Mabbett, K.

Wharton, D.A.

Horizon / Pleins textes

Cold-tolerance and acclimation in the 
free-living nematode, Panagrellus redivivus 
Kathryn MABBETT and David A. WHARTON* 
Department of Zoology, University College of Wales, Abeystwyth, Dyfed, U.K. 
SUMMARY 
The supercooling points of P. redivivus adults, acclimated at 22", are depressed by 2-3', 3-5 hours after transfer to lower 
acclimation  temperatures of 15", 10' and 5". At 15" supercooling  points  return  to  normal  levels  after 15 hours but at 10' and 5' 
the  depression of  supercooling  points  is  more  permanent.  After  24  hours'  exposure  there  is a sign ficant  negative  correlation  between 
supercooling  points  and  acclimation  temperature. The supercooling  points  of P. redivivus adults,  acclimated  at 10°, is  elevated  after 
transfer  to 22'. These  results  indicate  that P. redivivus adults  are  able  to  acclimate  to  changes  in  temperature  in  a  way  which  affects 
their  supercooling  point. The possible  rôle  of  cryoprotective  substances in  this  response  is  discussed. 
RESUME 
Tolérance et acclimatation  au  froid chez le nématode libre Panagrellus  redivivus 
Les  points  de  surfusion  des  adultes  de Panagrellus  redivivus acclimatés à 22' sont  abaissés  de 2 à 3", 3 à 5 heures  après  transfert 
aux  températures  d'acclimatation  de 15', 10' et 5'. A 15', les  points  de  surfusion  reviennent à la normale  après 15 heures,  mais 
à 10" et 5' l'abaissement  des  points  de  surfusion  devient  définitif.  Après  une  exposition  de  24  heures,  il  y  a  forte  corrélation  négative 
entre  les  points  de  surfusion et la  température  d'acclimatation.  Ces  points de surfusion  des  adultes  de P. redivivus acclimatés 2 10' 
sont élevés après le transfert à 22'. Ces résultats indiquent que les adultes de P. redivivus ont la capacité de s'acclimater aux 
variations  de  température  par un mécanisme qui affecte  leur  point  de  surfusion.  Le  rôle  possible  de  substances  cryoprotectrices 
dans  cette  réaction  est  discuté. 
A number of studies indicate that nematodes can 
survive sub-zero  temperatures (Anderson, Wang & 
Levine, 1966; Balasingham, 1964; Vrain,  1978), but 
until  recently the mechanisms involved were unknown. 
Arthropods which can survive sub-zero temperatures 
(cold-tolerant) can either  withstand extracellular ice 
formation (freezing-tolerant) or avoid freezing  by 
supercooling but die  once  the  body fluids  freeze 
(freezing-susceptible). An animal is said to supercool 
when it can  maintain  its body  fluids in a liquid  phase 
at temperatures below their  melting  point (Block, 
1982). Most nematode species investigated are 
freezing-susceptible but can avoid freezing  by 
supercooling. This mechanism  has  been  demonstrated 
in Trichostrongylus colubriformis (J 3), Ditylenchus 
dipsaci (J 4), P. redivivus (adults), Nenzatodirus battus 
(J 3) and Globodera rostochiensis (J 2) (Ash & Atkinson, 
1982; Wharton, Young & Barrett, 1984; Perry & 
Wharton, 1985). Supercooling points as low as - 28' 
have been recorded. Aphelenchoides ritzemabosi is the 
only species for which there is  any evidence for 
freezing-resistance (Asahina, 1959). 
Some arthropods can increase their cold-tolerance 
during winter by lowering their supercooling points 
(Somme, 1982). Nematodes  may also be able to do this. 
The ability of the infective juveniles of Trichinella 
spiralis to survive freezing at - 32' or - 45' is 
increased by preconditioning at - 15' (Smith, 1984). 
The mechanism of cold-tolerance in this species has  yet 
to be demonstrated. Chilling lowers the supercooling 
points of N. battus eggs (Ash & Atkinson, 1982). 
This  paper investigates the ability of the adult stage of 
P. redivivus to acclimate to falling temperatures by 
lowering  its  supercooling point. 
Materials and methods 
P. redivivus were cultured on moist  oats  at 22'. For 
the acclimation  studies cultures were  transferred to 1 5 O ,  
* Present  address : Department of  Zoology,  University of Otago, P.O. Box 56, Dunedin,  New  Zealand.' 
Revue Nématol., 9 (2) : 167-170  (1986) 167 
K. Mabbett & D. A. Wharton 
10' or 5'. Acclimation  temperatures were provided  by 
cooled incubators,  with an accuracy of & 05'. At 
intervals  after  transfer,  samples were removed and  the 
nematodes  cleaned  and  isolated by rapid  migration 
through tissue  paper. The supercooling  points of more 
than 30 adults were measured. To  investigate the effect 
of an increase in  temperature  on cold-acclimated 
nematodes  a  culture was acclimated  at 10' for  48  hours 
and  then transferred to 22'. Supercooling  points were 
again  measured at various  intervals  after  transfer. The 
same nematode culture was used for each series of 
experiments. 
Supercooling  points were measured by direct 
observation  using  a  thermoelectric cooling microscope 
stage. The design and operation of the  stage is described 
in detail by Wharton  and  Rowland (1984). A  drop of 
nematode  suspension was transferred to a small square 
of cellulose  acetate  sheeting  and the surface  water 
removed with filter paper. A drop of liquid paraffin 
was placed on top of the sample to prevent water 
evaporation. The water content of nematodes will 
remain  constant  for several days in this medium (Perry, 
1977). The specimen was placed on  top of the 
thermocouple in  the cooling  chamber of the microscope 
stage  and the  chamber sealed and  insulated. The stage 
was cooled rapidly to O' and  the  control  unit switched in 
to provide  a  controlled  rate of cooling of l'/min. Adult 
nematodes were observed as they were cooled from O' to 
- 40" and  the supercooling  point  recorded. The 
supercooling  point was  easily observed as a  marked and 
sudden decrease in transparency  as  the  nematode  froze. 
Preliminary  experiments  had shown that  clumps of 
nematodes had much higher supercooling points than 
individuals.  Only the supercooling  points of individual 
adults, not  in contact  with  other  nematodes, were noted. 
Results 
The supercooling  points of P. redivivus adults 
acclimated at 22' and after  transfer to acclimation 
temperatures of  15', 10' and 5' is shown in Fig. 1. There 
is a  marked  depression of the supercooling  points during 
the first 3-5 hours  after  t ansfer to  the lower 
temperature. At 15' the supercooling points return to 
their  original levels 15  hours  after  transfer, whereas at 
10' and 5' the lowering of the supercooling  point is more 
permanent. 
The supercooling  points of nematodes  acclimated a t  
10" were elevated after transfer to 22' (Fig. 2). The 
supercooling  points of nematodes  after 24 hours 
exposure to an acclimation temperature is shown in 
Fig. 3. Supercooling points are negatively correlated 
with acclimation  temperature  (r = - 0.455; df = 137; 
p < 0.001). 
168 
-271 10 O C  
-271 5 O C  
- 2 4 p  , , , , , , 
-23 
O 4 8 12 16 20 24 
Tirne (hrs) 
Fig. 1. The mean supercooling points of P. redivivzu adults 
after  transfer  from  an  acclimation  temperature of 22' to 15'' 
10' and 5'. (Vertical bars represent the standard error of the 
vnean of more than 30 individual measurements). 
Discussion 
After  transfer  from 22' to a lower acclimation 
temperature of 15', 10' or 5" the  adults of P. redivivus 
show a  clear  depression of their  supercooling  points in 
the order of 2-3'. After transfer to 15' supercooling 
points recover to their  original levels after 15 hours.  A 
decrease in temperature results in a lowering of the 
nematodes'  supercooling  points but this  decrease is  not: 
sustained at 15'. After, 24 hours' exposure there is a 
Revue  Nématal., 9 (2) : 167-170 (1986) 
Cold-tolerance and acclimation of Panagrellus  redivivus 
O 
Y c 
Q 
Kn 
C 
'0 -25- 
.- 
-24- 
O, 
O 
O, 
(O 
9 -23- 
-22 1 I 1 1 
O 4 8 12 1 6  2b $4 
Time ( h r s )  
Fig. 2. The mean  supercooling  points  of P. redivivus adults 
after  transfer  from  an  acclimation  temperature  of IOo to 22'. 
(Vertical bars  represent the standard error  of the  mean of  more 
than 30 individual measurements). 
c E 
I 
0) 
@ -23' 5 I Ib 15 20 25 
Acclimation temperature O C  
Fig. 3. The mean supercooling points of P. redivivus adults 
after 24 hours'  exposure to different  acclimation  temperatures. 
(Vertical bars represent the standard error of the  mean of  more 
than 30 individual measurements). 
significant  correlation  between  supercooling  points and 
acclimation  temperature. The elevation of supercooling 
points  after  transfer  from 10' to 22' indicates  that the 
nematodes  are not responding  toa change in 
temperature, regardless of whether it is a decrease or an 
increase, and that the change in supercooling points 
observed are  not due  to some  sort of ageing effect in the 
culture. 
The depression of supercooling  points in response to 
lowered acclimation  temperatures  in P. redivivus is 
comparable to  that observed in some  arthropods. 
Transfer of the antarctic terrestrial mite, Alaskozetes 
antarcticus from 0' to - 5' resulted in a 2-3' depression 
Revue Nématol., 9 (2) : 167-170 (1986) 
of supercooling  points  (Young & Block, 1980). 
Measurements of supercooling  points have been made 
using a constant rate of cooling of 1°/min. A 2-3" 
depression of supercooling  point  under  these  conditions 
may  reflect  a  greatly  increased  ability to survive  sub-zero 
temperatures well above  the  measured  supercooling 
point  and close to  those  encountered in  the field. The 
time of exposure is an important  factor  determining  the 
temperature at which  spontaneous  freezing  occurs 
(Samme, 1982). 
Most freezing-susceptible  arthropods  synthesise 
glycerol, or  other polyhydric alcohols, as a 
cryoprotectant (Duman et al., 1982). P. redivivus is 
known to synthesise glycerol as an end  product of 
anaerobic  catabolism  (Buttenvorth & Barrett, 1985). 
Preliminary results, however, indicate no correlation 
between glycerol concentrations  and  supercooling 
points (Mabbett, Wharton & Buttenvorth, unpubl.). 
Glycerol concentrations may be too low to produce 
significant cryoprotective effects (Wharton, Young & 
Barrett, 1984). Nematodes are known to be able to 
synthesise  other  polyhydric alcohols, including  sorbitol, 
ribotol and inositol (Barrett, 1981; Womersley, 1981). 
These are  known to act  or may be  able to act as 
cryoprotective  agents in arthropods (Duman et aL, 
1982). 
Trehalose, and  other sugars, have also been  suggested 
as cryoprotectants in arthropods (Duman et aL, 1982). 
During chilling, the trehalose  concentration of the eggs 
of N. battus increases and  the mean  supercooling  point 
decreases (Ash & Atkinson, 1982). Treha.lose may, 
however, have other rôles during  ovenvintering (Ash & 
Atkinson, 1983). The rôle of potential cryoprotective 
compounds  in  nematode cold-tolerance is uxlear  and 
merits further investigation. 
ACKNOWLEDGEMENTS 
We would  like to thank  the  Nuffield  Foundation for 
fiiancial support and Jean Clough for technical assistance. , 
REFERENCES 
ANDERSON, F. L., WANG, G. T. & LEVINE, N. D. (1966).  Effect 
of temperature on survival  of the free-living stages of 
Trichostrongylus colubriformis. J. Parasitol., 52 : 713-721. 
ASAHINA, E. (1959).  Frost-resistance in a  nematode, 
Aphelenchus ritzema-bosi. Low Temp. Sci., B 17 : 51-62. 
ASH, C. P. J. & ATIUNSON, H. J.  (1982). The possible  rôle  of 
trehalose in the survival of  eggs  of Nematodirus battus 
during  dormancy. Parasitology, 85 : LV. 
ASH, C. P. J. & ATKINSON, H. J. (1983). Evidence for a 
temperature-dependent  conversion  of  lipid  reserves to 
carbohydrate in quiescent  eggs  of the nematode, 
Nematodirus battus.  Comp.  Biochem. Physiol., 76 B : 
603-610. 
169 
K. Mabbett & D. A. Wharton 
BALASINGAM, E. (1964).  Comparative  studies  on  the  effects  of 
temperature  on  free-living  stages  of Plococonus lotoris, 
Dochtnoides  stenocephala and Ancylostoma  caninum.  Can J .  
BARRETT, J. (1981). Biochemisty of Parasitic  Helminths. 
London,  Macmillans, x f 308  p. 
BLOCK,  W.  (1982).  Cold hardiness  in  i vertebrate 
poikilotherms. Comp. Biochem. Physiol., 73 A : 581-593. 
BUTTERWORTH, P. E. & BARRETT, J. (1985).  Anaerobic 
metabolism  inthe  free-living  ematode Panagrellus 
redivivus. Physiol. Zool., 58 : 9-17. 
DUMAN, J. G., HORWARTH, K. C., TOMCHANEY, A. & 
PATTERSON, J. C. (1982). Antifreeze agents of terrestrial 
arthropods. Cornp. Biochenz. Physiol., 73 A : 545-555. 
PERRY, R. N. (1977). The water  dynamics  of  stages  of 
Ditylenchus  dipsaci and D.  ?nyceliophagus during  desiccation 
and  rehydration. Parasitology, 75 : 45-70. 
PERRY, R. N. & WHARTON, D. A. (1985). Cold tolerance of 
hatched  and  unhatched  second  stage  juveniles of the  potato 
cyst-nematode Globodera rostochiensis. Int. J .  Parasitol., 15 : 
ZOOL, 42 : 907-918. 
441-445. 
Accepté pour publication le 8 janvier 1986. 
SMITH, H. J. (1984). Preconditioning of Trichinella spiralis 
nativa larvae in musculature to low temperatures. Vet. 
Parasitol., 17 : 85-90. 
S0MME, L. (1982).  Supercooling  and  winter  survival in 
terrestrial  arthropods. Comp.  Biochevz. Physiol., 73 A : 
519-543. 
VRAIN, T. C. (1978).  Influence  of  chilling  and  freezing 
temperatures  on  infectivity of Meloidogyne  incognitaand M. 
hapla. J .  Nemafol., 10 : 177-180. 
WHARTON, D. A. & ROWLAND J. J. (1984). A thermoelectric 
microscope  stage for the measurement  of  the  supercooling 
points  of  microscopic  organisms. J.  Microsc., 134 : 299-305. 
WHARTON, D. A., YOUNG, S. R. & BARRETT, J. (1984).  Cold 
tolerance  in  nematodes. J. Comp.  Physiol., B  154 : 73-77. 
WOMERSLEY,  C.  (198  1).  Biochemical  and  physiological  aspects 
of anhydrobiosis. Co?np. Biochem. Physiol., 70  B : 669-678. 
YOUNG, S. R. & BLOCK, W. (1980).  Experimental  studies  on 
the  cold-tolerance  of Alaskozetes  antarcticus. J. Insect 
Physiol., 26 : 189-200. 
170 Revue Nématol., 9 (2) : 167-1 70 (1986) 


Cold-tolerance and acclimation in the free-living nematode, Panagrellus redivivus

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