Reversat, Georges

Horizon / Pleins textes

Effects of ageing and starvation on respiration 
and food reserve content in adult 
Hirschmamiella spinicaudata 
Georges REVERSAT 
Laboratoire de N&matologie, ORSXOM, B.P. 1386, Dakar, Sénttgal. 
SUMhfARY 
Females and males of Hirschmanniella spinicaudata, freshly extracted from infested rice roots, were kept in 
vitro at 280 in the dark. Just after extraction and thon every three weeks, batches of animals of the same sex were 
submitted to physiological measurements: survival, respiration and body content of lipids and carbohydrates. Num- 
bers of survivors decreased wit.h time to zero at 33 weeks for females and 36 weeks for males. Initial food reserves were 
1.5 pg of lipids and 0.7 pg of carbohydrates per fcmale and 0.9 pg of lipids and 0.5 pg of carbohydrates per male. 
After nine weeks of storage 86% of lipids and 65% of carbohydrates were consumed in females and 88% of lipids 
and 7170 of earbohydrates in males. Initial hourly rates of 0, uptake were 5.4 nl pcr female and 2.1 nl per male. 
Aftar twelve weeks of storage these rates dropped to 0.7 nl per female and 0.5 nl par male. 
RÉSUMÉ 
Eflets du vieillissement et du jeûne sur la respiration et la teneur en réserves nutrifives 
chez les adultes d’Hirschmanniella spinicaudata I 
Des femelles et des mâles d’HirschmannielZa spinicaudafa, fraichement extraits de racines infestées de riz, sont 
gard& in vitro à 280 à 1’obscurit.é. Juste aprè.s l’extraction, puis à des intervalles de temps de trois semaines, des 
lots d’animaux de chaque sexe sont testés pour plusieurs paramètres physiologiques : survie, respiration et teneurs 
en lipides et glucides. Le nombre des animaux survivants décroit avec. le temps et devient nul à 33 semaines pour 
les femelles et 36 semaines pour les mâIes. Les teneurs initiales de réserves nut,ritives sont de 1,û pg de lipides 
et 0,7 kg de glucides par femelle et. de Oj9 pg de lipides et 0,s yg de glucides par mile. Aprés neuf semailles de 
stockage, 86gh des lipides et 65 y(, des glucides sont consommés c.hez les femelles et 88% des lipides et 71 0/0 des 
glubides chez les m&les. La consommation horaire initiale d’oxyg&ne est de 5.4 nl par femelle et de 2,l nl par mâle. 
Apres douze semaines de st,ockagt?, ces consommations chutent à 0,7 nl par femelle et 0,5 111 par mâle. 
Unt.il the present time, quantitative stuciies 
concerning the effects of starvation on the 
c.hemic.al composition of infec.tive stages in plant 
parasitic nematodes have been confined to 
second stage juveniles of species fitted with egg- 
masses (Van Gundy, Bird & Wallace, 1967 ; 
Ogunfowora, 1978 ; Reversat, 1980, 1951). The 
Iack of information about other infec,t.ive stages 
is probably due to difflculties in obtaining 
sufEcient amounts of biological material needed 
for chemical determinations. Adults of the nema- 
tode Hirschmanniella spinicaudata are infective 
to rice roots (Merny, 1972) and characterized 
by their rather large size (Luc & Fortuner, 
1978). A female of H. spinicaudata is equivalent 
to 100 and a male to 60 juveniles of Meloidogyne 
javanica, according to the calculation of the wet 
weight by Andrassy (1956). Thus sampling of 
these animals for chemical determination is 
possible by individual handling. 
The present study was initiated to determine 
some ef’fects of ageing and starvat.ion on females 
Revrre Némaiol. 4 (1) : 125-130 (1981) 125 
G. Reversat 
and males of H. spinicazrdata. Survival, respir- 
ation and food reserves content were measured 
after different times of st.orage in vitro. 
Materials and methods 
NEMATODEPRODUCTION 
Populations of H. spinicaztdata were inc.reased 
in the greenhouse on rice (Oryza satina L.) C.V. 
MorobSkan. Pots containing ‘2.5 liters of 
steam sterilized sandy soi1 were sowed with 
six seeds and watered daily until the appearence 
of the fourth leaf (three weeks). At this date 
pots were placed in containers with sealed 
bottom, flooded with water and then mass 
inoculated with 2 000 individuals of mixed 
stages. Every two weeks from sowing t.o the 
yield, each pot was fertilized with 215 mg of 
urea and 175 mg of K H, PO,. 0nc.e a week 
pots were taken out of the containers, clrained 
for 48 hours and then replaced and flooded in 
the containers. These conditions of alternative 
flooding and drainage prevented the formation 
of toxic soluble sulphurs and favoured t,he 
development of H. spinicaudata populations 
(Fort,uner, 1976). Infested roots were harvested 
eight to twelve weeks after inoculat,ion, carefully 
cleaned and then macerat.ed in an aqueous 
solution. At the beginning of this work animals 
stored in water were attacked by an unident- 
ifieded fungus, which killed them a11 in a few 
weeks. Agallol )->, whic,h is a fungicide prevented 
this infection. Thus maceration of the roots and 
further storage of animals were carried out in a. 
solution of 125 ppm of Agallol SI in 4 mM sodium 
phosphate buffer, pH 7 (Gomori, 1955), called 
AP solution. Roots in AP solution were aerated 
by diffusion in a shallow t.ray or by bubbling in 
a jar, and kept at 280 in the dark. Every three 
days, roots were placed in fresh AP solution and 
nemat,odes leaving t.he roots were recovered in 
t.he solution. Nematodes were allowed t,o pass 
through two layers of kleenex @ paper mounted 
on a coarse plastic, sieve (aperture 1 mm) and 
clean nematodes were collected 24 hours later. 
At t‘his date, nematodes were considered at the 
zero time of the experiment whereas their 
trophic contact wit.h the plant had been inter- 
rupted since 2.5 & 1.5 days. Nematodes were 
126 
then stored in AP solution in 1 liter Roux 
bottles wit.h the deepness of the medium not 
exc.eeding 4 mm at 280 in the dark. 
SURVIVAL 
At the zero t.ime, 300 females and 300 males 
were divided in lots of 25 individuals of the same 
sex. Eac.h lot was placed in a perspex round box 
with a diameter of 3.5 cm wit,h 2.5 ml of AP 
solution. Boxes with a device preventing evap- 
oration were stored under the preceding c.on- 
ditions. Every three weeks each lot was tran- 
fered in fresh AP solution to a new box, by hand 
pic.king each individual. When handling, indi- 
viduals were tested for survival by checking 
activity, either spontaneous or when stimulat,ed 
with a probe. Inactive animals were isolated and 
examined for the possible alteration of the body 
content. due to death. Dead individuals were 
discarded and survivors were counted. 
CHEMI~A~~DETERMINATION~ * 
The day before the times 0, three, six and 
nine weeks the st.ored nematodes were placed 
on kleenex @ paper in AP solution as before for 
select.ion of the motile individuals. After one day 
the suspension was distributed in perçpex count- 
ing slides (Merny & Luc, 1969) and females ancl 
males were individually hand picked under the 
dissecting microscope and deposited in special 
cent,rifuge tubes (Reversat, 1976) filled with 
de-ionized water. For each of the storaçe times 
six t,ubes with 75 females and six tubes with 
100 males were prepared. In order to achieve a 
random sample each adult was picked following 
t,he rows of the slicle and deposited in a different 
tube unt.il a11 the tubes received the required 
number of individuals. Nematodes were settled 
by c.entrifugation and t,he excess water was 
clisc.arded. Tubes with nematodes in about 20 ~1 
of water were st.ored at -200 until use. 
For each of the carbohydrates and lipids 
determinations, three tubes were used. General 
procedures for chemical determinations (Rever- 
sat., 1976) were modified as follows. For c.arbo- 
hydrates, 0.25 ml of KOH 30 yo was added to 
each tube and then tubes were kept in a boiling 
Rezw.? NémaIol. 4 (1) : 125-130 (1981) 
Ageing, starvation and respiration of Hirschmanniella spinicaudat.a 
water bath for 1/2 hour during which nematodes 
were completely digested. After c.ooling, t.he 
content of tubes was macle up to 1 ml with 
de-ionized water. Carbohydrat,e content of t.he 
potassic solution was spec.trophotometrically 
determined with the anthron reagent against a 
standard of gluc.ose (Seifter cl al., 1950). For 
lipids, 2.5 ml of c.onc.entrated H$O, was added 
to eac.h tube and then tubes were kept* in a 
boiling water bath for ten minutes during which 
animals were c.ompletely digested. After c,ooling, 
lipid content of the sulfuric solution was spectro- 
photometrically determined with the sulpho- 
phospho-vanillic reagent, (Drevon S: Schmit, 
1964) against a standard of olive oil dissolved 
in concentrated H,SO,. Results are expressed 
in microgram per individual (&ind.). 
RESPIRATION 
Respiration rate was determined using glass 
stoppered Cart.esian divers (Reversat., 1975). 
Animals in the divers were kept in a solution 
cont.aining 7.5 ppm of 2-met.hoxyethyl mercury 
chloride (active ingredient of Agallol) in 20 mM 
sodium phosphate buffer, pH 7 (Gomori, 1955). 
Measurements commenced two hours after t,he 
introduction of animals into the divers and were 
made at 280 during six hours. From four to ten 
individuals were int,roduced in tbe 10 ~1 divers 
ac.cording to previous experiments. Results are 
espressed in nanoliter of oxygen per individual 
per heur (nl/ind./hr.). Respiration was measured 
on eight replications for each sex after 0, t.hree, 
six, nine and twelve weeks of storage. 
Results 
SURVIVAL 
Stored animals exhibited four survival stat,es : 
a) active, b) inactive with a twist.ed habitus, 
reacting well to a light touch, c) inact.ive with a 
straight habit.us, interna1 structure unchanged, 
reac.ting poorly ev-en to intense touch, generally, 
by bending at the nec.k level, d) inactive with a 
straight habitus, interna1 structures altered, 
never reacting t.o touch. When animals were 
stored individually, stages 0 and c were revers- 
Rwue Némafol. 4 (1) : 125-130 (1981) 
ible to stage a but stage d was not reversible. 
Thus stages n, b and c were counted as survivors 
throughout the experiment. The percentage of 
survivors decreased steadily in both sexes and 
became ni1 at the end of 33 weeks in females and 
36 weeks in males (Fig. 1). 
100 
FR 
Urvival 
Weeks of storage 
Fig. 1 : Survival çurves of adult. Hirschrnanniella 
spinicauduta (Each curve was determined on 300 
individuals, stored at 280 and examined individually 
every three weeks). 
CHEMICALCOMPOSITION 
Lipid content decreased rapidly in bot11 sexes 
during the first three weeks and then t.he rate 
of decrease slowed (Fig. 2.). At the end of nine 
weeks, consumption of lipids expressed as a 
percentage of initial content was 86 yo in females 
and 88% in males. In both sexes, carbohydrat,e 
content remained c.onstant during the first 
three weeks interval and then decreased (Fig. 3). 
At the end of nine weeks, consumption of 
carbohydrate was 65% in females and 71 Ojo in 
males. 
RESPIRATION 
In bath sexes, respiration decreased rapidly 
during the first three weeks and then remained 
almost constant (Fig. 4). 
127 
G. Reuersat 
iii 
8 
l- 
Lipids 
l.O- 
.5- 
0 
I I I 
0 3 6 9 
Weeks of storage 
Fig. 2 : Effect of storage time on t,he lipid content. of 
adult Hirschmanniella spinicaudata (For each storage 
time, three replicates were determined for each sex, 
females, open circles and malss, black circles. Straight. 
lines are joining mcane, mhich are not figured). 
0 3 6 9 
Weeks of storage 
Fig. 3 : Effect of storage time on the çarbohydrate 
content of adult. Hirschmanniella spinicaudata fspeci- 
fications as in Fig. 2). 
Respiration 
0 3 6 9 12 
Weeks of saorage 
Fig. 4 : Effect of storaga time on the respiration (rat.e 
of 0, consumpt.ion) of adult, Hirschmanniella spinicau- 
data (For each storage time, eight. replications were 
cletermined at 280 and the vertical line equals the 
c.onfidence interval at 95’$$). 
Discussion 
Animals for study were selected from a mass 
rearing and their actual age was heterogeneous 
when the experiment, start.ed. Synchronous 
rearing obtained by inowlating only second 
stage juveniles may provide a more precise 
approach. St.ages a, b, c. and d might be presu- 
mably t,he sequence of natural events leading 
animals to death. Assessment. of ac.tivity, either 
spontaneous or stimalated with a probe, has been 
usecl by some authors (Golden Lg, Shafer, 1960 ; 
Slack, Riggs & Hamblen, 1972) for survival 
determination. However, act.ivity, could be 
reversely interrupted by resting phases (Barrett, 
1969). The demonstration of degenerate body 
c.ont.ent as used by Davies and Fi&er (1976) and 
in tbe present study appears a more reliable test 
to azertain nematode death. 
Lipid c.ontent was greater than carbohydrate 
c.ontent in freshly estrac.ted nematodes and 
in t.he course of ageing, lipids were consumed 
before c.arbohydrates (Fig. 2 & 3). This has been 
observed in juveniles of Heierodera oryxae 
(Reversat, 1%X)), Meloidogyne jauanica (Rever- 
128 Reoue Némafol. 4 (1) : 125-130 (1981) 
dgeing, sfarvafion and respiration of Hirschmanniella spinicaudata 
sat,, 1981) and Sfrongyloides ratti (Barre& 
1969). Since the rat,e of lipid consumption was 
very high during the first three weeks (Fig. 2) it 
is suggested that feeding occured const.antly 
when animals were inside the roots. This could 
be correlated also with.the interruption of egg- 
laying in stored females. 
A decreasing rat.e of respiration with age bas 
been observed in several plant parasitic nema- 
todes (Rohde, 1960 ; Wallace & Greet, 1964 ; 
Bhatt & Rohde, 1970 ; Van Gundy, Bird & 
Wallace, 1967 ; Reversa& 1980). Atkinson (1976) 
suggested that in animais experienc.ing nor- 
mally prolonged absence of food, whiçh is true 
for H. spinicnudaiu, the observed decline of 
respiration rat.e may have an adaptat.ive signi- 
fic.ance. An answer to this question c.ould be 
obtained by refeeding animais after some weeks 
of starvation. 
Equivalence between results of respiromet.ry 
and results of chemical determinations may be 
tested by 1) integrating the curves of respiration 
rates (Fig. 4) for the period O-9 weeks 2) calc.u- 
lat.ing the required quantity of osygen for a 
complete oxidation of lipids (Fig. 2) and carbo- 
hydrates (Fig. 3) consumed during the period 
O-9 weeks. Suitable coefficients are 2 019 ml/g 
for lipids and 829 ml/g for carbohydrates 
(Polonowki et ~2.; 1966). There is a good c.orre- 
lat.ion for females : animais c.onsumed 2 760 nl 
of oxygen and the oxidation of reserves needed 
2 720 nl of oxygen. However males consumed 
less oxygen (1 250 nl) t.han required for oxidation 
of reserves (1 530 ni). This suggest,s that the 
met,abolism could be different in bot.h sexes : a 
c.omplete oxidation.of reserves in females and an 
incomplete oxidation of reserves probably 
balanced ‘by an excretion of organic substances 
in males. 
ACKNOWLEDGEMENTS 
The author thanks Bayer AG, Leverkusen (West 
Germany) for kindly supplying Agallol ‘B and 2 metho- 
xymet.hyl-mercury chloride and T. Maiga for assistance 
in t.he meking of illust.rations. 
REFERENCES 
ANDRASSY, 1. (1956). Die Rauminhalts- und Gewicht- 
bestimmung der Fadenwürmer (Nematoden). Act. 
ZOO~. hzzng., 2 : l-15. 
Revue Nfmafol. 4 (1): 125-130 (1981) 
ATKINSON, H.J. (1976). The respiratory physiology of 
nematodes. In : Croll, N.A. (Ed.) The organizafion 
of nemafodes, London, Acadrmiç Press : 243-272. 
BARRETT, J. (1969). The affect of ageing on the meta- 
bolism of the infec.tive larvae of Sfrongyloides raffi 
Sandground, 1925. Parasifology, 59 : 3-17. 
BHATT, B.D. Lf: ROH~E, R.A. (1970). The influence of 
environmental fac.tors on the respirat,ion of plant 
parasitic nematodes. J. Nemafol., 2 : 277-285. 
DAVIES, K.A. & FISHER, J.M. (1976). Duration of 
infect.ivity of second stage larvae of Heferodera 
avenae. Nemafoloyiea, 3v : 163-168. 
DREVON, B. & SCH~~IT, J.M. (1964). La r6action sul- 
fophosphovanillique dans l’ét.ude des lipides S&i- 
ques. Bull. Trau. Soc. Pharm. Lyon, 8 : 173-178. 
FORTUNER, R. (1976). Etude Ic~ologique des nématodes 
des rizières du Sé.négal. Cah. ORSTOM, SÉr. Biol., 
11 : 179-191. 
GOLDEN, A.M. CQ SHAFER, T. (1960). Survival of 
emerged larvae of the sugar beet nematode (Hete- 
rodera schachfii) in water and in poil. Nemafologica, 
5 : 32-36. 
GO~IORI, G. (1955). Preparat.ion of buffers for use in 
enzyme studies. In : Colowic.k, S.P. & Kaplan, N.O. 
(Eds.) Mefhods in enzymology, New-York, hcademic 
Prese. Vol. 1 : 138-146. 
Luc, M. & FORTUNER, R. (1975). Hirschmanniella 
spinicaudafa. CIH Descripfions of Plant Parasific 
Nemafodes, Set 5, No. 68, 3 p. 
MERNY, G. (1972). Les nématodes des rizihres inondées 
de Côte d’ivoire. III. Etude sur la, dynamique des 
populations de deux endoparasites H~rschmanniellu 
spinicaudafa et Heferoderu oryzae. Cah. ORSTOM, 
Sér. Biol., No. 16 : 31-87. 
MERNY, G. & Luc, M. (1969). Les techniques d’échan- 
tillonnage des peuplements de nématodes dans le 
sol. In : Lamotte, M. & Bourlibe F. (Eds) Problèmes 
d’écologie : l’échanfillonnage des peuplemenfs ani- 
maux des milieux terrestres. Paris, Masson, : %37- 
272. 
OGUNFOWORA, A.O. (1979j. Fact.ors affecting emer- 
gence, survival and infectivity of Meloidogyne naasi. 
Nemafologica, 24 : 72-80. 
POLONOWSKI, M., BOULANGER, P., MACHEBOEUF, M. 
& ROCHE, J. (1966). Biochimie mt!dicale, Fascicule II 
Enzymes ef métabolisme, Paris, Masson, 431 p. 
REVERSAT, G. (1975). IMéthodes pour la mesure de la 
consommation d’oxygène des nématodrs par la 
technique du ludion. Cah. ORSTOM, St?. Biol., 
10 : 169-157. 
REVER~AT, G. (1976). Etude de la composition bio- 
chimique globale des juvéniles des nt;matodes 
Meloidogyne javanica et, Heferodera oryrae. Cah. 
ORSTOM, Sér. Biol., 11 : 295-234. 
REVERSAT, G. (1980). Effect of in vit,ro storage t.ime 
on the physiology of second stage juveniles of 
Heferodera orytae. Revue Némafol., 3 : 233-241. 
REVERSAT, G. (1981). Consomption of food reserves 
by st,arved second-stage juveniles of AJeZoidogyne 
129 
G. Reversat 
javanica under condit.ions inducing osmobiosis. 
Nemafologica (in press). 
ROIIDE, R.A. (1960). The influence of carbon dioxide 
on respiration of certain plant. parasitic nematodes. 
Proc. helminfh. Soc. Cash., 17 : 160-164. 
SEIFTER, S., DANTON, S., NOVIC, R. &z MIINTWYLER, E. 
(1950). The estimation of glycogen with the anthrone 
reagent. ilrch. Biochem., 25 : 191-200. 
SLACH, D.A., RIGG~, R.D. & HA~~BLEN, M.L. (1972). 
The effect of temperature and moisture on the 
Accepté pour publication le 31 octobre 1980. 
130 
survival of Heferodera qlycines in the absence of a 
ho& J. Nemafol., 4 : C?k3-%ci. 
VAN GUNUY, S.D., BIRD, A.F. <It WALLACE, H.R. 
(1907). Ageing and st.arvat,ion in larvae of Meloido- 
dyne javanica and Tylenchulus semipenetrans. Phy- 
fopathology, 57 : 559-571. 
WALLACE, H.R. & GREET, D.N. (1964). Observations 
on the taxonomy and biology of Tylenchorhynchus 
nzacrzzrzzs (Goodey, 1932) Filipjev 1936 and T;ylen- 
chorhynchus icarus sp. nov. Purasitology, 54 : 119- 
144. 
Retrne Nématol. 4 (1) : 125-1x0 (1981) 


Effects of ageing and starvation on respiration and food reserve content in adult Hirschmanniella spinicaudata

Abstract

Similar works

Full text

Available Versions

Horizon / Pleins textes