Effect of laboratory containment on the nitrogen metabolism of termites

Insect Blochem Vol 15 No 4, pp 503-509, 1985

0020-1790/85 $300+000 Copyright r~ 1985 Pergamon Press Lid

Printed m Great Britain All nghts reserved

E F F E C T OF L A B O R A T O R Y C O N T A I N M E N T O N THE N I T R O G E N M E T A B O L I S M OF T E R M I T E S MARGARET LOVELOCK, R W O'BRIEN a n d M SLAYTOR Department of Biochemistry, The University of Sydney, New South Wales, 2006, Austraha (Recewed 13 June 1984, rettsed 18 October 1984)

A~tract--When Nasutttermes extttosus, Nasutttermes walkerl and Coptotermes lacteus were brought into the laboratory they rapidly lost within 24-48 hr, their abdlty to fix dinltrogen With N extttosuv and N wall, err the loss was hnear over the first 26-32 hr at a rate of aoout 3~,~o per hour N walkert completely lost its abdlty to fix dmltrogen and did not recover it dunng a further 11 days in the laboratory whereas N extttosus and C lacteus partially recovered their &mtrogen fixing ability to about 25-50°~o of the original rate During laboratory storage of up to 60 days both C lacteus and N extttosus gradually lost total mtrogen, whde at the same t~me the,r uric aod content increased The uric aod content of N walkert increased during 17 days m the laboratory whde total mtrogen remained essentmlly constant Xanthine dehydrogenase was not detected m freshly-collected N nalkert but was detectable after two days of laboratory storage and reached a maximum actlwty m 8-10 days The rate of dmltrogen fixation, total mtrogen and uric acid of field populations of N extttosus and N walkert (tested within 2 hr of collection) remamed within close lim*ts over a 6-8 week period, indicating that the changes m these parameters observed m populations kept in the laboratory &d not occur m field populations In field populations of N walkert the total nitrogen was about 1 4°0 of the fresh weight (6 7°0 of the dry weight) and the uric acid content was about 1 3°g of the fresh weight (6 6°0 of the dry weight), with the amount of total nitrogen present as uric acid being about 31°o In N ermosus these values were total nitrogen about 1 6°0 of the fresh weight (7 4°0 of the dry weight) uric acid about 0 6°0 of the fresh weight (2 9°~oof the dry weight), w~th uric acid accounting for about 13°o of total mtrogen When workers of N walkert were stored m a container near their nest they lost dimtrogen fixing abdlty to the same extent as workers brought into the laboratory, indicating that disruption of the nest was sufficient to affect dimtrogen fixation Key Word Index Termites, mtrogen metabolism, dmltrogen fixation, uric aod, xanthine dehydrogenase, Na~uttterrnes extttom~ Na~uttterrnes ~all~ert, Coptotermes lacteu~

INTRODUCTION Dinitrogen fixation m termites was first d e m o n s t r a t e d independently by Breznak et al (1973) and B e n e m a n n (1973) Since then a n u m b e r of other studies have confirmed dlnltrogen fixation in a wide range of termites (listed by Breznak, 1982 French et a l , 1976, Sylvester-Bradley et a l , 1978, Schaefer and W h l t f o r d , 1979, Prestwlch et a l , 1980, R o h r m a n n a n d R o s s m a n , 1980, Prestwlch a n d Bentley, 1981) The natural diet o f termites is i m p o r t a n t in determining whether they fix dinltrogen as shown by the work of Prestwich et al (1980) They f o u n d t h a t N a s u t t t e r m e s corntger feeding on woody litter low in fixed nitrogen h a d a higher dlnitrogen fixing activity t h a n R h y n c h o t e r r n e s p e r a r a m a t u s feeding on leaf litter with a higher fixed nitrogen content R o h r m a n n and R o s s m a n (1980) f o u n d that M a c r o t e r m e s u k u z n , which m a i n t a i n s a fungus garden, & d not reduce acetylene, a n d t h a t a soil-feeding C u b t t e r m e s sp reduced only slight a m o u n t s of acetylene They speculated t h a t the high chitin c o n t e n t of the fungus eaten by M ul~uztt, plus the presence o f chltinase m the gut o f this species, may provide sufficient nitrogen for the termite a n d thus repress dinitrogen fixation in the gut, C u b t t e r m e s sp was presumed to o b t a i n its m t r o g e n requirements from the soil It ingests The a b o v e findings are consistent with the earlier experiments of Breznak et al (1973) in which they showed repression of d l m t r o g e n fixation by feeding termites some form 503

of fixed nitrogen, such as a m m o n i u m salts, aspartlc acid or autoclaved Eschertchta colt cells T h u s repression was reheved when the termites were transferred to a diet low in m t r o g e n Despite the a p p a r e n t i m p o r t a n c e of dlnltrogen fixation in some species of termites there is n o w evidence to indicate t h a t the nitrogen m e t a b o l i s m of termites b r o u g h t into the l a b o r a t o r y from the field undergoes rapid a n d d r a m a t i c changes Prestwich et al (1980) showed that d m l t r o g e n fixation in N c o r m g e r and R p e r a r a m a t u s fell to zero after storage of the termites in the l a b o r a t o r y for two days a n d did n o t recover over a further five days of storage However, the rate at which d m l t r o g e n fixation dechned was not determined T h e uric acid c o n t e n t o f l a b o r a t o r y - m a i n t a i n e d termites increases with time o f storage (Potrlkus a n d Breznak, 1980a, N a z a r c z u k et a l , 1981) In the case of R e t t c u h t e r m e s f l a v t p e s the uric acid content of the termites rose from l 7 to 45 4~o over a 15 m o n t h period, with the increase being at the expense of non-uric acid nitrogen ( P o t n k u s a n d Breznak, 1980a) The work o f Prestwlch et al (1980) leaves unanswered several basic questions concerning dlmtrogen fixation in termites, for example is the loss of dlnitrogen-fixmg ability of termites b r o u g h t into the l a b o r a t o r y a general p h e n o m e n o n and, if so, h o w rapidly is it lost, does d m l t r o g e n fixing ability recover during l a b o r a t o r y storage, does the same p h e n o m e n a occur in field p o p u l a t i o n s and, finally, w h a t effect

504

MARGARET LO'~EL()(K et al

d o e s t h e loss o f d l m t r o g e n f i x a t i o n h a v e o n t h e g e n e r a l n i t r o g e n m e t a b o h s m o f t e r m i t e s 9 In th~s p a p e r we a t t e m p t to a n s w e r s o m e o f t h e s e q u e s t i o n s M A T E R I A L S AND M E T H O D S

Termtte~

Ground nests o1 ( optoterme~ la~ teus (Froggatt) v~ere collected in Ourlmbah and Olney State Forests N S W and of Nmuttterme~ e',lttosu~ (Hill) near Ku-rlng-gal National Park Sydney Nmuttterme~ t, all~ert (Hill) was collected from galler, es leading to an arboreal nest on a Eu~ahptm sp tree located on the Greenwich foreshore of Sydney Harbour The same nest was sampled for all experiments All species were maintained in the laboratory In their own nest material in polythene bins at ambient temperature (20-23 C) m the dark The termites were fed E u t a h p t m sp wood and the humidity was maintained with a d a m p towel placed over the nest material Worker caste termites were used in all experiments The fresh weight of termites was determined by weighing 100 workers and dry weight was determined after drying the same termites to constant weight at 90 C Dmltrogen fixation

The assay for dlnltrogen fixation was based on the acetylene reduction method of Hardy et al (1968) Fifty termites were removed from their nest material and were placed in a serum vial (6 nil) and capped with a Suba-seal Instrument-grade acetylene was injected into the zlal to give an atmosphere of 12°. acetylene at ambient barometric pressure Ethylene production ~ a s measured b) gas chromatograph2¢ using a Hewlett Packard 5710A gas chromatograph with a flame lonxsatlon detector A column (0 31 cm x t 8 m) of Poropak N (80-100 mesh, Alltech Associates, Deerfield, Ilhnols U S A ) was used at 60 C with nitrogen as carrier gas 130ml mln) and the peaks v~erc recorded on a He~lett Packard 3390 Integrator The ethylene peak heights were calibrated against samples from a serum ~lal of known ~olume containing 4#1 of ethylene Corrections were made for ethzlene in the acetylene and possible release of ethylene trom the Suba-seals by Including a no organism xlal in each determination Vials containlng termites but no acetylene were Included to correct for any endogenous ethxlene release by the termites To reduce release of ethylene [rom the Suba-beals they were rested for at least two weeks between use Acetylene reduction was calculated as nmol o[ eth,Aene produced/termite per hr and in this paper is considered sznon,cmous with dlnltrogen fixation Termites were routinely incubated for I hr at 24 C under these condltmns the rate of acetylene reduction by all species was linear For freshly-collected C lantern incubations were commenced immediately on collection and assays were made 3 5-5 hr later These assays were corrected for the non-linear rate of acetylene reduction between 1 5 and 5 hr For freshly-collected N e'~ltlO~m and & ~talaerl incubations were started 1 5 2 hr after collection D¢ l(~ltltlllallOll O/ Ioldl IllllO~¢ll

Ten termites ol each species were digested with sulphurle acid (3 ml) and potassium sulphate ( 1 6 g) and the a m m o n i a produced was estimated by the phenol,,hypochlorlte method (Kaplan 1965) OeD:,lmln¢ltlOll o] loll a~ld

Uric acid was extracted lrom whole termites with lithium carbonate and ,xa~ assayed with urlca~e as described bx Nazarczuk et al 119811 kantttme deh~drogenasc a~sal

Workers ol '¢ ,,all, eH were lmmoblhsed ,it 4 C and were

degutted with line-tipped lorceps The degutted bodies 16t/t and the guts (60) were homogenlsed separately in 2 ml ol 0 I M tNs HCI buffer pH 8 0 in a Ten Broeck homogenlsei at 4 C followed by eentrlfugatlon at 2 7 0 0 0 g lor 1 0 m m (Potrlkus and Breznak 19811 The supernatant lractlon~ wert. retained for the enzyme assa~ Xanthlne d e h \ drogenase (EC 1 2 1 ~ 7 1 activity was determined spe~trophotometrlcally b~y measuring the substratc-dependenl reduction of N ~ D + (Parzen and Fox 19641 The reaction mixture (hnal ~olume I 0ml) contained Tlls HCI buffer pH 8 0 83 l~lnol NAD 0 5ttmol h,cpoxanthlnc 0 15 ilmol and termite extract (about I) 9 mg protein1 Potassium c,¢anlde was omitted lrom the ,ls~a~ as Na~DH oxidation b', extracts was neghglble One unit ol cnz',me actlXlt5 1~ defined as the a m o u n t ol protein reducing 1 nmol ol NAD+ mln Specific actl~lt~ is defined as u m t s , m g ol protein Protein in termite extracts ~ a s determined b'~ the bluret method (Gornall et al 19491 using boxlne serum albumin as a standard R ES{ LTS

T h e effect o f l a b o r a t o r 3 s t o r a g e o n d l n l t r o g e n f i x a t i o n by N e x t t l o ~ m is s h o ~ n in F i g l a T w o s e p a r a t e p o p u l a t i o n s , p o p u l a t i o n 1 collected in A p r i l a n d p o p u l a t i o n 2 collected in A u g u s t 1983 were tested a n d b o t h s h o w e d a r a p i d fall in d m l t r o g e n f i x a t i o n o v e r 3 d a y s S o m e r e c o v e r ) o f activity bY b o t h p o p u l a t i o n s w a s o b s e r v e d at d a y 7 a n d in t h e case o f p o p u l a t i o n 1 t h e r e w a s a full r e c o v e r y o f activity at d a y 13 T e r m i t e s f r o m b o t h p o p u l a t i o n s e x h i b i t e d a f l u c t u a t i n g activity with p o p u l a t i o n 1 s h o w i n g g o o d activities (close to t h e o r i g i n a l actl~lty) at dayb 28 a n d 49, w h e r e a s t h e a c m l t y o f p o p u l a t i o n 2 r e m a i n e d at less t h a n 5 0 ° . o f t h e o r i g i n a l actwlt~ T h e decline o f d l n l t r o g e n f i x a t i o n in V e t m o s u ~ ~ a s h n e a r m e r a b o u t 32 h r for t ~ o p o p u l a t m n s f r o m different n e s t s (Fig 2a) R e g r e s s i o n a n a l y s i s o f t h e d a t a g a z e a c o r r e l a t i o n coefficmnt for b o t h p o p u l a t i o n s o f 0 99 with t h e decline in d m l t r o g e n fixation ol p o p u l a t i o n 1 b e i n g t h e e q m ~ a l e n t o f 0 034 n m o [ e t h , , l e n e p r o d u c e d : t e r m i t e p e r h r a n d for p o p u l a t i o n 2 b e i n g l) 024 n m o l e t h y l e n e p r o d u c e d t e r m i t e p e r h r T h i s w a s a loss o f a b o u t "~ 4",, o f d l m t r o g e n fixing a b l h t ) h r t o t b o t h p o p u l a t i o n s B~ e x t r a p o l a t i n g t h e lines to zero t i m e t h e p r e s u m e d v a l u e s for a c e t y l e n e l e d u c t l o n m t h e field p o p u l a t i o n s were 1 0 4 a n d 077nmol e t h y l e n e p r o d u c e d ' t e r m i t e p e r h r respective b T h e d l n l t r o g e n h x a t l o n in a n o t h e r n a s u t e t e r m i t e V Ila//,e~t ( b i g 2by, also d e c h n e d at a linear rate o v e r t h e hrst 26 29 h r o f l a b o r a t o r y s t o r a g e R e g r e s s m n a n a l ) s i s ( c o r r e l a t m n coefficient for b o t h p o p u l a t i o n s 0 99) a n d e,~trapolatlon o f t h e h n e to zero t i m e l n d m a t e d t h a t t h e field p o p u l a t i o n s w o u l d h a v e rates ol a c e t } l e n e r e d u c t m n ol 1 29 a n d 1 35 n m o l e t h y l e n e p r o d u c e d t e r m i t e p e r h r D l n l t r o g e n fl,~atmn m b o t h p o p u l a t i o n s w a s lost c o m p l e t e l y w i t h i n 48 h r a n d dld not recmer over a furthel 9 days of laboratory s t o r a g e T h e loss o f a c e t y l e n e r e d u c t i o n w a s 0 047 ( p o p u l a t i o n 11 a n d 0 043 ( p o p u l a t i o n 2) n m o l ethyle n e p r o d u c e d / t e r m i t e p e r h r ol a b o u t 3 5°o, h r A t h i r d e,~perlment with N uall~ell ( n o t s h o , ~ n ) g a z e r e s u l t s ( c o r r e l a t i o n coefficwnt f r o m r e g r e s s i o n a n a l ) sis o f 0 86) for a c e t y l e n e r e d u c t i o n at zero t i m e ( t h a t is ume o f collection) o f l 25 n m o l e t h y l e n e p r o d u c e d t e r m i t e p e r h r with ,l loss o f actl',lty ol

N~trogen metabohsm in termites

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Fig 1 Effect of laboratory storage on acetylene reduction (a), tot,,l mtrogen (b) and uric acid (c) of N extttosus ( 0 ) Population 1 (collected Aprd 26, 1983), ( i ) population 2 (collected August 8, 1983)

Populations were collected from different nests

0 047 nmol ethylene produced/termite per hr The acetylene reduction by a sample of N walkert placed m a container and left at the nest site for 24 hr was compared w~th that of a similar sample taken at the same txme and kept m the laboratory for 24 hr The values obtained were 0 22 and 0 24nmol ethylene produced/termite per hr, respectwely When C lacteus was brought into the laboratory it lost its ablhty to fix dmitrogen (Fig 3a) more rapxdly than did N exltlosus and N r*alkert (Fig 2) with about 90~o of the actwlty being lost within 24 hr The dlnltrogen fixation by population 1 recovered to some extent (usually less than 25% of the original actlwty), except at day 49 when the activity was about 40~o of the original value The dlmtrogen fixation by population 2 continued to dechne after day 1 and was not detected after day 14, the termites rapidly died after day 28 Another population of C la~teus gave virtually ldenhcal results as population 2 (data not shown) This population was collected at the same time and m the same area as population 2 Other parameters of mtrogen metabohsm, namely total mtrogen and uric aczd were measured m termxtes kept m the laboratory In the case of N e.~tttosus the total mtrogen (Fig lb) of both populations de-

chned Termites collected m April (populahon 1) had a higher mltlal nitrogen content than those collected m August (populahon 2) However, regression analys~s of total nitrogen versus t~me gave a low correlation coefficient of 0 54, with a rate of dechne of mtrogen of 0 32 pg N/termite per day In population 2 the dechne was 0 49 #g N/termite per day with a correlatxon coefficient of 0 9 The uric acid of both populations of N exttzosus (Fig lc) increased during laboratory storage, wxth population 1 showing a dechne in uric acid up to day 10 Regression analys~s showed that uric acid m population 1 (after day 10) increased at a rate of 0 56 pg/termlte per day (correlation coefficient 0 85), whereas that in population 2 increased at a rate of 1 18#g uric acid/termite per day (correlation coefficient 0 99) The effect of laboratory storage on the total mtrogen (Fig 3b) and uric acid (Fig 3c) of C lacteus was slmdar to that of N extt~osus With population 2 there was a rapid increase after day 14, a finding not observed m population 1 Since these termites &ed soon after day 28, whereas those of population 1 were still ahve after 56 days, the increase m uric acid concentration up to day 28 may have been lethal or

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Fig 2 Rate of decrease of acetylene reduction ot ,~ e~mo~us (a) and ~/ ~al~ert (b) (0) Population l (m) population 2 N exmosus was collected from two different nests on April 4 1984 N ~all~er~ was collected from the same nest on March 5 1984 (population 1) and March 12 1984 (population 2) The relationship between acetylene reduction and t~me of storage was e~aluated b) linear regression analysis and plotted b) the method of least squares The correlation coefficient for all populations was 0 99

been an Indicator of some rapid derangement of mtrogen mltabollsm, particularly since dlnltrogen fixation was zero (Fig 3a) Regression analysis of the data showed that the decline in total nitrogen was 049/~g N/termite per day (correlation coefficient 0 77) for population 1 and 0 29 # g N/termite per da~ (correlation coefficient 0 75) for population 2, with uric acid in population 1 increasing at a rate of 0 34/~g/termlte per day (correlation coefficient 0 85) and in population 2 at a rate of 1 63/~g/term~te per day (correlation coefficient 0 94) The changes in mtrogen metabohsm observed for the three species of termites, particularly the rapid fall in dimtrogen fixation raised the question of whether the parameters measured in this study (dlnltrogen fixation, total nitrogen and uric acid) remained constant in field populations The two Nasutttermes species were examined because they were located close enough to the laboratory for measurements to be made within 2 hr of collection One nest of each species was sampled over a period of 6 weeks for N e,ctttosus and 8 weeks for N walkerl The results of the analyses are shown in Table 1 and demonstrate that dlmtrogen fixation, total nitrogen and uric acid content of field population of both species remain within fairly close hmlts Dinltrogen fixation in N walkert was about four times greater than that In N e~lttosus when calculated on a per termite basis When the activity was calculated on a fresh or dry weight bas~s the value was about double that for N extuo~u~ Total nitrogen in N ~alkert (about 1 4°0 of

flesh weight or 6 7". of dD weight) was similar to that of N exttlosu~ (about 1 6",, of fresh weight or 7 4°° of dry weight) When calculated on a per termite basis the total nitrogen of ~ IialkerI was about double that of N e x m o s u s In N ~alkerl uric acid constituted about 1 3°0 of the fresh weight (about 6°° of the dry weight) with the amount of the total nitrogen present as uric acid being about 31°o The uric acid content of N e',tttosus was about 0 6°0 of the fresh weight (about 2 9°0 of the dry weight) and accounted for about 13°0 of the total nitrogen (calculations were based on the mean values in Table 1) An increase in the uric acid content of N walkert occurred during laboratory storage (Fig 4a) with rates of increase of 7 5 and 9 3 pg uric acid/termite per day This is equivalent to an increase on a fresh weight basis of about 2 6"../month for populations 1 and 2 respectively During the period of the experiments the total nitrogen was 123-135/lg N/termite (data not shown) Xanthine dehydrogenase (Fig 4b) was undetectable on the day of collection and for 2 days of captivity After this period the enzyme increased in activity reaching a maximum at about day 8 The enzyme was present in the bodies of the termite, but not the guts DIsCU%~ION

This study clearly demonstrates that the lower termite C la~teus and the higher termites ?v e \ ttto~uv and N ~alkert. rapldl? lose their dlnltrogen

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Fng 3 Effect o f l a b o r a t o r y storage on acetylene r e d u c t i o n (a), total m t r o g e n (b) a n d u n c acid (c) o f C ( 0 ) P o p u l a t i o n 1 (collected A p r i l 26, 1983), (Hi) p o p u l a u o n 2 (collected A u g u s t 8, 1983) P o p u l a t i o n s were collected from dtfferent nests

lacteus

Table 1 Acetylene reduction, total mtrogen and unc acid of freshly-collected termites* Termtte species

Test parameter

N exlllo$lIs

N walkerl

(n = 7)1"

(n = 6):~

0 33 + 0 08 78 + 19§ 351 + 86~

1 29 _+0 08 152 _+9 4¶ 714 + 44¶

69 _+4 164+10 738+45

121 + 10 142+1 2 667+56

27 ± 3 64+07 28 8 ± 3 2

113 + 15 13 3 + 1 8 59 9 + 8 4

Acetylene reductton

nmol C2Hdtermlte per hr nmol C2Hdg fresh wt per hr nmol C2H4/g dry wt per hr Total mtrogen tzg/termxte mg/g fresh wt mg/g dry wt Uric acid pg/termlte mg/g fresh wt mg/g dry wt

*Values are means + SEM for termRes tested 2 hr after collection tOne nest was sampled seven ttmes between August 1 and September 14, 1983 :[:One nest was sampled snx tames between January 12 and March 12, 1984 §Average fresh weight of N exmosu* was 4 2 rag/termite vath a ratto of fresh wenght to dry weight of 4 5 ¶Average fresh wenght of N walken was 8 5 rag/termite voth a ratao of fresh weight to dry weight of 4 7

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Fig 4 Effect of laborato D storage on uric acid (a) and xanthlne deh~drogenase (b) ol 'v )~all~erl ( 0 ) Population 1 (collected February 13 1984) (ll) population 2 (collected Februa D 27 1984) Both populations were collected from the same nest as that used for the data shown in Fig 2 The relationship between uric acid and time of storage as evaluated by linear regression analysis and plotted b) the method of least squares The correlation coefficient for population 1 was 0 97 and for population 2 was 0 99

fixing ability when they are b r o u g h t into the laboratory a n d thus they mimic the b e h a v ] o u r of N ~orntget a n d R p e r a t a m a t u s reported by Prestwlch et al (1980) Prestwlch a n d Bentley (1981) found that the rate of d m l t r o g e n fixation of whole nests of N cotntget was ten times greater t h a n for termites removed from their nests a n d tested m vials (Prestw]ch et a l , 1980) a n d they attributed this higher activity to a lack o f physical & s t u r b a n c e of the nest O u r results also indicate that removal of termites from their nest, r a t h e r t h a n bringing them into the laboratory, is the reason for the loss of & n i t r o g e n fixation T h u s termites which were removed from a nest of N . a l l s e r t a n d left m a c o n t a i n e r at the nest site for 2 4 h r before testing h a d lost dm]trogen fixation to the same extent as termites kept in the l a b o r a t o r y for 2 4 h r It is not obvious why the physical disruption of the nest should have such d r a m a t i c effects on d m l t r o g e n fixation An ~mportant finding of this study, a n d reported for the first time to our knowledge, is t h a t dlnltrogen fixation in field p o p u l a t i o n s of the two nasute species was r e m a r k a b l y c o n s t a n t over 6-7 weeks and thus these termites do not a p p e a r to lose dlnltrogen fixing ablhty as do termites b r o u g h t into the l a b o r a t o r y N e~tttosus a n d one p o p u l a t i o n o f C l a ( t e u s partially recovered thexr ability to fix d l m t r o g e n d u r i n g l a b o r a t o r y storage whereas. N walkert failed to recover activity d u r i n g the 16 days o f storage m the l a b o r a t o r y Prestwlch et al (1980) observed t h a t N

cornlger a n d R

p e r a r a m a t u s did n o t recover their dlnltrogen fixing ability during 5 days in the laboratory A similar decline in dmztrogen fixation occurred in R [taz lpes with the rate falling to a b o u t 25"° ot the original value over 9 m o n t h s (Potrlkus and Breznak 1980a) U n f o r t u n a t e l y ~t is not k n o w n whether there was a precipitate fall In d l m t r o g e n fixation m this species over the hrst 24 48 hr The results of this study now provide an explan a t i o n for the report by Nazarczuk et al (1981) t h a t keeping C la~teus u n d e r a r g o n oxygen instead of & n i t r o g e n / o x y g e n had no effect on its nitrogen metabolism The ability of these termites to fix dmJtrogen was probably lost or serlousl) reduced prior to the start of the experiment and thus a lack ol d m l t r o g e n in their e n v i r o n m e n t ~ a s of little consequence The rates o f dinltrogen fixation s h o w n b~ freshlycollected C la~teus and ~r exltlosus were higher t h a n the values quoted for these species by French et al (1976) The values for C lacteu~ reported herein are 10 16 times greater a n d for ~ exltlosus are two-fold greater The values reported by F r e n c h et al (1976) are similar to those we o b t a i n e d for termites kept in the l a b o r a t o r y for more than 24 hr A n unexplained finding by French et al (1976) was the absence ol d m l t r o g e n fixation m N e~ltto7us when fed on wood but not when the termite was fed on filter p a p e r N extttosu~ used in our experiments was fed o n wood Dlnltrogen fixation in ~v extttosu~ was a b o u t dou-

509

Nitrogen metabohsm m termites ble t h a t in N ~orntger (Prestwlch et a l , 1980), but o f the same order as t h a t m Nasutttermes spp found in B r a z l h a n forests (Sylvester-Bradley et a l , 1978), whereas the activity In N walkerl was constderabley higher, being a b o u t four times greater t h a n in N corntger a n d a b o u t double t h a t of the most active Brazdlan speoes W h e n the rate of d l m t r o g e n fixation in two p o p u l a t i o n s of C lacteus (Fig 2a) was calculated a s / l g N fixed/g fresh weight per day, assuming that nltrogenase has a three-fold greater affinity for ethylene t h a n for dlnltrogen ( H a r d y et a l , 1968), the values of 38 a n d 56 were o b t a i n e d These values are considerably higher t h a n the values given by m o s t lower termites (see list m Breznak, 1982) Storage of N e~tttosus a n d C lacteus in the l a b o r a t o r y resulted in the termites losing total nitrogen while at the same time increasing thexr uric acid c o n t e n t This contrasts with R f i a t lpes in which there was an increase in b o t h total nitrogen a n d uric acid when the termites were kept in the laboratory (Potrlkus and Breznak, 1980a) The uric acid c o n t e n t of N ~alkert appeared to be high being double that of N e-~mosu~ and o f workers of three other species of termites kept in captivity for one m o n t h (Potrlkus a n d Breznak, 1980a) In N walkerl the uric acid content constituted 31°o of the total nitrogen of the termite, again higher t h a n t h a t of N e,clttOSUS and the worker caste of other termites (Potrlkus a n d Breznak, 1980a) The increase in uric acid in N walkerl over a comparatively short t~me in the l a b o r a t o r y (17 days), during which the total mtrogen remained essentially constant, indicates t h a t its synthesis was at the expense o f some other m t r o g e n o u s c o m p o u n d s , a conclusion first reached by Potrlkus and Breznak (1980a) for R flavtpes One would assume that the a c c u m u l a t i o n of uric acid in termites shown in this a n d other stu&es ( P o t n k u s a n d Breznak, 1980a, Nazarczuk et a l , 1981) would be due to a n intake o f nitrogen m excess of the needs of the termite, as it is in Pertplaneta amerz~ana ( M u l h n s a n d C o c h r a n , 1975a) However, in c o n t r a s t to the cockroach ( M u l h n s a n d C o c h r a n , 1975b), there appears to be no m o b l h s a t l o n o f uric acad as such since termites do n o t a p p e a r to possess urlcase a n d do not excrete uric acid (Potrlkus a n d Breznak, 1980a) Potrlkus a n d Breznak (1981) as being present m termites, increases in actlwty after the termite loses the ability to fix dlnltrogen This, coupled with the finding t h a t xanthlne dehydrogenase Is present only in the body tissue and not the gut of N walkert, suggests that some derepresslon of xanthine dehydrogenase occurs as a consequence o f the loss of some m e t a b o h t e subsequent to the loss of & n i t r o g e n fixation This hypothesis is currently bemg investigated Acl~nowledgement~--We thank Ms Ludmda Tlttel for excellent technical assistance Th~s work was supported, m part, by a grant from the Austrahan Grants Scheme

REFERENCES

Benemann J R (1973) Nitrogen fixation in termites Scwnce 181, 164-165 Breznak J A (1982) Intestinal mlcroblota of termites and other xylophagous insects A Rev Mwrobtol 36, 323-343 Breznak J A , Brdl W J , Mertms J W and Coppel H C (1973) Nitrogen metabohsm m termites Nature, Lond 244, 577-580 French J R J , Turner G L and Bradbury J F (1976) Nitrogen fixation by bacteria from the hlndgut of termites J gen Mwroblol 95, 202-206 Gornall A G , Bardawdl C H and David M M (1949) Determination of serum proteins by means of the bluret reaction J blol Chem 177, 751-766 Hardy R W F , Holsten R D , Jackson E K and Burns R C (1968) The acetylene-ethylene assay for N 2 fixation laboratory and field evaluation Pl Phvstol 43, 1185-1207 Kaplan A (1965) Urea nitrogen and urinary ammoma In Standard Methods of Chmcal Chemtstrv (Edited by Meltes S ), Vol 5 pp 245-256 Academic Press, New York Mulhns D E and Cochran D G (1975a) Nitrogen metabohsm in the American cockroach--I An examination of positive nitrogen balance w~th respect to uric acid stores Comp Blochem Phkstol 50A, 489-500 Mulhns D E and Cochrane D G (1975b) Nitrogen metabohsm m the American cockroach--II An exammatlon of negative nitrogen balance w~th respect to moblhslng of uric acid stores Comp Bzochem Phvslol 50A, 501-510 Nazarczuk R A , O'Bnen R W and Slaytor M (1981) Alteration of the gut mlcroblota and its effect on mtrogen metabohsm m termites Insect Btochem 11, 267-275 Parzen S D and Box A S (1964) PurlficaUon of xanthme dehydrogenase from Drosophda melanogaster Btochtm btoph~s Acta 92, 465-471 Pomkus C J and Breznak J A (1980a) Uric acid in wood-eating termites Insect Btochem 10, 19-27 Potnkus C J and Breznak J A (1980b) Uric aoddegrading bacteria m guts of termites [Rettcuhtermes flavtpes (Kollar)] Appl Environ Mtcrobtol 40, 117-124 Potrlkus C J and Breznak J A (1980c) Anaerobic degradation of uric acid by gut bacterm of termites Appl Environ Mtcrobzol 40, 125-132 Potnkus C J and Breznak J A (1981) Gut bacteria recycle uric acid mtrogen to termites A strategy for nutnent conservation Proc natn 4cad Sct U S A 78, 4601-4605 Prestwlch G D and Bentley B L (1981) Nitrogen fixation by mtact colomes of the termite Nasutttermes corntger Oecologla 49, 249-251 Prestwlch G D , Bentley B L and Carpenter E J (1980) Nitrogen sources for neotroplcal nasute termites Fixation and selective foraging Oecologla 46, 397-401 Rohrmann G F and Rossman A Y (1980) Nutnent strategies of Macroterme~ ukuzn (Isoptera Termltldae) Pedobtologta 20, 61-73 Schaefer D A and Whltford W G (1979) Nitrogen fixation In desert termites Bull ecol Soc Am 60, 128 Sylvester-Bradley R , Bandelra A G and Ollvelra L A de (1978) Flxaqao de nltrog6mo (redugao de acetlleno) em cupms (Insecta Isoptera) da Amaz6ma Central Acta amazon 8, 621-627