ C, the colonies *were kept at room temperature (18-26 ~ C).

Origin of mushrooms: Termitomyces microcarpus (Heim) was collected betnveen 6th and 12th November 1980, i.e. a few days before the alates fleer. They grew on ftmgus comb fragments ,which were brought onto the soil surface by the ,workers of O. montarms, as has been observed in other Odontotermes sp, (S.~DS, 1969). Although the mushrooms (basidiocarps) usually grew~ on these fragments, in one ease only fungus nodules (Singer and L~UTHOLO, 1981)'which 'were also called mycot~tes (HEIM, 1942), vchite nodules of conidia (SAn0s, 1969) or spherules (BA~RA and BATRA, 1978), 'were brought up onto the surface and some of these grew ou~ to mushrooms. Basidiocarps of the Termitomyces sp. of M. michaelseni 'were no~ found during our observation time in the Kajiado area. Ho~ve.wer, in some mounds of M. michaeIseni beside the usual fungxts nodtfles elongated fungus nodules could be found during the rainy seasons. If fungus comb ~r such nodtfles 'was collected" and brought into the laboratory the development of basidiocarps could be induced in a few cases. However. these were uncharacteristically small (cap diameter not more than 1.5 cm) and the species 'was therefore not determined.

Collection and application o] spores : caps from both Termitomyces species were cut off and laid on filter paper to collect the basidiospores. Pieces of 3 x 3 mm plain filter paper (controls) or of fiIter paper with spores, ,were offered to the first foraging workers of both termite species. Subsequently the ~orkers ~aere fed on hay. In addition, basidiospores were collected in glass Petri dishes, suspended in tap water and dropp2d onto the inner surface of the coputarium of the incipient O. montanus colonies at the time the first ~vor, kers emerged. These colonies were also fed on hay as soon as the first ~vor, kers started foraging. For each experimental group 40 colonies nvere used.

R E S U L T S

D e v e l o p m e n t o f i n c i p i e n t co lon ies : one of the f i r s t b e h a v i o u r a l e l e m e n t s

r e c o g n i z e d in n e w l y e m e r g e d w o r k e r s o f M, m i c h a e l s e n i and O. m o n t a n u s was f eed ing on soil . Th is h a p p e n e d b e f o r e the h e a d c a p s u l e was p r o p e r l y s c l e r o t i s e d so tha t the pas sage o f so i l t h r o u g h the oesophagus c o u l d be

obse rved . This soi l w a s d e p o s i t e d as b r o w n semi - l iqu id f~eca[ pe l l e t s on

the b o t t o m o f the nes t . W h e n the f i r s t w o r k e r s s t a r t e d f o r a g i n g o n hay

these pe l l e t s b e c a m e g r e e n i s h . A l t h o u g h these p i l la rs w e r e g r o o m e d in the

s a m e w a y as f u n g u s c o m b in i n c i p i e n t co lon ies ( S I ~ E R a n d LEUTHOLD, i981)

they r e m a i n e d s te r i le , i.e. no fungus hyphm w e r e o b s e r v e d . The p i l l a r s w e r e

ea t en aga in by the w o r k e r s and u sua l l y n e w p i l l a r s w e r e p r o d u c e d . F i f ty

e igh t and 82 days a f t e r the e m e r g e n c e o f w o r k e r s o f M. m i c h a e t s e n i a n d

0 . m o n t a n u s r e spec t ive ly , w o r k e r s s t a r t e d dying. So ld i e r s a n d la rvm soon f o l l o w e d and f ina l ly the t w o r e p r o d u c t i v e s died. Af te r 105 a n d 146 days

in M. m i c h a e t s e n i a n d O. m o n t a n u s r e spec t ive ly , a l l the co lon i e s w e r e dead.

Feed ing forag ing w o r k e r s w i t h bas id iospores : w h e n a p i e c e o f f i l t e r

p a p e r w i t h o r w i t h o u t b a s i d i o s p o r e s was o f f e r ed to e i t h e r spec ies , i t was

e i t h e r ea ten , o r p a r t i a l l y c h e w e d and p l a s t e r e d w i t h m o i s t e n e d soil . I n t he

FUNGUS COMB I N M. MICHAELSENI 207

la t ter case the wet filter p a p e r w a s replaced to ensure tha t the worke r s had fed on basidiospores. Five days and nine days af ter the f i rs t provis ion of bas idiospores for M. michaelseni and O. montanus respect ively, some of the pil lars were part ial ly covered wi th a whi te layer of hyph~e. These pi l lars then became grey, and a f te r 15 days (M. michaelseni) and 21 days (O. mon- tanus) the oldest pa r t o f the fungus comb had changed to yel low and the f i rs t nodules were visible. A few days a f t e r the es tab l i shment of the fungus comb in M. michaelseni the w o r k e r s bui l t a ceil a round the fungus comb, and larva~ and eggs were main ly car r ied onto the comb. Thir ty six days af ter the first offer of bas id iospores to the foraging worke r s of M. michael- seni, 92.5 % of the colonies had fungus comb which could not be distin- guished f rom fungus comb in t roduced f r o m a field colony. The remain ing colonies died as descr ibed above. However , in 17.5 % of the colonies the new fungus comb ~vas comple te ly consumed dur ing the I00 days of obser- vat ion and since no new fungus c o m b was established, these colonies became weak and died. Similarly in O. montanus 85 % of the colonies had es tabl ished fungus comb within 53 days of the f irst appl ica t ion of basidiospores . During the 150 days of observat ion, the fungus d i sappeared again in 15 % of the colonies, i.e. 70 % of the colonies had fungus comb at the end of the experi- ment . In no case did colonies es tabl i sh fungus comb if they were fed on f i l ter p a p e r without basidiospores .

Application of basidiospores onto the inner nest surface : fungus c o m b was establ ished as descr ibed above when a bas id iospore suspens ion was appl ied to the inner surface of O. montanus nests. The f irst grey pil lars also appea red nine days a f t e r the worke r s s ta r ted to deposi t f~ecal pellets and the first comb with nodules was observed af ter 18 days. Fifty nine days a f te r the applicat ion of the bas id iospores 82.5 % of the colonies had fungus comb, while 10 % of the colonies had eaten the fungus c o m b and died.

D I S C U S S I O N

These investigations p rove the hypothes is of SAN~S (1960) that those macro te rmi f ines whose swarming alates do not ca r ry Termi tomyces conidio- spores f r o m the parenta l nest can es tabl ish fungus comb only if the f irst emerging worke r s collect app rop r i a t e bas id iospores . Since successful esta- b l i shment of fungus comb was obse rved a f te r young workers had fed on f i l ter pape r with bas id iospores bu t also on soil with bas id iospores , i t can b e a s sumed tha t the spores passed the te rmi te gut before they s tar ted to grow.

Alates m a y fly some dis tance f r o m thei r nest of origin, The eff icient dispersal of bas idiospores into the env i ronmen t mus t the re fore be an impor- tant cons t ra in t on the successfull sp read of new colonies. Although the presence of Termitomyces spores in soil r emote f r o m te rmi te nests has not

208 R. S I E B E R

been d e m o n s t r a t e d , it is k n o w n tha t some t e rmi tes p r o m o t e b a s i d i o s p o r e d i spe r sa l t h r o u g h the i r b e h a v i o u r a l r epe r to i r e . The worke r s of s o m e Odonto- t e r m e s spp. c a r ry f u n g u s c o m b f r a g m e n t s (SANDS, 1969), or f u n g u s n o d u l e s (p re sen t s tudy) , o n t o the soil su r face above the nest , where the m u s h r o o m s

grow. As n o t e d ear l ie r , f r u i t i n g T e r m i t o m y e e s were no t f ound on M. michae l .

s en i nes t s d u r i n g the p e r i o d of s tudy , a nd yet young field co lon ies we re deve lop ing success fu l ly a t tha t t ime. I t is therefore a s s u m e d tha t bas id io- spores were e i t he r t r a n s p o r t e d by w i n d s f rom a n o t h e r area, o r t he r e were long-l ived spores in the soil f r o m a n ear l ie r dispersal , o r b a s i d i o s p o r e s f rom T e r m i t o r n y c e s m u s h r o o m s of n e a r b y M a c r o t e r m e s s u b h y a l i n u s ( R a m b u r ) nes t s wh ich w e r e once o b s e r v e d a f t e r ra ins in F e b r u a r y 1981 w e r e be ing used. I t was in fac t poss ib le to inocu la t e combs in i n c i p i e n t l a b o r a t o r y co lonies of M. rn ichae l sen i w i t h f u n g u s c o m b f rom M. subhya l inus , a n d vice versa. These two t e r m i t e speetes m a y have the same T e r m i t o r n y c e s species in t he i r nes t , a n d t he re fo re be ab le to inocu la t e each o the r ' s i n c i p i e n t co lonies

f r o m bas id io spo re s in the field. These e x p e r i m e n t s give no f u r t h e r i n f o r m a t i o n as to w h e t h e r fo rag ing

w o r k e r s p u r p o s l y seek ou t bas id iospores , or w h e t h e r they a re co l lec ted inc iden ta l ly . F u r t h e r m o r e . it is st i l l no t k n o w n w h e t h e r b a s i d i o s p o r e s m u s t pass t h r o u g h the w o r k e r gu t be fore mycel ia l g rowth can begin , as it had b e e n a s s u m e d by JOH~SO~ et at., 1981.

ACKNOWIA~DGEMENTS, ~ This ~ork was supported by the Swiss Directorate fer Dcvelopment Co-operation and Humanitarian Aid, I am grateful to Drs N.M. Cor~r.[~s and R.H. LEUTHOLD for criticism of the manuscripL and to Dr E. ERNST for determining Odontoterrnes tt's

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BAT~.A LR., BATRA S ~r162 1978. w Termite Fungus Mutualism. In " Insect Fungus Sym- biosis ~. Ed. L.R. Batra, Montctair, N.J., Allanheld, 117-I63.

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