Demeure, Yves

Paycheng, Claude

Reversat, Georges

Souchaud, Bernard

Horizon / Pleins textes

A photographic technique to evaluate the consumption 
of food  reserves in individwal  starved  second  stage  juveniles 
of Meloidogyne . javanica 
Georges REVERSAT*, Yves  DEMEURE *, Bernard  SOUCHAUD * and Claude PAYCHENG* * 
*Laboratoire de Nématologie,  **Laboratoire de Chimie,  ORSTOM,  B.P. 13S6, Dakar,  Sénégal. 
SUMMARY 
Individual  nematodes, 1, 8, and  15-days-old, were fixed, stained  and  photographed.  Amounts of silver fixed on 
the prints were determined by atomic absorption. Results compared favorably with colorimetric determination 
uf lipids,  carbohydrates,  proteins  and  dry weight utilizing 5 30,000 individuals.  This  technique  may be useful to 
measure depletion of metabolic reserves in  small organisms in which large  quantities of specimens are difficult to 
obtain. 
RBSUMB 
Evaluation  individuelle  de  la  consorn~nation  des  réserves  nutritives 
chez les juvéniles du second stade de Meloidogyne javanica p a r  
une  technique  photographique. 
Des nématodes 5gés de 1 ,  8 et 15 jours sont fixés, colorés et  photographiés  individuellement.,  Sur des épreuves 
agrandies,  la  quantité  d’argent fixée est  dissoute  chimiquement  puis dosée par  absorption  atomique. Les résultats 
correspondent bien aux mesures du  poids sec et  aux dosages colorimétriques de  lipides,  glucides et  protéines, réalisés 
sur  des échantillons d’environ 30.000 individus.  Cette  technique  pourrait  s’appliquer A I’évaluation  de  la consom- 
mation des réserves métabolisables chez de petitS.organismes pour lesquels les quantités d’individus du même 
stade, nécessaires aux dosages colorimétriques,  ne  peuvent  être  obtenues. 
Many free-living nematodes must withstand 
more or less prolonged periods of starvation 
during which endogenous food reserves, mainly 
lipids,  are consumed (Cooper & Van  Gundy, 
1971). Ghemical techniques for quantitative 
determination of such  reserves  often  require 
several thousands of individuals  per  determina- 
tion  (Reversat,  1976).  Consumption of food 
reserves  in a few individuals  has  been  estimated 
using  camera  lucida  drawings of nematodes 
treated  with a lipid staining compound  (osmium 
tetroxide) after which al1 the stained portions 
were cut  out  and weighed (Van  Gundy, Bird & 
Wallace, 1967). In nematodes of increasing age 
there was a progressive reduction  in  stained 
areas which was attributed t o  the consumption 
of endogenous lipid reserves. The photographi,c 
R e v u e  Némafol. 3 (1)  : 101-105 (1980) 
technique described in this paper avoids indi- 
vidual  errors  in  drawing  and  cutting  and 
provides a more precise analysis of density of 
the stained area. On a photographic print of 
a stained  nematode, opaqueness of stained 
portions is represented by the darlcening of 
the  paper, which corresponds t o  immobilization 
of metallic  silver. Thus, opaqueness  can he 
measured by  determining  the  quantity of fixed 
silver in the photographic image. 
To demonstrate this principle infec.tive juve- 
niles of M e l o i d o g y n e   j a v a n i c a  were choosen 
because they  can be  obtained  in  large  quantities 
required for chemical  determination  and  because 
a relationship lzas been demonstrated between 
their infectivity and the amount of their food 
reserves (Van Gundy , Bird & Wallace, 1967). 
101 
G. Reversat,  P. Demeure ,  B. Souchaud LP: C. Paycheng  
' Materials and methods 
JUVENILES 
The juveniles used were from a single egg- 
nlass  population of M .  jauanica, maintained  on 
the roots of kenaf ( I l i b i s c u s  c a p a b i n u s ) .  Ten 
seeds of kenaf were sowed in 2.5 liters plastic 
pots, containing steam sterilized sandy soil, 
and watered  aily.  After  four weeks, each 
pot was inoculated  with  20,000  freshly  hatched 
juveniles, then pots were kept in a greenhouse 
with an average temperature of 26-280. Five 
weeks after  inoculation,  infected  roots were 
washed to remove soil and dehris and placed 
over a funnel collector in a misting chamber, 
where juveniles hatched from egg-masses. Ne- 
matodes  obtained  uring the first 24 hours 
were discarded and nematodes for experiment 
were c,ollected 24 hours later. Nematodes were 
placed on four  layers of .tissue  paper which 
remove debris and  inactive  nematodes  and 
allowed the  active ones to migrate  through. 
One day  later clean and ac.tive nematodes were 
collected as one day old juveniles.  Juveniles 
were stored  in  Petri dishes, in  phosphate buffer 
pH 7 a t  a  conc,entration of 4 mM, at. 280, in  the 
dark. After one and two weeks of storage  active 
juveniles were selected, by  the tissue  paper 
method described above. At the time 1, 8 and 
15 days  the sample of active  juveniles was 
divided in two parts : the first of several hun- 
dred individuals for the photographic method, 
the second of several hundred  thousand indivi- 
duals for c,hemical determinations. 
PHOTOGRAPHIC METHOD 
Juveniles were fixed (Netscher & Seinhorst, 
1969) and  then  stored  in  4 % formalin for obser- 
vation. Fixed  juveniles were tranferred t o  a 
lipid staining  solution,  Sudan  Black (Merck 
ref. 1387) saturated  in  60 % ethanol  (Gabe, 
1968) for 72 hours a t  280. The  stained nemat>odes 
were mounted  in glycerine on microslides 
(Seinhorst,  1959).  Ten  juveniles of each age 
were photographed  individually  with  a  Leitz 
Orthoplan microscope equiped with  an  automa- 
tic  Orthomat camera and Kodak Panatomic X 
film of 16 Din sensibility.  Three prints on  paper 
- - . . ~  Kodabrom G5, (X600)  were  made of each of 
. ~ -  - -  - - - _ _  . .~ 
102 
the juveniles  with the same  exposure and deve- 
lopment times and at  the same temperature. 
Each. of the 90 images was cut  into pieces 
approximately 1 cm long and placed in  a 250 ml 
erlenméyer flaslc containing 25. ml of 6.5 y. 
nitric acid. Flasks were heated until the disap- 
pearence of the image by chemical dissolution 
of the silver. Each of the 90 solutions  was 
adjusted to 250 ml by weighing with addition 
of distilled  wat,er,  filtered,  and  measured for 
silver content using an Atomic Absorption 
Spectrophotometer  (Instrumentation  Labora- 
tory Type IL 151) and reference solutions of 
silver nitrate in 0.65 y. nitric acid. 
CHEMICAL DETERMINATIONS 
At each harvest  time, juveniles were divided 
into  eight  equivalent  samples of & 30,000 indi- 
viduals  (Reversat,  1980). For dry weight deter- 
minations  juveniles were tranferred  with  a 
very  small  amount of water in  preweighed 
aluminium foi1 cups. After two hours of desi- 
ccation a t  1100 cups were weighed again with 
an  accuracy of f 0,005 mg.  For  chemical 
determinations, juveniles were first digested in 
an appropriate chemical (Reversat, 1976) and 
then colorimetric  reactions were used : with 
the  anthrone  reagent for total  carbohydrates 
(Seifter et al., 1950), with the sulfophosphova- 
nillic reagent for tota1,lipids  (Chabrol & Charon- 
nat, 1937) and with the Folin phenol reagent 
for total proteins (Lowry et al., 1951). 
Results 
The  progressive clearing of the  stained nema- 
todes with age, observed on prints (Fig. l ) ,  is 
correlated with  a decrease of the silver content 
of the nematodes prints (Tabl. 1). There was a 
significant difference at  the 0.1 % level for the 
quantities of silver  measured  between  means of 
juveniles 1 and 8 days old and between means of 
juveniles 8 and 15 days old. 
Two facts  are  noted  with respect t o  the relia- 
bilit4y of the  method.  First,  statistical  study 
within the results of each age group (Tab. 1) 
showed that  variability between the  three 
replicate prints of the same  individual  was very 
slight compared to  the variability between t,he 
Recrue NPmatol. 3 ( 1 )  : 101-105  (1980) 
- .~ - .__  - - - - - - - - - -  - - . .. 
Photographic  fechnique to evaluate food reserves  consurnption 
Table 1 
Evaluation of food reserve  consumption by Meloidogyne  javanica juveniles  during in vitro storage  by  silver  method 
and by chemical determination. Dates are expressed as means f standard deviation. Silver method : results in 
10-o g of silver  per  print.  Ten juveniles  for  each of the  three ages and  three  prints for  each  juvenile.  Chemical deter- 
mination : results in g of substance per juveniles. For each age, two replications for each of the four determi- 
nations  and f 30,000 juveniles for each replication. 
Determination 1 day 8 clays 15 days 
Silver method  juv no 1 
juv no 2 
juv no 3 
juv no 4 
juv no 5 
juv no 6 
juv no 7 
juv no S 
juv no 9 
juv no 10 
mean f SD 
Chemical determination 
Dry weight 
Total  carbohydrates 
Total  lipids 
Total  proteins 
,847 f 14 
860 f 28 
904 f 18 
741 f 52 
740 f 50 
876 & 34 
856 f 76 
977 f 35 
802 f 18 
669 f 49 
827 5 90 
23.9 f 0.2 
1.7 f 0.2 
9.2 f 0.2 , 
9.0 -+ 0.2 
452 f 14 
626 & 34 
538 f 55 
498 f 45 
308 f 20 
581 & 16 
524 f 9 
473 1 30 
572 f 20 
553 & 26 
512 f 89 
16.7 i 0.6 
1.5 f 0 
3.4 f 0.2 
7.3 f 0.4 
282 f 8 
307 f 2 
283 f 7 
368 f 11 
359 f 29 
169 f 12 
132 f 15 
160 f 9 
134 f 32 
244 f 90 
244 f 12 
14.3 f 0.2 
1.1 f O 
1.4 f 0.2 
7.5 f 0.3 
Analysis of variance for silver  method : 
Within each age : F = 15 for 1 day, F = 22.5 for 8 days, F = 79 for 15 days with a F limit of 5.24 at  the 1 o joo  
level. 
Between ages : t = 7.6 between 1 and 8 days, t = 6.7 between 8 and 15 days with a t limit of 3.92 at  the 1 ojoo 
level. 
head  
0.3 m m  
O 
tail 
1 d a y  8 d a y s  15 d a y s  
Fig. 1 : Photographie prints of stained Meloidogyne javanica juveniles of three different ages. (Only 
five of the  ten juveniles used in the  study,  are  represented.  Note  reduction of the overall black area 
with increasing age, i.e. lixed silver on the photographie paper). 
Revue  Nématol. 3 (1) : 101-105 (1980) 
G. Reversat,  Y .  Demeure ,  B. Souchaud dl- C. Paycheng  
ten individuals of the same age. Moreover, the 
increasing  value of F with age demonstrated 
that  the  rate of the food reserves consumption 
varied widely between  individuals,  as  had  been 
previously  observed  (Van  Gundy,  Bird & 
Wallace,  1967).  Secondly, the  photographic 
image of a one-day-old juvenile  is saturated 
t o  only  1/4 of the  maximum  saturation of silver 
content. As a  matter of fact solar exposed and 
developed photographic  paper  Kodabrom G5 
exhibit a nletallic silver content of 156 y/cln2 
(100 y. of saturation).  The corresponding value 
for the one-day-old juvenile image was 37.6 y/ 
cm2 (24 % of saturation), for the background of 
the  prints 1.6 y/cm2 (1 % of saturation  )and for 
unexposed  and developed paper 1.2 y/cm2, 
(0.8 y. of saturation). Therefore, although one- 
day-old juveniles appear t o  be very  opaque 
(Fig. l),  it is possible to  detect  individual 
variations  (Tab. 1). 
Chemical determinations confirnled that lipids 
are  the  main food reserves consumed during 
starvation,  and  carhohydrates  and  proteins 
were less consumed (Tab. 1). 
Discussion 
In Caenorhabditis briggsae, ageing is charac- 
terized by  the progressive development of 
dark colored ' granules of lipofuc,sine, called 
age pigment,  and  located  in  the  intestinal 
epithelium  (Epstein,  Himmelhoch & Gershon, 
1972). On a photographic print, this darkening 
could compensate the progressive disappearance 
of stained food reserves. If suc.h age pigment 
was present  in the  intestine of M .  javanica 
juveniles, its  contribution t o  the  opaqueness 
was very  reduced, since unstained  areas  in 8 and 
15 days old juveniles,  located in  the  gut region, 
were very  similar t o  the background of the 
prints (Fig: 1). 
When, plotted as percentage of initial values 
on the  same  graph,  dry weight,  lipids and silver 
content of M. jauanica juveniles decreased with 
related rates (Fig. 2). In conclusion, it appears 
possible t o  quantify in a reproducible manner 
nematode  opaqueness by silver determination 
(Tab. 1) and that there is a direct relationship 
between the  nematode opaqueness and  its 
- reserve .. content . .  (Fig. 2). Thus,  this  photographic . . _. - -  _ ,  . ._ 
technique  can  actually  evaluate  consumption of 
food reserves. 
days 
O I I I 
1 8 15 
Fig. 2. Effect. of storage  time on Melo idogyne   j avan ica  
juveniles : decrease of dry weight, silver content of 
photographic prints and lipid content as percentage 
of initial values. 
Since this  technique  requires  only  ten indivi- 
duals of Meloidogyne jauanica juveniles whereas 
chemical  determinations  are  made  upon samples 
of 30,000, it could be applied t o  t'he study of 
nematode species in which large  numbers of 
individuals of the same stage are dificult to  
obtain. Further, we suggest that  this technique , 
can  be applied t o  the  study of other small 
Invertebrata  exhibiting, as nematodes, soft  
and  transparent  integuments,  as rotifers  and 
enchgtraeids; application to others groups, as 
tardigrades, collembola and acarii, would pro- 
bably require particular  adaptation  due  to  the 
thickness of their integument. 
ACKNOWLEDGEMENTS 
We thank Dr D.P. Taylor  €or  helpful discussion and 
c0mment.s and for assistance  in  preparation of thc 
English text and M. Luc for reviewing the text. - . -  . . 
Revue Nématol. 3 (1) : 101-105 (1980) 104 
Photographi t   technique €0 evaluate  food  reserues  corlsumption 
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105 


A photographic technique to evaluate the consumption a food reserves in individual starved second stage juveniles cf Meloidogyne javanica

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