Distribution of Termitomyces and other fungi in the nests and major workers of several Nigerian macrotermitinae

Soil Bid. Biockm. Vol. 19. No. 3. pp. 335-341, 1987 Printed in Great Britain. All rights reserved

Copyright C

0038-0717/87 $3.00 + 0.00 1987 Pcrgamon Journals Ltd

DISTRIBUTION OF TERA4lTUMYCES AND OTHER FUNGI IN THE NESTS AND MAJOR WORKERS OF SEVERAL NIGERIAN MACROTERMITINAE REBECCA J. THOMAS* Tropical Development and Research Institute, College House. Wrights Lane, London W8, U.K.

(Accepfed I Ocrober1986) Sammary-The distribution of the symbiotic fungus Termiromyces Heim and other fungi in the nests and major workers of the termites Macrotermes subhyufinus (Rambur), Microrermes grussei Ghidini, Microwmes &pidus Sjiistedt, Microrermes subhyalinus Silvestri, Microlermes sp. nov.. Odonrolermes smea~hmani (Fuller) and Ancisrrorennes cuuirhorax (Sjbstedt) was investigated. The isolation of Temuiomycesfrom forager faecal pellets indicated that Microrermessubhyulinus and Ancirtrolermes incorporated Termitomycesinto the foraged food material within the gut. Mucrolermes subhyafinur had a different strategy for incorporating Termiromyces into the food material. as Termilomyces was not found in forager gut. Termitomyces was found in the digestive tract of nurse workers of all species examined. Other fungi found were mostly common soil fungi, which showed a reduction in number in Mucrorermes subhyafimu from nest structure, to fresh comb, to old comb. The similarity of the fungal flora in soil, termites and fungus combs suggests that the fungi, other than Termilomyces and XyIuriuare brought into the nest as contaminants by the termites.

INTRODUCIJON

A brief account of the relationship between termites of the sub-family Macrotermitinae and their symbiotic fungus, Termitomyces, was given by Thomas (1987) with particular reference to Mucrotermes belliCOWS(Smeathman). The aim of this study was to determine how the pattern of distribution of fungi in nests and workers varies for Macrotermitinae with differing nesting and feeding biology and to compare the results with those from a more comprehensive study of M. bellicosus. Termitomyces grows on fungus combs, which are constructed from the termites’ primary faecal pellets which consist of poorly digested food (Sands, 1960; Grass& 1978; Badertscher et al., 1983). The termites eat the older parts of the fungus comb while building it up on the top and sides with fresh material. This work was carried out in the southern Guinea savanna vegetation zone of Nigeria near Mokwa (9” 18’ N, 5” 04’ E). The following species were studied: Mucrotermes subhyalinus (Rambur): in this area it builds low multi-domed mounds, with fungus combs found in chambers closely associated with each other. Unlike M. belficosus it has no foodstore, no central hive and only two zones can be distinguished in the fungus combs. The fungus combs are large, convoluted, irregularly shaped structures. The newly deposited material (fresh comb zone) is darker in colour. The rest of the comb (old comb zone) is lighter in colour with the asexual reproductive structures of Termitomyces (mycot&s) present.

Microtermes spp: there is a complex of seven sympatric Microtermes species in the area. They are reproductively isolated by their differing swarming flight times and pair-forming behaviour (Wood, 1981). During this study they could not be assigned a species name, so were given code letters, as in Wood (1981). and identified later. The species studied were Microtermes lepidus Sjiistedt (A), Microtermes grussei Ghidini (B), Microtermes sp. nov. (C) and Microtermes subhyulinus Silvestri (G and R). The Microtermes subhyulinus termites and fungus combs, designated R, were not from Mokwa but were collected from a narrow belt of forest fringing floodplains of the River Niger at Rabba (9”12’ N, S”O4’ E). All Microtermes have diffuse subterranean nests, consisting of chambers containing single fungus combs up to Scm dia. The chambers are linked by narrow galleries. The freshly deposited material is lighter in colour than the older which is dark grey. The faecal pellet structure is obvious in the fresh material but not in the older. Species of Microtermes are common in primary savanna woodland and forest, and some species increase in abundance under cultivation to become important pests. Odontotermes smeuthmuni (Fuller): this species has entirely subterranean nests, consisting of fungus combs in separate chambers in the soil. The newly deposited portions of the comb are darkest in colour. Ancistrotermes cuuithorux (Sjiistedt): this species has subterranean polycalic nests with the fungus combs in individual chambers. The fungus combs are fairly small (3-5 cm dia). MATERIALS AND METHODS

lPrcsmt address: ICAP. Cranfield Institute of Technology, Cranfield. Redford MK43 OAL. U.K.

Isolation media used were a general soil fungus medium (SF) containing glucose (log I-‘), yeast 335

REBECCA

336

J.

when the fungi identification.

extract (0.5 g I-‘) and novobiocin (0.1 gl-‘1, and a selective medium (SEL) for Termitomyces which contained three fungicides, gallic acid and ox bile (Thomas, 1985). Distribution

THCMAS

were coded

and

for

RESULTS

Distribution of fungi in the nest

offungiin the nest

Fungi were isolated from fungus combs, and from the soil nest structure of Macrotermes ~hyuijn~ by (I) direct isolation and (2) dilution plates (Thomas, 1987). (1) Samples were taken from both fresh and old comb zones of M. subhyalinus and A. cavithorax, but from whole comb only for M. grassei, M. fepidus, Mi~rotermes subhyalinus, Microtermes sp. nov. and 0. smeathmani. (2) One mound of M. subhyafinus used to set up three replicate dilution series on SF. Five plates were prepared for each material and series. Due to the smaiI amount of material available it was not possible to carry out separate dilution plate experiments on fresh and old Microtermes comb material, and no replicate dilution series could be made. Five plates were set up on SF, and five on SEL from comb frotn Mi~rotermes grassei, Mierotermes lepidus and Micro termes sp. nov.

The numbers of fungi obtained from different parts of the M. subhyalinus nest system are given in Table 1. The nest structure had signi~~ntiy higher numbers of fungal colonies than fresh comb (P c 0.01) and old comb (P < O.OOl), and the fresh comb had significantly higher numbers of colonies than the old comb (P < 0.001). The numbers of fungi obtained from the Microtermes fungus combs are given in Table 2. Comb from Microtermes sp. nov. had signiticantiy higher numbers of fungai colonies (P
In contrast to M. ~e~l~co~~ (Thomas 1987) simila numbers of foragers and nurse workers of M. sub hyafinus had a boius in the crop (Table 4). However the boius in nurse workers was light in coiour am appeared to be fungus comb, while that of the Foragers was dark and was composed of soil and/o the foraged material as in M. bellicosus foragers. No Termitomyces was obtained from the digestivt tract of M. subhyafinus foragers, whereas 13% of the nurse workers had Termitomyces (P c 0.05) (Tab11 4). There is no significant difference in the number o Termitomyces colonies obtained from the faecai pei lets of soil and food-carrying foragers and nursl major workers Microtermes subhyalinus (Table 51 Ancisfrofermes cavithorux foragers also had Termi tomyces in their faecat pellets. The species of fungi obtained on both media fror the fungus combs and termites of ail the specie investigated are given in Table 6.

~so[ation offungi from the termite digestiue tract

Foraging major workers (foragers) and major workers from fungus comb (nurse workers) of M. subhyalinus were surface sterilized in Milton’s fluid. The digestive tract was dissected out in sterii water and ptaced on SF and SEL plates. The presence or absence of a boius in the crop was recorded. Microtermes and A. cavithorux major workers were squeezed with forceps causing them to eject a faecai pellet which was picked up with a sterile needle and spread on SF and SEL plates. Microtermes subhyalinus foragers were divided into two groups, those with dark gut containing soil, and those with lighter coloured gut carrying food. Some intermediates were also found, but not used. Ail plates were incubated at 2&3O”C, and the fungi developing counted after 2 days, and after 5 days

Table I. (a) Mean number of fungal colonies per g dry wt of different parts of the hf. rubhyafinur nest system. Results of the dilution plate experiment on SF medium expressed as counts x IO” + SE, n = 5)

Fresh comb Old comb Nest structure

isolated

A

B

C

Mean

7.58 2 I .93 I.38 I: 0.65 10.21 & 0.61

IS.52 f 3.58 2.76 f I .03 25.00 $q2.38

15.28 * 2.20 6.54 f 1.36 21.29 k 3.79

12.80 3.56 18.83

Table I. (b) Analysis of variance on the numbers of fungal colonies found in different parts of the nest system Degrees of freedom

Mean squares

Parts of nest Between samples Interaction Error

2 2 4 36

887.61 320.495 56.518 25.065

Total

44

Source of variation

Variance ratio (F) 35.41*12.79.‘* 2.25NS

Fungi in termite nests Table 2. (a) Mean number of fungal colonies per g dry wt of different hficmermes spcciafungus combs. Results of the dilution plate experiment on SF medium (all numbers x Id)

MicrorermeJ sp. nav.

hfirroremwr grarsei 89.46 36.84 42.10 47.36 65.78

105.X 68.39 1IS.29 I IS.29 as.90

77.51 53.04 53.04

44.88 44.80

56.31 + 9.62

Mean 98.09 * 9.15 colonies + SE

Microrermes lepidw

54.67 f 5.99

Table 2. (b) Analysis of variance on the numbers of fungi obtained from the fungus comb of different Microrermes species

k=-

source of variation

of freedom

Mean squam

Variance ratio (F)

2

3028.66

8.56”

Between Microrermes spp Error

I2

TdZd

IA

353.67

Table 3. (a) Mean number of Termiromyres colonies per g dry wt of different Microtermes fungus combs. Results of the dilution plate experiment on selective medium. (All numbers x IO’) Microtermes ~0. nav.

Microlermes arassei

Microrermes IePiduJ

125.06 54.72 87.94 117.25 91.84

18.42 31.57 44.73 7.89 10.52

16.32 34.68 87.71 14.28 12.24

22.63 c 6.89

33.05 f 14.24

Mean 95.36 + 12.41 colonies +SE

Table 3. (b) Analysis of variance on the numbers of Termiromyce$ colonies obtained from the fungus combs of different hficrorermes species source of variance

Degrm of freedom

Mean squares

Variance ratio (F)

I I .48*’

Between Microrermes sp Error

2

7735.32

12

673.83

Total

14

Table 4. Presence of a bolus in, and the isolation of Temiromyces from. the put of M. subhvaiinus maior workers (SEL medium)

Occurrence of bolus (n = 60) Isolation of Termiromvces(n = 30)

Foragers

Nurse workers

38%

40%

0%

13%’

337 DISCUSSION

The association of fungi with termites and their nests All the fungi isolated from the fungus comb and workers of the various termite species (Table 6) are commonly found in Mokwa soils (McDonald, 1968), except for Termitomyces and Xylaria. hf. subhyalinus nests were similar to those of M. beflicosus (Thomas, 1987) with most s found in the nest structure in the di-y”‘” ution being plate experiment, and the number of different species declining from fresh to old comb (Table 6). There was also a drop in the total number of fungi from fresh to old M. subhyulinus comb (Table I). The majority of fungi obtained on the Microtermes fungus comb dilution plates were, in contrast to M. subhyulinus, Termitomyces. The small Microtermes combs are subject to the influence of variations in the soil conditions, whereas the much larger Macrotermes combs are in stable conditions of temperature and moisture which could favour the survival of other fungal species. In Microtermes subhyalinus foragers the total number of fungi and number of species was highest in those carrying soil in the gut (Table 6). Nurse workers had a lower number of fungi and species than foodcarrying foragers. This, together with the similarity of the fungal flora in soils, termites and fungus combs, suggests that the fungi, other than Termitomyces and Xyluriu, are brought into the nest as contaminants by the termites, both mixed with the food material and adhering to their bodies. Other authors have also reported a wide range of fungi from termites. Ruyooka (1979). working in East Africa, found a range of Fungi Imperfecti in the gut of all M. subhyalinus castes except minor soldiers. Three genera, i.e. Aspergillrcs, Curvularia and Fusarium, amongst others, commonly obtained from the termites investigated here, were found in workers of Odontotermes obesus (Rambur) (Das et al.. 1962). with other species being recorded in the gut of workers (Rajgopal et al., 1979, 1981), workers and soldiers (Bose, 1923) and minor workers (Batra and Batra, 1979). Spores of soil fungi have been found in the combs of 0. obesus (Das et 01.. 1962; Mohindra and Mukerji, 1982), Odontotermes redemanni (Wasmann) (Petch, 1906), and from comb. nest material and workers’ gut of Odontotermes gurdaspurensis Holmgren, K. and N. (Batra and Batra, 1966). Many species, especially Aspergillus were obtained from the exterior of 0. obesus and 0. redemanii (Singh et al., 1978). Association of Termitomyces with Jiigus combs and termites M. subhyulinus appears to have a similar strategy

xl = 4. P c 0.05.

Table 5. Presence (% isolation) of Termiromyces in the faecal pellets of the different classes of Microrermes major workers. and Ancisrrowrmes ccmirhorax major workers Microrermes subhvalinw (R)

Ancirrrorcrmes covi~horax

Nurse workers

Nurse workers

Foragers

83 29

71 7

60 10

hficrolermes subhvalimu

Classes of major worker Termitomyres n

Foragers Soil Food carriers 90 30

93 29

6(a).

Specirs

3 5

II

+i-+

++++

2 5

+++ 2 5

+

++++

7

it

7 30

++

++t+

+

++

t

++++

++t+

+

+

f +t++

J.O-t4.99 208 >

8 30

7 30

+

tit

+

*

+++ t

+

2 5

ffc

t+t* ii

3 5

++

++++

3 6

++

++t

+++

2 6

+t

t++t

3 6

tit

++

++++

I 6

ct+t

I I”

++t

++++

nests rnd !crmitcs, and from fungus comb and major workers of other teamile species

(no. 8 dry WI-’ x IO’)

[email protected]

--x.99

DiMion-

7 30

t

f

+

++

++t ++++

*

++

+

++

4

f

+

f

*+

0-14‘9 Is-2Y.Y 34.0-49.9 50.0..,

3 3

++

Isolation inda

13

t t

++++

+t

+

t

t-f

t++t

+

c

+

++

f

f t

+++

f f

+ ++I‘ +

+

++

++4 ++++

+t

directly (isoln) or by dilution pIales (dp) from different parls of M. subhydinw

+

+ f

t-f +

f

t

fungi isolated

+ ++

of

Frqwncy of fun@

Toul No. isolation%

Table

22

Fungi

: +

iu termite nests

+

+

+

*

=

e

i+

c

*

+

: *

*

:

+

+

c

c

i+++ + o*g 1 i

340

REBECCA J. T~~OMAS

to M. belficosus (Thomas, 1987) for incorporating Termiromyces into the food material. M. subhyufint4s foragers did not carry Termizomyces (Table 4). and few carried it in M. bellitosus. In M. bellicosus no Termiromyces was isolated from the foodstore, indicating inoculation has not occurred at this stage, and so must occur either on transfer of food material from foodstore to fungus comb, or by growth of Termitomyces from older comb. M. su~hyuI~us does not have a foodstore in the mound, although Darlington (1982) found subterranean foraging pits in the closely related species ~acrufermes mjchuelse#i (Sjiistedt) where at Ieast part of the foraged plant material was deposited. The digestive tract of M. subhyalinus foragers does not contain Termitomyces so its incorporation into the food material must, similarly to M. bellicosus, either occur on transfer from foodstores outside the mound, if they occur, to fungus comb, or by growth into the freshly deposited material by Termitomyces mycelium from the older sections of the comb. The latter appears likely as the isolation frequency from freshly deposited comb is much lower than from older comb. Similarly, Rohrmann (1978) found fresh comb of Macrotermes ukutii Fuller lacked the hyphae of the older comb. Microtermes subhyalinus and A. cauithorux employ a different strategy for incorporating Termi~omyces into food material. The Microtermes ~bhyufinus major workers, including soil-manipulating foragers, and A. c&thorax foragers, have Termitomyces in the gut, suggesting they must have fed on it before leaving the corpb. All other species of Microtermes examined have had Termitomyces in the gut of foraging workers. This means that the majority of faecal pellets will contain Termitomyces when placed on the fungus comb, which is confirmed for A. cucithorux by the similar isolation frequency of Termitomyces from the fresh and old comb zones. Although the isolation data is given for whole comb for the Microtermes species in Table 6, isolations (but not the dilution plate experiments due to the small amount of material) were carried out separately on fresh and old comb, with Termitomyces being of similar or higher isolation frequency on fresh and old comb. This strategy would appear to be necessary in Microtermes and A. cutGthorax where combs are small units in pockets in the soil and more isolated from other combs than in centralized nest systems. Unlike the Macrotermes species, M~crotermes uses up the fungus comb completely, with the comb being atmost totally consumed by the end of the dry season (Wood and Johnson, 1978). The depth at which combs are found changes through the season too. This also indicates that the source of inoculum for new combs would have to be the termite. In addition to interspecific differences in the carriage of Termitomyces there are also differences between castes. In M. be~t~cos~ the young workers have a higher percentage of Termitomyces in the gut than nurse workers, perhaps indicating feeding differences. Abo-Khatwa (1978) suggested that some of the cellulase components found in the digestive system of M. subhyalinus came from Termitomyces, and Martin and Martin (1978, 1979) explained the nut~tionai dependence of Macrotermes rtataiensis (Haviland) on its fungus gardens was due to Termitomyces being the

source of C, -enzymes, active against crystalline cellulose. Doflein (1906) found that the crop of alI larvae and nymphs of Odonfotermes obscwiceps ~~rnann) was ii&d with Termiromyces, but not of the workers. These differences in feeding between castes may relate to the nutritional demands of the different castes. Ackno~le~e~nts-us work was carried out as part of a CASE studentship funded by NERC and the Centre for Overseas Pest Research. Thanks an due to the Director of the Institute of Agricultural Research and the Officer-in-Charge, Mokwa, for providing facilities at the geld-station at Mokwa.

REFERENCES Abo-Khatwa N. (1978) Cellulase of fungus-growing termites: A new bathos on its origins. Exprientia 34, 559460. Badertscher S., Gerber C. and Leuthold R. H. (1983) Polyethism in food supply and processing in termite colonies of Macrotermes s~h~uf~~ (isoptera). Rehmriourai Ecology & Sociobiology 12, I S-1 19. Batra L. R. and Batra S. W. T. (1966) Fungus-growing termites of tropical India and associated funs. Journal of the Kansas Entomological Society 39, 125-738. Batra L. R. and Batta S. W. T. (1979) Termite-fungus mutuaIism. In Insect-Fungus ~~~~0s~ (L. R. Batra. Ed.), pp. 117-163. Allanh~d, Montclair, N.J. Bose S. R. (1923) The funai cultivated by the termites of Barkuda. .Reco;d o/the Indian Museum*rS, 253-258. Darlington J. P. E. C. (1982) The underwound passages and storage pits used in foraging by a nest of the termite Macrotermes michaelseni in Kajiado, Kenya. Journai of Zoology (London) 198, 237-247. Das S. R., Maheshwari K. L., Nigam S. S., Shukla R. K. and Tandon R. N. (1962) Micro-organisms from the fungus garden of the termite Odontotermes obesus (Rambur). In Proceedings of theSymposium on Termites in the Humid Tropics, New Delhi, 1960, pp. 163-166. Doflein F. (1906) Termite trufffes. Spoiiu Zeyhmico 3, 203-209. Gras& P. P. (1978) Sur la v&table nature et ie rdle des meules B champignons construites par les termites Macrotermitinae (Isoptera, Termitidae). Compre Rendu Hebdomadaire des SpOnces de l’dcademie &s Sciences, Paris (Series D) 287, 12234226. McDonaId D. (1968) A list of fungi associated with groundnuts in Nigeria. Samaru ~~cellaneo~ Pnper NO. 27. Martin M. M. and Martin J. S. (1978) Cellulose digestion in the midgut of the fungus-growing termite Mucrotermes nutule~~: the role of acquired digestive enzymes. Science 199, 1453-1455. Martin M. M. and Martin J. S. (1979) The distribution and origins of the cellulolytic enzymes of the higher termite, Macrotermes natalensis. Physiological Zoology 52, 11-21. Mohindra P. and Mukeji K. G. (1982) Fungal ecology of termite mounds. Revue &Ecologie et de Biofogie du Sol w(3). 351-363. Petch T. ( 1906) The fungi of certain termite nests, .&nab of the Royal Botunic Gardens of Perudeniya 3, 18.5-270. Rajgopal S., Rao D. R. and Varma A. K. (1979) Assoeiation of fungi in termite gut. Current Science 48(22), 998-999. Rajgopal S., Rao D. R. and Varma A. K. (1981) Fungi of the worker termite-gut, Odontotermes obesus (Rambur) from Northern India. Nova Hedwigia 341-2),97-100. Roh~ann G. F. (19781 The o&in. structure and nutritional importan& of the comb ‘;I ;wo species of Macrotermitinae (Insecta, Isoptera). Pedobiologia 18, 89-98.

Fungi in termite nests Ruyooka D. B. A. (1979) Fungi associated with Macrotermes near subhyalfnw. Report, Internationaf Centre of insect Phys~~y and Ecdgy 6(197@, 32-33. Sands W. A. (1940) Initiation of fungus comb construction in laboratory colonies of Rncirtrotermes gutiteensis (Silvestri). Xnrectes Sociaux 7, 251-259. Singb 0. R., Singh J. and Singh I. D. (1978) Microbiz association with the termites in a tropical deciduous forest at Varanasi. Tropical Ecology D(2). 163473. Thomas R. J. (1985) Selective medium for isolation of Termitomyces from termite nests. Transactions OJ the British Mycological Society 84 519-526. l%omas R. J. (1987) Distribution of Termitomyces Heim and other fungi in the nests and major workers of

WI

Macfotermes befIicosw (Smeathman) in Nigeria. Soil Biofogy & Biochemistry 19, 329-333. Wood f, G. (1981) Reproductive isolating mechanisms among species of Microtermes (Isoptcra, Tcrmitidae) in the Southern Guinea savanna near Mokwa. Nigeria. In Biosystematics of Social Insects (P. E. Howse and J.-L. Clement. Eds), pp. 309-325. Systematics Association Special Volume 19. Academic Press, London and New York. Wood T. G. and Johnson R. A. (1978) Abundance and vertical distribution in soil of Mfcrotermes (Isoptera, Termitidae) in savanna woodland and agricultural ecosystems at Mokwa. Nigeria. ~e~orabiIia Zoofogica 29, 203-213.