J. therm. Biol. Vol. 23, No. 2, pp. 75±80, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0306-4565/98 $19.00 + 0.00
SURVIVAL OF STEINERNEMATID NEMATODES EXPOSED TO FREEZING I. M. BROWN1* and R. GAUGLER1 Department of Entomology, Rutgers University, New Brunswick, NJ 08903-0231, U.S.A.
(Received 15 March 1998; accepted in revised form 4 May 1998) AbstractÐ1. Entomopathogenic nematodes Steinernema riobravis, S. carpocapsae and S. glaseri survived prolonged exposure to freezing at ÿ48C. LT50 values were 2.1, 1.8 and 0.6 days respectively. 2. Steinernema riobravis and S. carpocapsae were still pathogenic after 6 days of freezing and S. glaseri showed poor pathogenicity over 4 days of freezing at ÿ48C. 3. Incubation in 20% glycerol for 48 h prior to freezing at ÿ208C enhanced survival and pathogenicity of S. carpocapsae. Survival increased to 30 days; nematodes were still pathogenic after 24 days of exposure. # 1998 Elsevier Science Ltd. All rights reserved
Key Word Index: Nematode; entomopathogenic; steinernema; cryopreservation; cryobiology; glycerol; freezing injury; prolonged exposure
posures at low temperatures in insects and also found a scarcity of time-temperature studies. The majority of entomopathogenic nematode infective juveniles (Rhabditida: Steinernema and Heterorhabditis) are freezing tolerant and freeze when the surrounding environment freezes (Brown and Gaugler, 1996; Brown, unpublished results). Schmiege (1963) reported that 70% of Steinernema sp. infective juveniles survived freezing for 18 h at ÿ108C. Only Heterorhabditis zealandica has been found to be freeze avoiding (Wharton and Surrey, 1994). Acclimation to lower temperatures prior to freezing has been shown to enhance freezing survival of both Heterorhabditis bacteriophora and Steinernema feltiae (Brown and Gaugler, 1996). Entomopathogenic nematodes can be eective biological control agents of insect pests (Bedding et al., 1993; Georgis and Manweiler, 1994). Brie¯y, the nematode lifecycle occurs within the insect cadaver. When resources are depleted third stage juveniles develop into nonfeeding infective juveniles (dauer juvenile). The infective juveniles migrate from the cadaver and ®nd another insect to infect. Once inside a new host, the infective juveniles release a symbiotic bacteria which kills the insect and begins a new lifecycle. The infection process is a critical step in the nematode lifecycle and is easily compromized by a reduction in overall infective juvenile ®tness. Therefore, pathogenicity provides
Nematodes from a wide variety of habitats survive freezing conditions. These include the Antarctic (Wharton and Block, 1993), temperate pastures and soils (Wharton and Allan, 1989) and even parasite hosts (Tyrrell et al., 1994). Nematodes surviving freezing utilize two major strategies: freezing tolerance and freeze avoidance. Most freezing tolerant nematodes freeze as exogenous ice nucleation penetrates the cuticle or body ori®ces when the surrounding water freezes (Wharton, 1995). On thawing these nematodes continue their normal lifecycle. Freeze avoiding species possess a barrier (i.e. sheath, eggshell or cyst wall) which prevents inoculative freezing, allowing the nematode to avoid freezing by supercooling it's body ¯uids below the ¯uid's freezing point (Wharton, 1995; Wharton and Allan, 1989). Freezing occurs at the supercooling point and is lethal. Prolonged freezing is the eect of time on the physiology and biochemistry of the frozen organism. Brown (1994), Wharton and Brown (1991) and Brown and Gaugler (1996) have examined the eect of prolonged freezing conditions in nematodes. Sùmme (1996) reviewed the eect of prolonged ex* To whom correspondence should be addressed. Tel.: (732) 932-9459; Fax: (732) 932-7229; E-mail: [email protected]
I. M. Brown and R. Gaugler
an eective index of overall ®tness and can be used to assess freezing injury. The lack of a long-term storage method limits entomopathogenic nematode commercialization. To this end several cryopreservation studies (Curran et al., 1992; Smith et al., 1990; Popiel and Vasquez, 1989) and patents (Popiel et al., 1988) have assessed the potential of cryopreservation as a long-term storage method. Entomopathogenic nematode strain collections are already routinely stored in liquid nitrogen for long periods (Curran et al., 1992). However, liquid nitrogen is only feasible for the storage of small stock cultures and not for large commercial quantities of nematodes. This study is an eort to combine cryobiology with cryopreservation (i.e. the enhancement of existing natural physiological and biochemical mechanisms by arti®cial cryopreservation techniques). The impact of prolonged freezing on nematode ®tness was assessed using the nematode's ability to cause host mortality (pathogenicity) and establish itself in the host (infectivity) as the ®tness index. The ability of the cryoprotectant, glycerol to enhance ®tness was then investigated. Understanding the cryobiology of entomopathogenic nematodes may lead to the development of novel and inexpensive cryopreservation techniques that enhance the natural freezing tolerance of these nematodes at higher temperatures (i.e. ÿ208C).
MATERIALS AND METHODS
Nematode survival The duration that Steinernema riobravis, S. carpocapsae (all strain) and S. glaseri (NC strain) could survive freezing at ÿ48C was determined using the method described in Brown and Gaugler (1996). Brie¯y, approximately 5000 infective juveniles in 2 ml of tap water were allowed to cool passively from 208C to ÿ48C in an incubator set at ÿ48C. Freezing was standardized by triggering freezing at ÿ0.58C by the addition of small ice crystals with a spatula. After 12 h, and every 24 h afterwards six replicates were removed and thawed passively at 258C. The cooling and warming regime, while not directly controlled, was replicated accurately. Survival was counted 24 h after thawing.
by vacuum suction. The moistened discs were placed in 50 mm Petri dishes and 10 active, last instars of the greater wax moth, Galleria mellonella, were added. After 72 h incubation at 258C, G. mellonella mortality was assessed. Cadavers were dissected to check for nematode infection (Woodring and Kaya, 1988). Cryopreservation Steinernema carpocapsae infective juveniles were incubated in 20% glycerol (w/w) for 48 h at 258C. Samples of 5000 infective juveniles in 5 ml of glycerol solution were placed in a freezer at ÿ208C and allowed to cool passively. The freezing event was standardised in the same way as above. After 24 h and every 48 h afterwards six replicates were removed and thawed passively at 258C. Survival was assessed 24 h later. Pathogenicity was assessed in the same way as above. Control nematodes were frozen in water at ÿ208C. Infectivity Infectivity studies were carried out on Steinernema carpocapsae infective juveniles incubated in 20% glycerol w/w for 48 h before being exposed to freezing at ÿ208C. Infectivity was de®ned as the ability of infective juveniles to successfully establish an infection in a host insect. Infective juveniles were frozen at ÿ208C as above. Control nematodes were held in distilled water at 258C. After 1, 10 and 20 days nematodes were thawed and survival counted 24 h later. Infectivity was determined using the method developed by Caroli et al., (1996). Brie¯y, 200 infective juveniles were applied to each of 10 G. mellonella larvae in 10 mm well plates lined with ®lter paper. After incubation at 258C for 48 h the larvae were digested in pepsin and the nematodes counted. Statistics The survival time for 50% of the infective juveniles exposed to freezing at ÿ48C (ST50) and the time to reduce nematode pathogenicity to 50% (LT50) were both determined by probit analysis (SAS, 1987). The infectivity studies carried out on S.carpocapsae were analysed by ANOVA. The level of signi®cance used was a = 0.05 (SAS, 1987).
Nematode pathogenicity The impact of prolonged freezing at ÿ48C on the infective juvenile pathogenicity was determined using nematodes from the survival experiment above. For each replicate, all nematodes were separated from the water onto 50 mm ®lter paper discs
Nematode survival Infective juveniles of Steinernema riobravis, S. carpocapsae and S. glaseri showed decreased survival with time exposed to freezing at ÿ48C (Fig. 1).
Survival of steinernematid nematodes exposed to freezing
Fig. 2. Pathogenicity of steinernematid infective juveniles on G. mellonella larvae after prolonged exposure to freezing in water at ÿ48C. Nematode pathogenicity was measured as % host mortality. SE = standard error of the mean. N = 10. Fig. 1. Survival of steinernematid infective juveniles exposed to freezing at ÿ48C in water. Freezing was initiated at ÿ0.58C. SE = standard error of the mean. N = 6.
The ST50 was 2.06 2 0.14 days for S. riobravis, 1.80 2 0.19 days for S. carpocapsae and 0.62 2 0.03 days for S. glaseri. Steinernema riobravis and S. carpocapsae infective juveniles survived up to 19 and 15 days respectively at ÿ48C, whereas S. glaseri were all dead after 3 days. Survivors of all species frequently exhibited sluggish behavior and extensive vacuolization, suggesting sublethal freezing injury. Freezing injury in dead nematodes ranged from gross internal distortion to body rupturing and evisceration. Host mortality All three species showed decreased pathogenicity with time exposed to freezing conditions at ÿ48C (Fig. 2). The LT50 was 3.09 2 0.11 days for S. riobravis, 0.53 2 0.13 days for S. carpocapsae and 0.38 2 0.08 days for S. glaseri. Cryopreservation Cryopreservation in 20% glycerol increased the time period which nematodes could withstand freezing conditions, whereas all nematodes died in water at ÿ208C. The survival time for 50% of S. carpocapsae infective juveniles was 10.52 2 0.19 days (Fig. 3(A)). All control nematodes were dead at 12 h. Exposure to freezing reduced pathogenicity to 50% after 8.85 2 0.32 days (Fig. 3(B)).
Infectivity The infective juveniles that were exposed to freezing at ÿ208C had no signi®cant eect on infectivity (Fig. 4). Freezing signi®cantly (p < 0.05) reduced nematode infectivity on day 10 and 20 but not day 1. Infectivity for both frozen and control declined signi®cantly between day 1 and day 10 (p < 0.01) but not between day 10 and day 20. DISCUSSION
Steinernema riobravis and S. carpocapsae survived freezing at ÿ48C for at least twice as long as all other entomopathogenic nematodes species tested to date (Brown and Gaugler, 1996). S. glaseri survival however was comparable to that of other entomopathogenic nematode infective juveniles. H. bacteriophora, S. feltiae and S. anomali all showed 95% mortality after 3 days at ÿ48C (Brown and Gaugler, 1996). We calculated the LT50 values to be 0.51, 0.76 and 0.62 days for S. feltiae, S. anomali and H. bacteriophora respectively. Extensive prolonged freezing survival has been reported in the Antarctic species Panagrolaimus davidi which survived more than 150 days encased in ice at ÿ808C (Brown, 1994; Wharton and Brown, 1991). Freezing survival decreases with decreasing temperature and increasing exposure. Freezing tolerant species survive freezing without injury above their lower lethal temperature. Below this temperature, mortality increases with time. The alpine carabid beetle Pelophila borealis survived freezing at ÿ38C
I. M. Brown and R. Gaugler
Fig. 4. Infectivity of Steinernema carpocapsae after prolonged freezing at ÿ208C, q Unfrozen control maintained at 258C in water. Q Frozen at ÿ208C in 20% glycerol. Freezing was initiated at ÿ0.58C. SE = standard error of the mean. N = 10).
Fig. 3. The in¯uence of the cryoprotectant glycerol on Steinernema carpocapsae infective juveniles. (A) Survival after exposure to prolonged freezing conditions at ÿ208C and (B) pathogenicity after prolonged exposure to freezing conditions at ÿ208C. Infective juveniles were incubated in 20% glycerol at 258C for 48 h prior to freezing at ÿ208C. SE = standard error of the mean. N = 6.
for >64 days, whereas only 5% survived 48 days at ÿ58C and all died in 10 min at ÿ108C (Sùmme, 1996). All entomopathogenic nematodes (Brown and Gaugler, 1996) and P. davidi (Wharton and Brown, 1991) also demonstrated a decrease in survival with increasing exposure. In addition, all freezing tolerant nematode species tested so far have shown some freezing mortality at all temperatures examined (Sayre, 1964; Wharton and Allan, 1989; Wharton and Brown, 1991; Wharton and Block, 1993; Tyrrell et al., 1994). However, since most of these species are known to survive freezing for long periods, some of the freezing mortality is probably due to the use of sub-optimal freeze/thaw regimes (all papers cited above used a 18C/min cooling and warming regime) and not indicative of a species poor freezing tolerance ability. For example, P. davidi is capable of 51% survival after 276 days encased in ice at ÿ808C and yet only 60% of nematodes acclimated under the same conditions survived freezing at ÿ58C for one minute after cooling at 18C/min (Brown, 1994). Environmental factors such as the presence or absence of water, the thermal history and culture conditions of the nematode are also known to be important in¯uences on both short and long-term freezing survival (Wharton and Brown, 1991; Brown and Gaugler, 1996; Surrey, 1996).
Freezing injury results from the disruption of physiological and biochemical processes (Asahina, 1969; Muldrew and McGann, 1994; Storey and Storey, 1988), reducing the overall ®tness in the thawed animal. In nematodes freezing injuries range from sluggish behavior to small cuticular ruptures and severe rupturing (Haight et al., 1975; Brown, unpublished observations). Freezing damage to cell membranes has been quanti®ed by measuring the leakage of primary amines (O'Dell and Crowe, 1979). Microscopy has been used to assess the degree of granular appearance, vacuole formation and organ damage from thawed nematodes (Smith et al., 1990; Bosher and McKeen, 1954). We observed increasing sluggish behavior and vacuolization with increasing freezing exposure time in surviving infective juveniles of all species examined. Further work is required to quantify the relationship between microscopical observations, freezing injury and survival in these three steinernematid nematode species. The ability of infective juveniles to kill their host (pathogenicity) and establish within the host insect (infectivity) is easily compromised by any factor which reduces infective juvenile ®tness. Our results show that prolonged exposure to freezing at both ÿ48C and ÿ208C reduces infective juvenile pathogenicity and infectivity, indicating that freezing injury had occurred. However, the relationship between freezing and infectivity may only give an estimate of ®tness reduction caused by freezing injury. Caroli et al., (1996) found that at most 45% of the inoculum of four steinernematid species were found in cadavers. They suggest that steinernematid populations may only contain a proportion of individuals that are infective and the rest are quiescent
Survival of steinernematid nematodes exposed to freezing
or enter the cadaver after the initial infection. Futhermore, only one nematode is required to kill a Galleria (Miller, 1989), therefore high infection levels may occur even when a large proportion of the population has severe freezing injury. The incubation and freezing of S. carpocapsae in 20% glycerol greatly enhanced freezing survival and infectivity. Nematodes frozen in water at ÿ48C survived 15 days, however they could not kill insects after 6 days freezing. Whereas in glycerol at ÿ208C, survival increased from 15 to at least 30 days and nematodes could still kill insects after at least 24 days at ÿ208C. No glycerol experiments were carried out at ÿ48C because 20% glycerol solutions do not freeze at ÿ48C. Glycerol is widely used as a cryoprotectant because it is relatively nontoxic and its low molecular weight enables it to penetrate cell membranes. It is thought to prevent freezing injury by reducing cell membrane shrinkage and expansion as osmotic forces alter the cell water volume during freezing and thawing thereby preventing membrane damage (Pegg, 1987). However, the cuticles of most nematode species are relatively impermeable. Glycerol, therefore, probably functions as a nonpenetrating cryoprotectant by removing body water osmotically. Smith et al. (1990) observed shrinking of infective juveniles in glycerol. Several workers have developed cryopreservation methods for long term storage of entomopathogenic nematodes. Popiel and Vasquez (1989) successfully cryopreserved S. carpocapsae and Heterorhabditis bacteriophora using a two-step glycerol and methanol procedure prior to rapid emersion and storage in liquid nitrogen. Curran et al. (1992) modi®ed Popiel and Vasquez's method to store 167 isolates of S. carpocapsae, S. feltiae, S. glaseri and H. bacteriophora. Nugent et al. (1996) developed and optimised methods to store a wide range of heterorhabditid species. Smith et al. (1990) conducted an extensive series of tests on cryoprotectant toxicity and freezing regimes for S. feltiae ®rst stage and infective juveniles. Only infective juveniles appeared to have been successfully cryopreserved, however no long term survival data is given. Our infectivity results for the unfrozen controls and the samples frozen at ÿ208C for 24 h are comparable to the S. carpocapsae infection rates obtained by Caroli et al. (1996), suggesting that little or no further freezing damage has occurred after 24 h at ÿ208C. Whereas, after 10 and 20 days at ÿ208C infectivity was reduced to two infective juveniles per insect, indicating that substantial freezing damage must have occurred. The infectivity data contained large variations between insects and is probably the reason for there being no signi®cant
eect of time on infectivity between 1 and 10 days. Large variations in freezing survival data are common as freezing injuries are usually numerous and diverse in nature. Further work to determine whether survivors suered sub-lethal freezing injury and the nature of the damage is required. The above studies have all preincubated infective juveniles in a cryoprotectant (usually glycerol or dimethyl sulphoxide) prior to immersion and storage in liquid nitrogen at ÿ1968C. We have investigated the possibility of storing nematodes at much higher freezing temperatures than ÿ1968C. The aim is to develop a less expensive method which will store bulk nematodes at temperatures higher than ÿ1968C. Riga and Webster (1991) report that Caenorhabditis elegans is routinely stored in 15% glycerol at ÿ708C using a modi®ed Brenner (1974) technique. S. riobravis and S. carpocapsae infective juvenile freezing survival have shown potential for the development of a storage method at higher freezing temperatures such as ÿ208C, the temperature of a domestic freezer. Furthermore the substantial increase in S. carpocapsae survival and maintenance of pathogenicity by a simple cryopreservation procedure is even more promising. Future eorts in the cryopreservation of S. carpocapsae and S. riobravis maybe more successful if the focus is on the understanding and arti®cial enhancement of the natural freezing tolerance strategies of these nematodes. AcknowledgementsÐThis is New Jersey Agricultural Experiment Station Publication No. D. 08256-05-98 supported by state funds and the U.S. Hatch Act.
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