Comp. Biochem. Physiol. Vol. 78B, No. 3, pp. 737-739, 1984 0305-0491/84 $3.00 + 0.00 Printed in Great Britain © 1984 Pergamon Press Ltd

MAJOR VOLATILE METABOLITES PRODUCED BY TWO ISOLATES OF APHELENCHUS A VENAE U N D E R AEROBIC

A N D ANAEROBIC CONDITIONS

A. H W. MENDIS* and A. A. F. EVANS Imperial College of Science and Technology, Silwood Park, Ashurst Lodge, Ascot, Berkshire SL5 7 D E

U.K.

(Received 18 November 1983)

A b s t r a e t - - l . Greater quantities of glycerol were detected in aerobic incubates ofA. avenae isolate M than in isolate F. No ethanol was detected in aerobic incubates of either isolate.

2. In anaerobic incubates both isolates produced ethanol. 3. 10-20 times more glycerol was detected in anaerobic incubates as opposed to aerobic incubates of

both isolates M and F. 4. Isolate M produced more glycerol (ca. ten-fold) than isolate F under identical conditions. 5. The possible interplay between the A. avenae ct-glycerophosphate-oxidase complex and the glycolytic

sequence in glycerol production, and the survival value of the latter to this soil dwelling nematode is discussed.

INTRODUCTION

The numbers and amounts of metabolic end products produced by an organism are indicative of its peculiar physiology and frequently reflect the biochemical pathways which may be involved.

Caenorhabditis briggsae released a large variety of x4C-labelled products (especially amino acids) follow- ing incubation with various labelled substrates (Roth- stein, 1963). It was later shown that large amounts of 14C-glucose, trehalose and glycerol were produced from '4C-acetate by C. briggsae (Rothstein, 1969). Of these, glycerol was the major product if C. briggsae was incubated with 2 '4C-acetate in "whole (axenic) medium", whereas incubation in water produced little glycerol, glucose and trehalose being the major products (Rothstein, 1969). Turbatrix aceti and Panagrellus redivivus behaved similarly when incu- bated in water, but in "Whole medium" other neutral products, presumably sugars, were produced in addi- tion to glycerol" (Rothstein, 1969).

Incubation solutions of a mixed population of Aphelenchus avenae (Californian isolate) were anal- ysed by gas-liquid chromatography for organic acids, alcohols, glycols and ketones (Cooper and Van Gundy, 1970) and under anaerobic conditions, incu- bates contained ethanol which increased to an equi- librium concentration at 40hr and thereafter de- creased. No acetic acid or acetaldehyde was detected under these conditions. In the same study Cae- norhabditis sp. produced acetaldehyde during the first 12-18hr of anaerobiosis which declined to non- detectable levels after 30 hr. After this only ethanol was detected, reaching an equilibrium concentration at 34hr. An unidentified 4-C alcohol which ac- counted for 5-10% of catabolized glycogen was also found.

Investigating the metabolic changes during the induction of and recovery from anhydrobiosis in A. avenae, Madin et al. (1978) reported that trehalose

*Present Address: The Wellcome Research Laboratories, Langley Court, Beckenham, Kent BR3 3BS, UK.

and glycerol increased dramatically during desic- cation. The changes on rehydration were essentially the reverse of those during dehydration. They con- cluded that A. avenae synthesizes glycerol at the expense of lipid, probably via the glyoxylate path- way. Although this pathway is well documented in micro-organisms it has not been extensively studied in eukaryotic animals most of which do not seem to possess the appropriate enzymes. However, a number of nematodes have been shown to possess the key enzymes and Madin et al. (1978) cited evidence that the glyoxylate pathway is operative in A. avenae.

Another pathway common in certain parasitic protozoa, the ~-glycerophosphate-oxidase complex of certain Trypanosoma sp., produces glycerol in association with the glycolytic pathway. On the evi- dence of the sensitivity of oxygen consumption to the inhibitor salicyl hydroxamic acid (SHAM), Mendis and Evans (in press) suggested the existence of an ~-glycerophosphate-oxidase complex (Hill, 1976) in some isolates of Aphelenchus avenae. This paper reports on volatile end products secreted into the aqueous incubation media by 2 isolates of A. avenae.

MATERIALS AND METHODS

Mass cultures of A. avenae isolates M & F (culture temperature 25°C) were harvested at 35 days as described previously (Mendis and Evans, in press, and 1984). To favour detection of metabolites, approximately 3 g wet wt of nematodes were suspended in 20 ml of sterile distilled water. Two such groups were prepared for each treatment.

To one group 100 units penicillin and 100/~g strep- tomycin sulphate per ml were added to the incubation medium. The suspensions were bubbled with sterile O2-free N 2 for 1 hr in 150 ml flat-bottomed flasks, which were then sealed and incubated in a sealed desiccator flushed with O2-free N 2. The desiccator was incubated at 27°C in a controlled temperature cabinet for 36 hr.

Similar suspensions from both isolates were bubbled with humidified sterile air for 1 hr and incubated under aeration (10 ml/min) at 27°C for 30 hr.

Following incubation, the bathing solutions (incubates) were carefully removed and rapidly introduced into chilled

737

738 A . H . W . MENDIS and A. A. F. EVANS

Table l. Volatile organic metabolism in whole worm incubates of A. avenae isolates M and F following 30 hr aerobic or anaerobic incubation at 27 C

Detected Retention compound time (secs)

Solvent (water) 9.6 Ethanol 19.2 (ppm) Unidentified compounds P 33.6 Q 81.6 R 158.4 S 168.0 T 187.2 X 48.0 Y 100.8 Glycerol

(ppm) 624.0

Aerobic incubation Anaerobic incubation F M F M

3350 6700 4000 8000

+ + + +

+ + +

+ + +

I1 15 60 75 109 114 136(~139

+, compound detected: compound not detected.

( - 20°C) sterile quick-fit tubes and stored at 70C until analysis.

Aliquots of the frozen samples were analysed directly from the aqueous phase using gas-liquid chromatography on a 200 cm x 3.2 mm Porapak-P :~ (80-1000 mesh) column. The flame ionization detector zone temperature was 240C; the column oven temperature was 190°C. Carrier gas was N2 (30/min). Direct analysis of the aqueous incubates was possible using Porapak-P ~ because water elutes more rapidly than the compounds of interest (i.e. ethanol, acetaldehyde and glycerol (Cooper and Van Gundy, 1970).

Qualitative and quantitative measurements were based on retention times and areas under the curves using authentic standards.

RESULTS

During aerobic incubation fewer compounds were accumulated than under anaerobiosis (Fig. 1). In addition to small amounts of unknown compounds X

._m

a_

IsoLate M

SoLvent water

E thano l

S

0 I0 20

and Y, isolate F produced small but measurable amounts of glycerol. More glycerol (approx. 5 x ) was produced by isolate M but only compound P was found in addition.

In anaerobic incubates both isolates had produced ethanol, and from 10-20 times more glycerol than under aerobic conditions together with a further 4 unidentified compounds (Q, R, S and T) (Fig. 1). Again isolate M produced more glycerol (approx. 10 x ) than isolate F.

No ethanol was detected in the aerobic incubates of either isolate, but unidentified compound P (retention 33.6secs.) detected in anaerobic incu- bates of isolate F, was also observed in aerobic incubates of isolate M but not in aerobic incubates of isolate F. Compounds Q, R, S and T did not occur in aerobic incubates. Even under aerobic conditions isolate M produced 6-7 times more glycerol than isolate F.

Water

I so la te E

E thano l

~ eroL P

0 S

1 1 I 30 40 0 I0

Retention t ime (rain)

G l y c e r o L

20 30 40

Fig. 1. GLC analysis of volatile metabolites of two isolates of A . a v e n a e following anaerobic incubation at 25"C. Perkin-Elmer programmable GLC; attenuator setting 8; Column*: 200cm x 3.3mm i.d.: stationary phase*: Porapak-P a' (80-100 mesh); injector zone temperature: 240"C; oven temperature 190C;

detector: flame ionization; carrier gas: N_,, 30 ml/min; solvent: water.

Nematode produces glycerol and ethanol 739

Incubates of nematodes containing penicillin and streptomycin + nystatin (an antifungal) showed no detectable differences in the observations.

DISCUSSION

The production of fewer compounds under anaer- obic conditions suggests that in both isolates most end products were metabolized when oxygen was available. Under anaerobiosis ethanol and glycerol were produced in large amounts. Production of the unidentified compounds also varied with the condi- tions. Ethanol production by both isolates under anaerobic conditions only agrees with Cooper and Van Gundy (1970) but the fact that isolate M pro- duced about twice the ethanol of isolate F suggests different glycolytic or fermentative capacities between the isolates.

However, the major difference was the tenfold greater production of glycerol by isolate M compared with isolate F.

The synthetic pathways of glycerol postulated by Rothstein (1969) for T. aceti, C. briggsae and P. redivivus, envisaged the switching off and on of glycerol synthesis in response to glucose in the exter- nal medium whereas Madin et al. (1978) suggested dehydration as the stimulus for glycerol and trehalose synthesis in A. avenae.

Present observations suggest that the trace amounts of glycerol synthesized by both isolates of .4. avenae under normal environmental conditions may be of significant survival-value for such a soil- dwelling organism, giving some protection against the loss of bound water during desiccation. Madin et al. (1978) envisaged that glycerol synthesis in desic- cating A. avenae from California was favoured by a change in the activity of isocitrate lyase and malate synthase because activity only increased when pellets of nematodes were dried slowly at 97~ r.h. Rothstein (1969) had linked glycerol biosynthesis in C. briggsae with gluconeogenesis because he recognised that syn- thesis from acetate was unfavourable energetically.

Earlier studies (Mendis and Evans, 1980; 1984) have shown the presence of SHAM and rotenone sensitive components of ct-GP-oxidation (21 and 38~o mean sensitivity respectively) in A. avenae mito- chondrial fractions. In addition SHAM and rotenone refractory ct-GP-oxidase activity (accounting for 79 and 62~o of ct-GP induced mitochondrial state-3 oxygen uptake) was also demonstrated suggesting that a non-classical terminal oxidase capable of ~t-GP-oxidation (i.e. an ct-GP-oxidase complex or ~t-GPO) was present in two A. avenae isolates. A similar GPO-complex has been demonstrated in cer- tain trypanosome species (Opperdoes and Borst, 1976).

Although the specificity of SHAM inhibition ol this ~-glycerophosphate-oxidase-complex (in try-

panosomes) claimed by Opperdoes and Borst (1976) has been questioned (see Palmer, 1981). The activity of such a complex in conjunction with the glycolytic pathway via the NADH linked :t-glycerophosphate dehydrogenase would provide glycerol from DHAP (via a phosphatase) as a metabolic end product of A. avenae respiration under aerobic conditions and lead to greater glycerol production under anaerobic condi- tions, as was found.

It is perhaps significant that the Malawi isolate (M) released more glycerol than isolate F, the former having possibly adapted to resist desiccation. It is interesting to note that a similar pathway appears to act in the halophilic alga Dunalliela sp. (Ben-Amotz and Avron, 1981) in response to increasing salt concentrations.

Acknowledgements--This work formed part of the thesis submitted by A. H. W. Mendis for the degree of Doctor of Philosophy, University of London, Imperial College and was generously supported by financial assistance from his family.

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