Dalmasso, Antoine

Janati, A.

Name not available

Further observations on chromosomal processes during oogenesis of Meloidogyne hapla (Nematoda)

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Further observations on chrornosomal  processes 
during  oogenesis of Meloidogyne haplu (Nematoda) 
Ahmed JANATI * and Antoine DALMASSO 
INRA,  Station  de  Recherches sur les  Nématodes,  123 Bd  Franc i s   Me i l land ,  06600 Antibes ,   France.  
SUMMARY 
Oogenesis of two  populations (A and B) of Meloidogyne  hapla was  studied  using  the  Giemsa C banding  technique. 
Population  A  underwent  a  normal  meiotic  process  and  had 2 n =’ 30 somatic  chromosomes  while  population B 
showed  a  single maturation division of equational  type  and 2 n  somatic  karyotype = 45. In  both cases, the Giemsa C 
technique  provided good figures, even of the  “pseudointerphase” or “diffuse stage” wliich takes place  during  oocyte 
growth and which previously has been designed by this term because the poor staining of chromosomes when 
Schiff or orcei,n techniques were used. In population A, this phase corresponded to diplotene. In population A 
30 chromosomes  could  be  distinguished during oogonial  mitosis, but  only  nine or ten  pairs were  observed during 
diplotene.  At  dialtinesis  lifteen  bivalents  were  present.  Maturation  was  normal : the  first  polar  body  with  fifteen 
chromosomes  was  rejected and  a second mitotic division  followed. The  nine or ten  pairs  observed  during  meiotic 
prophase  were  thought  to  be  relictual  chromosome  feature  and  the  higher  number  observed  in  the  other  stages 
could  be the  result of some  chromosome  breaks.  The  variation of the chromosome  number  previously  signalized  for 
M .  hapla (n  = 14-15-16-17) would be related to this phenomenon. Population B possessed 45 chromosomes. At 
the early prophase of the single maturation division, chromosomes were not paired, contrary to  population A. 
At th’e end of the prophase  some of them  assembled  forming  large  chiasmatic or aggregate  chromosomes ; chro- 
matin  reduction did not  take place a t   the  end of the  division. 
RÉSUMÉ 
Nouvelles Observations sur les processus chromosolniques pendant l’ovogénèse de Meloidogyne hapla ( N e m a t o d a )  
L’ovogenèse  de deus populations A e t  B de Meloigogyne  hapla a  été  étudiée  en  utilisant  une  technique  de Giemsa 
C banding.  La  population A subit  une méiose normale  et  présente 2 n =. 30 chromosomes, tandis  que  la  popula- 
tion B n’a  qu’une  seule  division  de  maturation  de  type  équationnel  et 2 n = 45. Dans  les  deus  cas  la  technique 
employée  a  donné  d’excellentes figures, même  pour  la  pseudointerphase ou stadê diffus qui  prend  place  pendant 
la  croissance  de  l’ovocyte  et  qui  a.été  ainsi  nommée  antérieurement à cause  de  la  non  colorabilité  de  la  chromatine ’ 
par les  techniques  traditionnelles  au  Schiff ou à l’orcéine.  Cette  phase  correspond  pour  la  population A au  diplothe. 
Chez cette  population A on observe 30 chromosomes pendant la  mitose oogoniale, mais  seulement neuf ou dix  paires 
sont observées pendant le diplotène alors qu’on compte quinze bivalents à la diacinèse. La  maturation  est  nor- 
male : le premier globule polaire avec quinze chromosomes est rejeté et  la seconde division mitotique suit. Les 
neuf ou dix paires  observées pendant  la  prophase  méiotique  traduisent  peut-être  un  aspect  relictuel  des  chromo- 
somes, le’nombre plus élevé observé dans les autres stades pouvant résulter de fragmentations stabilisées. Les 
’ variations  du  nombre  de  chromosomes  antérieurement  signalées  pour M .  hapla (n = 14-15-16-17) pourraient  être 
liées à ce phénomène.  La  population B possède 45 chromosomes. Au début  de  la  prophase  de  l’unique  division de 
maturation, les  chromosomes  ne  s’apparient  pas,  contrairement à la  population A. A  la fin de  la  prophase  quelques- 
uns  sont assemblés, formant des  agrégats ; la  réduction  chromatique  ne se produit  pas à la fin de la  division. 
Gametogenesis and lraryotypes of nematodes 1963,  1966). I t  was demonstrated  that M .  hapla 
belonging to  the genus Meloidogyne were first reproduced  either by  facultative  nleiotic  parthe- 
studied  using orcein as a  stain  (Triantaphyllou, nogenesis, or by obligatory  “mitotic”  partheno- 
* Present address : Direction de la Recherche Agronomique, Station de Phytiâtrie, B.P. 415, Rabat, Maroc. 
Revue Néma.to1. 4 ( 2 ) :  221-226 (1981) 221 
A. Janati & A .  Dalmasso 
genesis. In  populations  undergoing meiosis, 
somatic  chromosomes  varied  in  number : 2  n = 
30,  32 or 34. In  mitotic  populations,  45 chromo- 
somes  were  visible in female gamete  and a single 
non  reducing  division  took  place. Two other 
species, M.  rzaasi and M .  graminicola which 
were found t o  have 2 n = 36 in oogonia and 
n = 18 chromosomes a t  diakinesis, reproduced 
by  facultative  parthenogenesis, while some other 
species  like M .  arenaria, &I, ja.vanica or M .  
incognita reproduced  by  obligatory  partheno- 
genesis and possessed high and variable chro- 
mosome numbers  (Triantaphyllou,  1969). On 
the  other  hand  the  basic  number of Heterodera, 
a closely related genus, was n = 9. Rec,ently 
two  hypothesis  have  been  formulated by Trian- 
taphyllou  (1966, -1969) : i )  meiotic M. hapla 
(like  most  meiotic species of Meloidogyne) is 
diploid.  The  basic  number n = 18 is sometimes 
altered by aneuploidy ; i i )  the species is te tm-  
ploid,  possibly l?Ieloidogyne ancl Neteroderahave 
a common ancestor ; individuals with 45 chro- 
mosomes are  pentaploid.  Yet  molecular  data 
(Dalmasso & Bergé,  1978) were in  favour of 
hypothesis that the meiotic form of M .  hapla 
is  diploid.  Recently  synaptonemal complexes 
and recombination nodules were demonstrated 
during  the  early  prophase  in  meiotic  populations 
of &/S. Izapla while they were not  present  in 
mitotic  population  (Goldstein & Triantaphyllou, 
1978). 
The  present  study  was  an  attempt  to  obtain 
new informatmians  on  the  reproduction  and chro- 
mosomes  changes in  this species by  means of the 
C Giemsa technique. 
Materials  and  methods 
Two populations of M .  hapla were grown on 
tomato  cv.  Marmande  in a greenhouse a t  200 : 
population A from Le  Beausset (France) had a 
meiotic  mode of reproduction  with  n = 15 chro- 
mosomes in the post meiotic oocytes ; popula- 
tion B from Saulcy (France) was mitotic with 
2 n = 45. Chromosomes  were stained  using  the 
Feulgen tec.hnique. 
Giemsa C banding  technique was adapted for 
nematode gonads and oocytes from Summer’s 
- -  technique  (1972)  for  staining  the  centromeric . -  
222 
heterochromatin. Gonads of Meloidogyne  from 
adult female  were  dissected  and  soaked in a 
hypotonic  solution of sodium  citrat,e  for 15 min. 
(0.85%)  in conic glass Eppendorfs.  The  material 
was centrifugated  at 200 g for 10  min.  and  the 
supernatant  gently removed subsequently.  Fixa- 
tive (ethanol, ,acetic ac.id : 3/1) was added and 
the  tube placed at  40 for 3 h.  The  material was 
centrifugated  again  at 200 g for 10  min.  and  the 
supernatant removed.  Gonads  and cells were 
suspended  in  the  fixative  and  dropped  on a cold 
glass  slide  previously c,overed by a film of water. 
Entire  gonads were also directly  treated  with a 
hypotonic solution and fixative on slides con- 
served in a moist chamber between each step. 
The  slides  were  air  dried,  soaked  in 5% barium 
hgdroxide at 500 for 15 min.  and  washed  in 
water. Then they were incubated in 2 x ssc 
(0.3 M sodium chloride : 0.03 M sodium  citrate) 
a t  600 for 1 h.  The slides were  rinsed  and  stained 
in Giemsa (4%  in pH 6.8 buffer) for 20-30 min. , 
and  mounted  after  dehydratation  in  Canada 
balsam or DPX. 
Re sults 
ASPECTS OF THE MEIOSIS IN POPULATION A 
(from Le Beausset) 
Oogonia  divided normally  (Fig. 1 a, b ) ,  
30  chromosomes  (Fig. 1 c) could be distinguished. 
Meiosis was  preceded  by a brief interphase 
(Fig. 1 d )  as in Ascaris  (Bogdanov,  1977). 
During  zygotene  chromatin was concentrated 
at  the  proximity of the nuclear  enveloppe  show- 
ing characteristic “bouquet” conformations. In 
the suhsequent pachytene chromosomes appea- 
red paired. Curiously the number of chromatic 
filaments  (9 or 10) was less than  expected  from 
observations on oogonia (Fig. 1 e).  Then  during 
the long period of growth of the oocyte 1, a 
large nucleus was present. The chromatin was 
not  very poorly colored by Schiff or  orcein 
techniques at  this  stage, called pseudointerphase 
in nematodes (Nigon, 1949) or diffuse stage in 
other  organisms  (Wilson,  1925 ; klasterska, 
1977).  The Giemsa C technique allowed a better 
coloration of this stage which corresponded to 
the  diplotene  (Fig. 1 f ,  g, h).  It is characterized 
Revue Nématol. 4 ( 2 ) :  221-226 (1981) 
Chromosornul process  during  oogenesis of Meloidogyne hapla 
Fig. 1. a, h : Qogenesis in Meloidogyne  hapla meiotic lorm ( x 3200) ; a, b : Oogonial 
interphase ; c : Oogonial metaphase (30 chromosomes) ; d : Late  premeiotic  interphase ; 
e : Pachytene ; f,. g,  h, i : 9-10 pairs  with  centromeres,  diplotene  sequences ; j : 9 pairs 
clearly visible ; k : Early  metaphase of meiosis (15  bivalents) ; 1 : Typic  metaphase of 
meiosis showing 15 chromosomes. 
A. Janati h A. Dalmasso 
by  many nucleolus-like  vesicles probably  result- 
ing  from  intense  RNA  synthesis.  Chromatic 
filaments were double ; later during diplotene, 
the  chromatic  filaments  piralised  (Fig. 1 i). 
Centromeres,  chiasmata  became  discernible, 
multiple regions of heterochromatin were seen. 
Chromosome pairs were very long  and  still 
numbered 9-10. Subsequently chromosomes  con- 
tracted  and became  dense (Fig. 1 j ) .  Schiff 
and orcein  colorations  did  not  stain  chromo- 
somes,  therefore  their  low  number  had  escaped 
attention. Following this  stage the nuclear 
membrane  disappeared,  and at  diakinesis 15 bi- 
valents were distinctly visible (Fig. 1 k, 1). 
DGring anaphase 1, bivalents  separated  and  two 
sets each of 15 chromosomes could be counted. 
That is the reduced number of chromosomes 
observed  during  oogonial  division. 
A second maturation division took  immediatly 
place afterwards. If sperm occurred, two polar 
bodies were emitted, if not, the diploidy was 
restored by fusion of the second  polar body 
with  the female pronucleus. 
ASPECTS OF THE MATURATION OF OOCYTES IN 
POPULATION B (from Saulcy) 
45  chromosomes  were counhed in  the oogonial 
zone with difficulty  due to the small size of 
cells.(Fig.  2 a).  At  the beginning of the  mataura- 
tion, chromosomes became filamentous forming 
a discrete  network ; later chromosomes shortened 
and  thickened  (Fig. 2 b). Generally  pairing  was’ 
not  observed  excepted  in a small region but 
we suspected  this t o  be fortuitous.  Then  multiple 
c.entromeric heterochomatin  centers  appeared 
along  chromosome filaments,  no  coupling of 
chromosomes was observed at  this  stage which 
c,orresponded t o  the period of stxong growth of 
the cells (diffuse chromatin  stage  according clas- 
sical Feulgen and orcein techniques) (Fig. 2 c). 
The  number of chromosomes appeared  un- 
changed,  this confirms that  no  pairing  took 
place  during  zygotene.  Later,  the  chromatic 
mat,erial  c.ondensed. Punctiform  elements aligned 
forming  composive  chromosomes (Fig. 2 d). 
Finally  about  ten t o  fifteen  masses  with  different 
size  were  visible at  he end of this  pecial 
prophase  (Fig.  2  e).  At  early  metaphase  disjonc- 
tion  occurred  and 45  chromosomes  were  observed ~~ 
224 
~~ 
which  gave  rise to  two  anaphase  sets  each  with 
45  chromosomes. As reported by Triantaphyllou 
(1966) a single equational division  occurred. 
1 
Discussion 
Referin,g ta  Triantaphyllou’s  hypothesis, meio- 
tic forms of A!!. hapla may be either  tetraploid or 
diploid. Recently, this author inclined for the 
second possibility considering 45 chromosomes 
populations as triploid.  The  basic  number of the 
genus Meloidogyne would  be n = 18, changes in 
chromosome number in M .  hapla (n = 15, 16 
and  17) would have  resulted  from  aneuploidy. 
The Giemsa technique applied to a meiotic 
and a mitotic  population of M. hapla,  supplied 
additional  information as i t  was  possible to  stain 
chromosomes during  the diffuse stage of the 
prophase in  both  populations.  Observations 
could also be  made  in  the zone of the oocyt4es. 
Counting of chromosomes  at,  diplotene  in  popula- 
tion A showed a abnormally Jow number of 
pairs. The eventuality of some supernumerary 
element  (trisomy)  seems  to be  dismissed  for 
only couple have  been  observed.  Another  expla- 
nation would attribute to chromosome breaks 
the higher numbers in metaphases 1. Meiotic 
prophase chromosomes should be considered as 
a relic chromosome entity. This would not be 
so surprizing for  similar  breaks  have  been 
reported in Ascaris but in somatic cells during 
embryogenesis.  Chromosome  breaks  nlay  be 
correlated  with  polycentromeric  struckures of 
diffuse centromeres, already reported in nema- 
todes  by  Boveri (1888) and  several  authors. 
Thus  chromosome  numbers  in Meloidogyne 
species may  result  on  the  first place  from breaks 
which would have given a new basic number 
n = 18 and secondly from polyploidy and in 
some  cases,  from  loss of chromosomes (aneu- 
ploidy). In  reality  these losses  would only 
concerne  pieces of ancestral chromosomes. In 
population B, chromatic figures tended ta con- 
firm that normal pairing does not occur. The 
number of chromosomes  remained  unchanged 
al1 long the  prophase of the single  division. Yet, 
just before metaphase, we have observed some 
apparently  disordered assemblage of these  chro- 
mosomes. This is a fugacious  event  and  rapidly 
Reoue Ntmabol. 4 (2): 221-226 (1981) 
. .  
Chrornosomal process during oogenesis of Meloidogyne hapla 
Fig. 2.  a, e : Oogenesis in M. hapla “mitotic”  form ( x 3200) ; a : Oogonial 
nuclei ; b : Early  prophase ; c : Prophase ; d,  e : Late  prophase of the single 
maturation division showing composite elements and  then  the  only 12 to 
15 chromatin bloclcs visible. 
A. J a n a t i  dl. A. Dalmasso 
45 units were again  observed at  the  metaphase. 
It could be  possible that some chromatid 
exchange  occurred  during  this  short  period. 
Further  ultramicroscopic  investigations  are 
needed to answer this question. What is most 
probable is that  the 45 clîromosomes represent 
a triploid  state as demonstrated  by DNA dosage 
(Lapp & Triantaphyllou, 1972). 
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226 Revue Nénzatol. 4. ( 2 ) :  221-226 ()981) 


Further observations on chromosomal processes during oogenesis of Meloidogyne hapla (Nematoda)

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