- Email: [email protected]
Cold tolerance of hatched and unhatched second stage juveniles of the potato cyst-nematode Globodera rostochiensis
COLD TOLERANCE OF HATCHED AND UNHATCHED SECOND STAGE JUVENILES OF THE POTATO CYST-NEMATODE GLOBODERA ROSTOCHIENSIS R. N. PERRY* and D. A. WHARTON* *Nematology Department, Rothamsted Experimental Station, Harpenden, Hertfordshire, AL5 254, U.K. *Department of Zoology, University College of Wales, Penglais, Aberystwyth, Dyfed, SY23 3DA, U.K.
(Received 24 J&y 1984) Abstract-PERRY R. N. and WHARTOND. A. 1985. Cold tolerance of hatched and unhatched second stage juveniles of the potato cyst-nematode, Gioboderu rostoch~e~~. I~ter~ationai ~oar~al for Parasitoiogy 15: 441-445. Hatched second-stage juveniles of Gtoboderarostochiensiscan survive sub-zero temperatures by supercooling when not in contact with water. When frozen in water free juveniles cannot survive ice seeding across the cuticle and concomitant freezing of their body contents. Unhatched juveniles can survive in water, probably because the egg-shell protects the juvenile from ice seeding across from the medium; in this state juveniles survive by supercooling. INDEX KEY WORDS: Nematode; cold tolerance; supercooling; egg-shell; Globoderu rostochiensis. INTRODUCTION IN MANY REGIONSof the world with extremely low
temperatures for several months of the year poikilothermic species have developed survival mechanisms. Cold-hardy arthropods that can survive temperatures below the freezing point of their body fluids fall into two main groups, termed freezing-tolerant and freezing-susceptible (Salt, 1969). Species in the first group can survive ice formation in their tissues while freezing-susceptible species die if frozen and therefore must avoid freezing by the ability to supercool. Sayre (1964) suggested that nematodes able to survive freezing may have either of these mechanisms. Wharton, Young & Barrett (1984) showed that thirdstage juveniles of Trichostrongylus colubriformis, fourth-stage juveniles of Ditylenchus disptzciand Punagrellus silusae adults are freezing-susceptible, their mean supercooling points being -3O”C, -21.FC and -20.7”C respectively. Asahina (1959) suggested that A~heiencho~de~ ~~t~e~ahosi can survive extracellular ice formation. Globoderu rostochiensti has spread to almost all parts of the world (Evans & Stone, 1977) including many where winter soil temperatures are below freezing. The indigenous distribution of potato cystnematode is in the high Andean region where frost can occur for a substantial number of days in a year. Therefore, G. rostochiensk must have evolved cold protection mechanisms. This paper examines (a) the ability of hatched second-stage juveniles to survive freezing, and (b) the effect of the egg-shell independently and in conjunction with the cyst wall on low
temperature survival. Exposure to the natural hatching stimulus, potato root diffusate (PRD), renders the unhatched juvenile more susceptible to desiccation (Perry, 1983), probably because the juvenile relies on the physical protection afforded by the eggshell and the trehalose content of the egg fluid for survival, and the initial stages of the hatching sequence involves the removal or alteration of these factors (Clarke, Perry & Hennessy, 1978). Therefore, we have also examined the survival of unhatched juveniles exposed to temperatures below freezing after treatment with PRD. MATERIALS AND METHODS Cysts of G. rostochiensis Ro 1, grown on potato cv. Arran Banner in pot cultures, were from a single generation harvested in 1982 and then stored at room temperature (20°C) for 18 months. They were soaked for 1 week at 20°C in artificial tap water (ATW) (Greenaway, 1970). Secondstage juveniles were obtained by exposing the cysts to PRD obtained by the method of Fenwick (1949) and diluted with ATW one in four by volume; only those juveniles which had hatched within the previous 48 h were used for experimentation. Free eggs were obtained from soaked cysts by cutting the cysts open with a scalpel. Approximately 100 free juveniles and 100-200 eggs were used for each low temperature trial. The design and operation of the thermoelectric microscope stage used for survival experiments and the determination of supercooling points have been described (Wharton & Rowland, 1984). The microscope stage, cooled by a Peltier module in conjunction with a refrigerated circulator, allows specimens to be observed as they are cooled
0 Crown Copyright 1985. 441
442
R. N.
PERRY
I.I.P.
and D. A. WHARTON
at a constant rate of 1”Clmin and the temperature at which individuaIs freeze (su~rcooling point) can be observed directly. Two procedures were followed to examine the effects of freezing on hatched juveniles and free eggs with and without previous exposure to root diffusate. In the first series of experiments juveniles and eggs were not in contact with water. A suspension of hatched juveniles was rinsed in ATW and transferred to a small square of cellulose acetate sheeting. ATW was removed using slivers of filter paper which prevented coiling and clumping of the nematodes (Perry, 19770). Immediately all surface water had been removed, liquid paraffin was added to cover the nematodes; this medium prevents evaporation and ensures that the nematode water content remains constant (Perry, 1977b).The acetate square was mounted 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 0°C before the control unit was switched in to give a cooling rate of 1“C min-l down to a set temperature within the range -5°C to -40°C. Where possible the supercooling point was determined by observing the temperature at which the nematodes became opaque (Wharton et al., 1984). As soon as the desired low temperature had been reached, the cooling unit was switched off and the nematodes were allowed to return to ambient temperature. The blackness of nematodes which had frozen reaches a maximum between -10°C and -5°C; the percentage of frozen nematodes was recorded at -5OC. Revival was assessed over the following 48 h after the nematodes had been rinsed several times and then immersed in ATW; the criterion for revival was movement of the nematode. The same procedure was followed with free eggs; rinsing with ATW may not have removed all the liquid paraffin surrounding the egg-shells which could have interfered with the action of PRD in subsequent hatch trials so juveniles were artificially hatched by rupturing the egg-shells (Ellenby & Perry, 1976) before revival assessment in PRD. For experimenis on free eggs previously exposed to PRD, eggs were immersed in PRD for 17 h, then rinsed in ATW which was removed before covering the eggs with liquid paraffin. A 17-h exposure to PRD is sufficient to initiate the hatching sequence but not long enough for hatching to occur (Perry & Beane, 1982). In the second series of experiments, with juveniles and eggs in contact with water, the cooling chamber was filled with a suspension of nematodes or eggs. Experiments were carried out as above and the temperature at which water froze was noted. After the experimental run, eggs were immersed in PRD for 10 days and the percentage hatch recorded. Experiments were also carried out on batches of 25-30 cysts; after the freezing/warming cycle cysts were set to hatch in PRD for 5 weeks, counts of hatched juveniles being taken weekly when fresh PRD was added. Total hatch as a percentage of cyst contents was determined at the end of each test.
RESULTS Figure 1 shows the percentage frozen and the percentage recovery of free juveniles exposed to subzero temperatures when not in contact with water. The mean supercooling point of ten juveniles was -29S”C with a range of -25.0 to -33=3”C.
20
0
;
o___“___o_--o__-o___d I 0
I -10
I
-20
,’ a’
VOL.
1985
i-20
!_
,’
L \
I -30
15.
La
l
t -40
-lo
Fru. 1. The percentage recovery (#) and percentage frozen (0) of hatched G. rostocniensis J2 after exposure to subzero temperatures when not in contact with water. S is the range of observed supercooling points. However, because of the size of G. rostochiensis juveniles, it was not always easy to determine the supercooling point. The percentage of nematodes frozen, as indicated by an increase in opacity during warming (Fig. 2), increased from 0% at -25°C to
100% at -40°C and this coincides with a marked decrease in the percentage recovery. The effect of low temperature on the freezing and recovery of unhatched juveniles is similar whether the eggs were previously exposed to PRD (Fig. 3) or not (Fig. 4). Unhatched juveniles freeze at temperatures beiow -30°C after treatment with PRD and below -35°C when there was no PRD pre-treatment. Probit analysis (Finney, 1970) of the results for percentage recovery in the three groups shows that the lines do not have significantly different slopes, so a comparison of the temperature at which 50% of the nematodes were killed ( TSO)is valid. The TAOfor free juveniles is -31*8”C (CL at 95% = -30.8 to -32aS”C) while for unhatched juveniles the r30 is -37.9”C (CL at 95% = -36.7 to -3&9”C) for those not exposed to PRD and -37*1”C (CL at 95% = -35.9 to -38*2’C) for
those eggs previously exposed to PRD. Thus, free juveniles not in contact with water can avoid freezing by supercooling, and survival is significantly enhanced in unhatched juveniles; prior treatment with PRD for 17 h has no significant effect on low temperature survival of unhatched juveniles. Results from experiments with free and unhatched juveniles frozen in water are given in Fig. 5. Free juveniles cannot survive freezing which occurs between -5.3 and -6,O”C; 100% revival occurred when juveniles were exposed to low temperatures above freezing but below the freezing point juveniles seem unable to prevent ice seeding across the cuticfe and do not survive freezing of their body contents. Unhatched juveniles in contact with water can survive freezing; after exposure to -WC, the percen-
I.J.P.
VOL.
15.
1985
Cold tolerance of G. rostochiensti
443 100
r
loo-
I L
50-
*go;
I 0 :
40-
t i20
20-
L
Lo
O-’
“-;o
-50
0
Temp.rat”r*
%
FIG. 3. The percentage recovery (0) and percentage frozen (0) of unhatched G. rosrochiensis 52, with previous treatment with potato root diffusate, after exposure to subzero temperatures when not in contact with water.
100
l-100
o--c
7
0”
40
I
I
-40
-30 hnp*rature
I -50
‘c
FIG. 4. The percentage recovery (0) and percentage frozen (0) of unhatched G. rostochiensis 52, with no previous treatment with potato root diffusate, after exposure to subzero temperatures when not in contact with water.
Fro. 2. Photomicrographs of G. rostochiends J2 Cj) and eggs(e) during a freeze/thaw cycle. consisting of cooling from + 20°C to -50°C and rewarming to + 2OC. The tip of the thermocouple can also be seen(t). A, unfrozen at t 20°C; B, frozen after cooling to -5O’C and rewarming to -10°C; C, melted after rewarming to +2”C.
tage hatch of eggs with and without previous treatment with PRD was 54 and 65% respectively. The percentage hatches of unhatched juveniles pretreated with PRD before exposure to 0 and -1OT are surprisingly low. This contributes to the difference in slope of the lines obtained by probit analysis of results from the two batches of unhatched juveniles and thus a comparison of T5e values would not be valid. The percentage hatch from cysts frozen in water varied considerably (Fig. 6). Although unhatched juveniles in cysts survived better at -30°C and -40°C than did unhatched juveniles in eggs isolated from cysts, the hatches at 0, -10 and -20°C did not exceed 50% of the cyst contents.
444
R. N. PERRYand D. A.
L
: ; I
i-60 ;
60
:
\.
240
;-y,\
(
20
t
‘0
;40
5
g
;I-
\
c20 L
;.___---.____---.___--__ a
oI
0
I
-10
I
-20 T*mp*rat”re
I
-30
LO I -40
I -50
‘C
FIG. 5. The percentage hatch from G. rostochiensis eggs, with (0) and without (0) previous treatment with potato root diffusate, after exposure to sub-zero temperatures when in contact with water. F is the range of temperatures at which the water in the suspension froze. - - is the percentage recovery of hatched juveniles.
100
1
1 60
5
1
FIG. 6. The percentage hatch of G. rostochiensis 52 from cysts after exposure to sub-zero temperatures when in
contact with water. Each point is the total percentage hatch after four weeks in potato root diffusate. F is the range of temperatures at which water in the cyst suspensions froze.
DISCUSSION
At temperatures below freezing nematodes are exposed to the danger of lethal ice formation in their body fluids. Hatched second-stage juveniles of G. rostochiensis in contact with water cannot survive freezing or, unlike T. colubriformis and D. dipsaci (Wharton et al., 1984), prevent inoculative freezing. When not in contact with water, juveniles can survive sub-zero temperatures by supercooling in the manner of cold-hardy insects classified as freezing-susceptible. There was a rapid decrease in the percentage recovery of juveniles in liquid paraffin after
WHARTON
I.J.P. VOL. 15. 1985
exposure to sub-zero temperatures within the range of their supercooling points; the TAO of -31.8”C is very close to the mean supercooling point of -29.5“C. Although free juveniles can avoid freezing by supercooling when not in contact with water this situation is unlikely to arise in the soil environment. The ecological importance of the egg-shell and possibly the egg-fluid surrounding the juvenile is paramount in freezing survival of this species. The results show that unhatched juveniles, whether immersed in water or liquid paraffin, can survive sub-zero temperatures. The percentage recovery declines with decrease in temperature below zero but even in contact with water at -20°C over 50% survive. The contribution of the egg-fluid to freezing survival is difficult to determine. Exposure of the eggs to PRD, with concomitant loss of trehalose from the egg-fluid, does not seem to hinder survival. The main uncertaintpconcerning the results in Figs. 3 and 5 is that a 17-h exposure to PRD may be too short for complete leakage of trehalose. Ellenby & Perry (1976) showed that, in isolated eggs, 24 h was necessary for maximum juvenile water content and, by inference, maximum trehalose leakage. Thus, the albeit diluted trehalose could still act as an antifreeze agent. However, the importance of trehalose is likely to be minimal. The mean of five determinations of the freezing point of 0.34 M trehalose-the concentration in unhatched, unstimulated eggs (Clarke et al., 1978)-was -6.4 & 0.4”C and this does not differ significantly (t-test, P > 0.01) from the mean freezing point of water determined in this work (-6.2 + Oa4”C). In contrast to free juveniles, unhatched juveniles can survive at temperatures much lower than the freezing point of water in the suspensions. It is likely that the egg-shell acts by preventing ice seeding across from the medium to the juvenile. The egg-shell has a similar protective function in desiccation survival where it slows down the rate of water loss from the unhatched juvenile and so enhances survival (Ellenby, 1968). The difference between the mechanisms for survival of desiccation and low temperature is that the egg-fluid has an important role in conjunction with the egg shell in desiccation survival; loss of trehalose and alteration of egg shell permeability on exposure to PRD renders the unhatched juveniles more susceptible to desiccation than are similar juveniles not exposed to PRD (Perry, 1983). It might be expected that the cyst wall would provide additional protection against low temperatures. The marked reduction in the percentage of juveniles hatching after exposure to -40°C compared to the hatch after exposure to -30°C agrees with the range of observed supercooling points for juveniles in liquid paraffin (-25 to -35*5’C) and the Tso for unhatched juveniles not exposed to PRD (-37el”C). However, hatches at 0, -10 and -20°C are much lower than those from isolated eggs at these temperatures. We cannot yet explain this. Possible changes in the physiology of unhatched juveniles, associated
I*I.Px
VQL.
15. 1985
Cold toterance of G. rostochiensis
with exposure to few tern~rat~e~ whi& delay or alter their respouse to the hatching stimulus would be un~jke~~ to affect euqsted eggs but not eggs removed from cysts. However, this does not detract from the main Gonclusion that, if unhatch~, G. ~o~~~~~e~~~ secand-stage juveniles can survive exposure to subzero temperatures; in this state juveniles are freezing-susceptible (Salt, 1961) and avoid freezing by supercooling. The ability to survive temperatures below freezing in t.he field may be an important factor in the virtually world-wide distribution of this parasite. The importance of the cyst as au ecological unit (Ellenby, 194.6)is an accepted concept. The ~ontr~b~t~o~ of the egg-shell to rhe survival of the unhatched juvenile and its central role in the hat~m~ mech~~sm fPerry & Chtrke, 1981j make the egg an equally ~rn~ort~t b~o~o~~~a~ unit. Any novel control programmes which aim to batch G ~~~o~~~e~~ juveniles in the absence of the host plant can be formulated in the knowledge that free juveniles are susceptible to environmental extremes. Acknowledgements-We thank Mr. T. Dixon for the statistical analyses and Mr. R. Moore far technical assistance. R. N. Perry thanks Professor J. Barrett for provision of laboratory facilities to carry out this work.
ALYMFME. I%% Frost-resistance in a nematode ~~~~~~~ c&&&s r~tzema-bag. Low ~~perat~re Scierwe BXX 51-62. CLARKEA. f,, PERRYR. N. L HEXNESSY J. t978. osmatif stress and the hatching of OIaaodera rosiocftie&s. Netnatotogica21: 343-392. &,EENBVC. 1946. Ecology of the eelworm cyst. Nature 157: 302-303. EI*LENBV C. 1968. Desiccation survival in the plant parasitic nematodes, Heterodera rostochiensis Wollenweber and Ditytenchus dipsuci (Kiihn) Filipjev. Proceedings of the Royaf Society B169: 203-213.
445
Esmuw C. 84 Pmim R. N. 1976. The influence of &he hr&bing factor on the water uptake of the second stage hiTa of the pot&o cyst nematode, ~e~~~~~ rclstrt~fti~f&~ ~o~r~~4 o~~~~~rne~~~~ ~~o~o~ 64: 141-147. Evarrs K=& STME A, R. 1977. A review of the d~st~butio~ and biology of the potato cyst-nematodes G~o~~e~ rostochiensis and G. paitida. Pest Articfes and News Summaries 23: 178-189. FENWICKD. W. 1949. Investigations on the emergence af larvae from cysts of the potato root eelworm Heteradera rostochiensis. I. Techniques and variability. Journa! of Heimintholagy 23: 1S7-170. FINNEYD. J. 1970. Probit Anaiysis. Cambridge University Press, Cambridge. GREENAWAY P. 1970. Sudium regulation in the freshwater moIlnsc Lintnrrea ~#~~~~i~(L.) (Gastropoda : Puimonitt;n).J~urna~of~~r~rnanta~~~o~o~ 53: $47~153. PEalzY R. N. 197%. Desiccatian survival of larvaf zmd adult stages of the plant parasitic nerzx&des* ~#~~C~ d&s& and L). rn~~~i~~~~g~s. Par~~to~ogy74: t 3% f 48. PERRY R. N. t971b, The: water dynamics of stages of Mtyfenchus dipsaci and D. myCe&ophagus during desiccation and rebydration. Parasitology 75: 45-70. PERRYR. N. 1983. The effect of potato root diffusate on the desiccation survival of unhatched juveniles of Globodwa rostochiensb Revue de Nematologie 6: 99-102. PERRYR. N. & BBANEJ. 1982. The effect of brief exposures to potato root diffusate on the hatching of Globdera FOSfOCh~enS&, ~evtie de Nemufologie % 2&Z%. PERKY R. N. & C~.ARKE A. 3. i9gl. Hatching mechanisms of nematodes. ~~r~~f~~~~ 83: 435-449.
SALT R. W. f969. Principles of insect ~~d-h~~~s~ Arm&& Review of~~famo~~gy Q: 55-74. %YRE R. M. J964. ~~~~-~~~~~§ in nematodes. f. Effects of rapid freezing on the eggs and larvae of ~e~o~~~~e incognita and M. hupia. Nemutofogica IO: 16%179. WHARTOND. A, & RMVMND J. J. 1984. A thermoelectric microscope stage for the measurement of the supercooling points of microscopic organisms. Journal of Microscopy X43: 299-305. WHARTOND. A., You~a S. R. & BARRETTJ. 1984. Cold tolerance in nematodes. Journal of Comparative PhysiokIgy 3154: 73-77.
(Received 24 J&y 1984) Abstract-PERRY R. N. and WHARTOND. A. 1985. Cold tolerance of hatched and unhatched second stage juveniles of the potato cyst-nematode, Gioboderu rostoch~e~~. I~ter~ationai ~oar~al for Parasitoiogy 15: 441-445. Hatched second-stage juveniles of Gtoboderarostochiensiscan survive sub-zero temperatures by supercooling when not in contact with water. When frozen in water free juveniles cannot survive ice seeding across the cuticle and concomitant freezing of their body contents. Unhatched juveniles can survive in water, probably because the egg-shell protects the juvenile from ice seeding across from the medium; in this state juveniles survive by supercooling. INDEX KEY WORDS: Nematode; cold tolerance; supercooling; egg-shell; Globoderu rostochiensis. INTRODUCTION IN MANY REGIONSof the world with extremely low
temperatures for several months of the year poikilothermic species have developed survival mechanisms. Cold-hardy arthropods that can survive temperatures below the freezing point of their body fluids fall into two main groups, termed freezing-tolerant and freezing-susceptible (Salt, 1969). Species in the first group can survive ice formation in their tissues while freezing-susceptible species die if frozen and therefore must avoid freezing by the ability to supercool. Sayre (1964) suggested that nematodes able to survive freezing may have either of these mechanisms. Wharton, Young & Barrett (1984) showed that thirdstage juveniles of Trichostrongylus colubriformis, fourth-stage juveniles of Ditylenchus disptzciand Punagrellus silusae adults are freezing-susceptible, their mean supercooling points being -3O”C, -21.FC and -20.7”C respectively. Asahina (1959) suggested that A~heiencho~de~ ~~t~e~ahosi can survive extracellular ice formation. Globoderu rostochiensti has spread to almost all parts of the world (Evans & Stone, 1977) including many where winter soil temperatures are below freezing. The indigenous distribution of potato cystnematode is in the high Andean region where frost can occur for a substantial number of days in a year. Therefore, G. rostochiensk must have evolved cold protection mechanisms. This paper examines (a) the ability of hatched second-stage juveniles to survive freezing, and (b) the effect of the egg-shell independently and in conjunction with the cyst wall on low
temperature survival. Exposure to the natural hatching stimulus, potato root diffusate (PRD), renders the unhatched juvenile more susceptible to desiccation (Perry, 1983), probably because the juvenile relies on the physical protection afforded by the eggshell and the trehalose content of the egg fluid for survival, and the initial stages of the hatching sequence involves the removal or alteration of these factors (Clarke, Perry & Hennessy, 1978). Therefore, we have also examined the survival of unhatched juveniles exposed to temperatures below freezing after treatment with PRD. MATERIALS AND METHODS Cysts of G. rostochiensis Ro 1, grown on potato cv. Arran Banner in pot cultures, were from a single generation harvested in 1982 and then stored at room temperature (20°C) for 18 months. They were soaked for 1 week at 20°C in artificial tap water (ATW) (Greenaway, 1970). Secondstage juveniles were obtained by exposing the cysts to PRD obtained by the method of Fenwick (1949) and diluted with ATW one in four by volume; only those juveniles which had hatched within the previous 48 h were used for experimentation. Free eggs were obtained from soaked cysts by cutting the cysts open with a scalpel. Approximately 100 free juveniles and 100-200 eggs were used for each low temperature trial. The design and operation of the thermoelectric microscope stage used for survival experiments and the determination of supercooling points have been described (Wharton & Rowland, 1984). The microscope stage, cooled by a Peltier module in conjunction with a refrigerated circulator, allows specimens to be observed as they are cooled
0 Crown Copyright 1985. 441
442
R. N.
PERRY
I.I.P.
and D. A. WHARTON
at a constant rate of 1”Clmin and the temperature at which individuaIs freeze (su~rcooling point) can be observed directly. Two procedures were followed to examine the effects of freezing on hatched juveniles and free eggs with and without previous exposure to root diffusate. In the first series of experiments juveniles and eggs were not in contact with water. A suspension of hatched juveniles was rinsed in ATW and transferred to a small square of cellulose acetate sheeting. ATW was removed using slivers of filter paper which prevented coiling and clumping of the nematodes (Perry, 19770). Immediately all surface water had been removed, liquid paraffin was added to cover the nematodes; this medium prevents evaporation and ensures that the nematode water content remains constant (Perry, 1977b).The acetate square was mounted 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 0°C before the control unit was switched in to give a cooling rate of 1“C min-l down to a set temperature within the range -5°C to -40°C. Where possible the supercooling point was determined by observing the temperature at which the nematodes became opaque (Wharton et al., 1984). As soon as the desired low temperature had been reached, the cooling unit was switched off and the nematodes were allowed to return to ambient temperature. The blackness of nematodes which had frozen reaches a maximum between -10°C and -5°C; the percentage of frozen nematodes was recorded at -5OC. Revival was assessed over the following 48 h after the nematodes had been rinsed several times and then immersed in ATW; the criterion for revival was movement of the nematode. The same procedure was followed with free eggs; rinsing with ATW may not have removed all the liquid paraffin surrounding the egg-shells which could have interfered with the action of PRD in subsequent hatch trials so juveniles were artificially hatched by rupturing the egg-shells (Ellenby & Perry, 1976) before revival assessment in PRD. For experimenis on free eggs previously exposed to PRD, eggs were immersed in PRD for 17 h, then rinsed in ATW which was removed before covering the eggs with liquid paraffin. A 17-h exposure to PRD is sufficient to initiate the hatching sequence but not long enough for hatching to occur (Perry & Beane, 1982). In the second series of experiments, with juveniles and eggs in contact with water, the cooling chamber was filled with a suspension of nematodes or eggs. Experiments were carried out as above and the temperature at which water froze was noted. After the experimental run, eggs were immersed in PRD for 10 days and the percentage hatch recorded. Experiments were also carried out on batches of 25-30 cysts; after the freezing/warming cycle cysts were set to hatch in PRD for 5 weeks, counts of hatched juveniles being taken weekly when fresh PRD was added. Total hatch as a percentage of cyst contents was determined at the end of each test.
RESULTS Figure 1 shows the percentage frozen and the percentage recovery of free juveniles exposed to subzero temperatures when not in contact with water. The mean supercooling point of ten juveniles was -29S”C with a range of -25.0 to -33=3”C.
20
0
;
o___“___o_--o__-o___d I 0
I -10
I
-20
,’ a’
VOL.
1985
i-20
!_
,’
L \
I -30
15.
La
l
t -40
-lo
Fru. 1. The percentage recovery (#) and percentage frozen (0) of hatched G. rostocniensis J2 after exposure to subzero temperatures when not in contact with water. S is the range of observed supercooling points. However, because of the size of G. rostochiensis juveniles, it was not always easy to determine the supercooling point. The percentage of nematodes frozen, as indicated by an increase in opacity during warming (Fig. 2), increased from 0% at -25°C to
100% at -40°C and this coincides with a marked decrease in the percentage recovery. The effect of low temperature on the freezing and recovery of unhatched juveniles is similar whether the eggs were previously exposed to PRD (Fig. 3) or not (Fig. 4). Unhatched juveniles freeze at temperatures beiow -30°C after treatment with PRD and below -35°C when there was no PRD pre-treatment. Probit analysis (Finney, 1970) of the results for percentage recovery in the three groups shows that the lines do not have significantly different slopes, so a comparison of the temperature at which 50% of the nematodes were killed ( TSO)is valid. The TAOfor free juveniles is -31*8”C (CL at 95% = -30.8 to -32aS”C) while for unhatched juveniles the r30 is -37.9”C (CL at 95% = -36.7 to -3&9”C) for those not exposed to PRD and -37*1”C (CL at 95% = -35.9 to -38*2’C) for
those eggs previously exposed to PRD. Thus, free juveniles not in contact with water can avoid freezing by supercooling, and survival is significantly enhanced in unhatched juveniles; prior treatment with PRD for 17 h has no significant effect on low temperature survival of unhatched juveniles. Results from experiments with free and unhatched juveniles frozen in water are given in Fig. 5. Free juveniles cannot survive freezing which occurs between -5.3 and -6,O”C; 100% revival occurred when juveniles were exposed to low temperatures above freezing but below the freezing point juveniles seem unable to prevent ice seeding across the cuticfe and do not survive freezing of their body contents. Unhatched juveniles in contact with water can survive freezing; after exposure to -WC, the percen-
I.J.P.
VOL.
15.
1985
Cold tolerance of G. rostochiensti
443 100
r
loo-
I L
50-
*go;
I 0 :
40-
t i20
20-
L
Lo
O-’
“-;o
-50
0
Temp.rat”r*
%
FIG. 3. The percentage recovery (0) and percentage frozen (0) of unhatched G. rosrochiensis 52, with previous treatment with potato root diffusate, after exposure to subzero temperatures when not in contact with water.
100
l-100
o--c
7
0”
40
I
I
-40
-30 hnp*rature
I -50
‘c
FIG. 4. The percentage recovery (0) and percentage frozen (0) of unhatched G. rostochiensis 52, with no previous treatment with potato root diffusate, after exposure to subzero temperatures when not in contact with water.
Fro. 2. Photomicrographs of G. rostochiends J2 Cj) and eggs(e) during a freeze/thaw cycle. consisting of cooling from + 20°C to -50°C and rewarming to + 2OC. The tip of the thermocouple can also be seen(t). A, unfrozen at t 20°C; B, frozen after cooling to -5O’C and rewarming to -10°C; C, melted after rewarming to +2”C.
tage hatch of eggs with and without previous treatment with PRD was 54 and 65% respectively. The percentage hatches of unhatched juveniles pretreated with PRD before exposure to 0 and -1OT are surprisingly low. This contributes to the difference in slope of the lines obtained by probit analysis of results from the two batches of unhatched juveniles and thus a comparison of T5e values would not be valid. The percentage hatch from cysts frozen in water varied considerably (Fig. 6). Although unhatched juveniles in cysts survived better at -30°C and -40°C than did unhatched juveniles in eggs isolated from cysts, the hatches at 0, -10 and -20°C did not exceed 50% of the cyst contents.
444
R. N. PERRYand D. A.
L
: ; I
i-60 ;
60
:
\.
240
;-y,\
(
20
t
‘0
;40
5
g
;I-
\
c20 L
;.___---.____---.___--__ a
oI
0
I
-10
I
-20 T*mp*rat”re
I
-30
LO I -40
I -50
‘C
FIG. 5. The percentage hatch from G. rostochiensis eggs, with (0) and without (0) previous treatment with potato root diffusate, after exposure to sub-zero temperatures when in contact with water. F is the range of temperatures at which the water in the suspension froze. - - is the percentage recovery of hatched juveniles.
100
1
1 60
5
1
FIG. 6. The percentage hatch of G. rostochiensis 52 from cysts after exposure to sub-zero temperatures when in
contact with water. Each point is the total percentage hatch after four weeks in potato root diffusate. F is the range of temperatures at which water in the cyst suspensions froze.
DISCUSSION
At temperatures below freezing nematodes are exposed to the danger of lethal ice formation in their body fluids. Hatched second-stage juveniles of G. rostochiensis in contact with water cannot survive freezing or, unlike T. colubriformis and D. dipsaci (Wharton et al., 1984), prevent inoculative freezing. When not in contact with water, juveniles can survive sub-zero temperatures by supercooling in the manner of cold-hardy insects classified as freezing-susceptible. There was a rapid decrease in the percentage recovery of juveniles in liquid paraffin after
WHARTON
I.J.P. VOL. 15. 1985
exposure to sub-zero temperatures within the range of their supercooling points; the TAO of -31.8”C is very close to the mean supercooling point of -29.5“C. Although free juveniles can avoid freezing by supercooling when not in contact with water this situation is unlikely to arise in the soil environment. The ecological importance of the egg-shell and possibly the egg-fluid surrounding the juvenile is paramount in freezing survival of this species. The results show that unhatched juveniles, whether immersed in water or liquid paraffin, can survive sub-zero temperatures. The percentage recovery declines with decrease in temperature below zero but even in contact with water at -20°C over 50% survive. The contribution of the egg-fluid to freezing survival is difficult to determine. Exposure of the eggs to PRD, with concomitant loss of trehalose from the egg-fluid, does not seem to hinder survival. The main uncertaintpconcerning the results in Figs. 3 and 5 is that a 17-h exposure to PRD may be too short for complete leakage of trehalose. Ellenby & Perry (1976) showed that, in isolated eggs, 24 h was necessary for maximum juvenile water content and, by inference, maximum trehalose leakage. Thus, the albeit diluted trehalose could still act as an antifreeze agent. However, the importance of trehalose is likely to be minimal. The mean of five determinations of the freezing point of 0.34 M trehalose-the concentration in unhatched, unstimulated eggs (Clarke et al., 1978)-was -6.4 & 0.4”C and this does not differ significantly (t-test, P > 0.01) from the mean freezing point of water determined in this work (-6.2 + Oa4”C). In contrast to free juveniles, unhatched juveniles can survive at temperatures much lower than the freezing point of water in the suspensions. It is likely that the egg-shell acts by preventing ice seeding across from the medium to the juvenile. The egg-shell has a similar protective function in desiccation survival where it slows down the rate of water loss from the unhatched juvenile and so enhances survival (Ellenby, 1968). The difference between the mechanisms for survival of desiccation and low temperature is that the egg-fluid has an important role in conjunction with the egg shell in desiccation survival; loss of trehalose and alteration of egg shell permeability on exposure to PRD renders the unhatched juveniles more susceptible to desiccation than are similar juveniles not exposed to PRD (Perry, 1983). It might be expected that the cyst wall would provide additional protection against low temperatures. The marked reduction in the percentage of juveniles hatching after exposure to -40°C compared to the hatch after exposure to -30°C agrees with the range of observed supercooling points for juveniles in liquid paraffin (-25 to -35*5’C) and the Tso for unhatched juveniles not exposed to PRD (-37el”C). However, hatches at 0, -10 and -20°C are much lower than those from isolated eggs at these temperatures. We cannot yet explain this. Possible changes in the physiology of unhatched juveniles, associated
I*I.Px
VQL.
15. 1985
Cold toterance of G. rostochiensis
with exposure to few tern~rat~e~ whi& delay or alter their respouse to the hatching stimulus would be un~jke~~ to affect euqsted eggs but not eggs removed from cysts. However, this does not detract from the main Gonclusion that, if unhatch~, G. ~o~~~~~e~~~ secand-stage juveniles can survive exposure to subzero temperatures; in this state juveniles are freezing-susceptible (Salt, 1961) and avoid freezing by supercooling. The ability to survive temperatures below freezing in t.he field may be an important factor in the virtually world-wide distribution of this parasite. The importance of the cyst as au ecological unit (Ellenby, 194.6)is an accepted concept. The ~ontr~b~t~o~ of the egg-shell to rhe survival of the unhatched juvenile and its central role in the hat~m~ mech~~sm fPerry & Chtrke, 1981j make the egg an equally ~rn~ort~t b~o~o~~~a~ unit. Any novel control programmes which aim to batch G ~~~o~~~e~~ juveniles in the absence of the host plant can be formulated in the knowledge that free juveniles are susceptible to environmental extremes. Acknowledgements-We thank Mr. T. Dixon for the statistical analyses and Mr. R. Moore far technical assistance. R. N. Perry thanks Professor J. Barrett for provision of laboratory facilities to carry out this work.
ALYMFME. I%% Frost-resistance in a nematode ~~~~~~~ c&&&s r~tzema-bag. Low ~~perat~re Scierwe BXX 51-62. CLARKEA. f,, PERRYR. N. L HEXNESSY J. t978. osmatif stress and the hatching of OIaaodera rosiocftie&s. Netnatotogica21: 343-392. &,EENBVC. 1946. Ecology of the eelworm cyst. Nature 157: 302-303. EI*LENBV C. 1968. Desiccation survival in the plant parasitic nematodes, Heterodera rostochiensis Wollenweber and Ditytenchus dipsuci (Kiihn) Filipjev. Proceedings of the Royaf Society B169: 203-213.
445
Esmuw C. 84 Pmim R. N. 1976. The influence of &he hr&bing factor on the water uptake of the second stage hiTa of the pot&o cyst nematode, ~e~~~~~ rclstrt~fti~f&~ ~o~r~~4 o~~~~~rne~~~~ ~~o~o~ 64: 141-147. Evarrs K=& STME A, R. 1977. A review of the d~st~butio~ and biology of the potato cyst-nematodes G~o~~e~ rostochiensis and G. paitida. Pest Articfes and News Summaries 23: 178-189. FENWICKD. W. 1949. Investigations on the emergence af larvae from cysts of the potato root eelworm Heteradera rostochiensis. I. Techniques and variability. Journa! of Heimintholagy 23: 1S7-170. FINNEYD. J. 1970. Probit Anaiysis. Cambridge University Press, Cambridge. GREENAWAY P. 1970. Sudium regulation in the freshwater moIlnsc Lintnrrea ~#~~~~i~(L.) (Gastropoda : Puimonitt;n).J~urna~of~~r~rnanta~~~o~o~ 53: $47~153. PEalzY R. N. 197%. Desiccatian survival of larvaf zmd adult stages of the plant parasitic nerzx&des* ~#~~C~ d&s& and L). rn~~~i~~~~g~s. Par~~to~ogy74: t 3% f 48. PERRY R. N. t971b, The: water dynamics of stages of Mtyfenchus dipsaci and D. myCe&ophagus during desiccation and rebydration. Parasitology 75: 45-70. PERRYR. N. 1983. The effect of potato root diffusate on the desiccation survival of unhatched juveniles of Globodwa rostochiensb Revue de Nematologie 6: 99-102. PERRYR. N. & BBANEJ. 1982. The effect of brief exposures to potato root diffusate on the hatching of Globdera FOSfOCh~enS&, ~evtie de Nemufologie % 2&Z%. PERKY R. N. & C~.ARKE A. 3. i9gl. Hatching mechanisms of nematodes. ~~r~~f~~~~ 83: 435-449.
SALT R. W. f969. Principles of insect ~~d-h~~~s~ Arm&& Review of~~famo~~gy Q: 55-74. %YRE R. M. J964. ~~~~-~~~~~§ in nematodes. f. Effects of rapid freezing on the eggs and larvae of ~e~o~~~~e incognita and M. hupia. Nemutofogica IO: 16%179. WHARTOND. A, & RMVMND J. J. 1984. A thermoelectric microscope stage for the measurement of the supercooling points of microscopic organisms. Journal of Microscopy X43: 299-305. WHARTOND. A., You~a S. R. & BARRETTJ. 1984. Cold tolerance in nematodes. Journal of Comparative PhysiokIgy 3154: 73-77.