- Email: [email protected]
Genetic improvement of the desiccation tolerance of the entomopathogenic nematode Heterorhabditis bacteriophora through selective breeding
Biological Control 31 (2004) 218–226 www.elsevier.com/locate/ybcon
Genetic improvement of the desiccation tolerance of the entomopathogenic nematode Heterorhabditis bacteriophora through selective breeding O. Strauch, J. Oestergaard, S. Hollmer, and R.-U. Ehlers* Department for Biotechnology and Biological Control, Institute for Phytopathology, Christian-Albrechts-University Kiel, Raisdorf 24223, Germany Received 6 January 2004; accepted 12 March 2004 Available online 6 May 2004
Abstract The commercial exploitation of the entomopathogenic nematode Heterorhabditis bacteriophora in biological control is limited due to its relatively short shelf life, that is related to its low tolerance of environmental extremes such as desiccation. Desiccation is a result of evaporation at low humidity or hypertonic osmotic conditions. For storage and transportation infective dauer juveniles (DJs) are mixed with clay minerals. To maintain DJ quality their metabolism is reduced by transfer of the DJs into a quiescent state, which is induced by moderate desiccation. If the nematodes were more resistant to desiccation stress, they could be further desiccated to prolong DJ survival and shelf life of nematode-based products. The reduction of the nematode metabolism and the survival in the quiescent stage could be enhanced by an increased resistance to desiccation stress. The objective of this investigation was to determine the genetic variability of the desiccation tolerance of H. bacteriophora and to exploit this variability for an enhancement of desiccation tolerance by breeding. A hybrid strain resulting from crosses of eight H. bacteriophora isolates from different geographic origins was used in our investigation. The desiccation stress was adjusted by hygroscopic polyethyleneglycol (PEG 600) solutions. By lowering the water activity (aw -value) of this solution, the removal of water from the DJs is enhanced. The influence of an adaptation phase on the desiccation tolerance was investigated. The lowest mean tolerated aw -value (0.85) was achieved with an adaptation phase of 72 h at an aw -value of 0.96. The variance of the desiccation tolerance increased with the reduction of the aw -value during adaptation. The heritability of the trait, determined by using homozygous inbred lines, was 0.46 for non-adapted and 0.48 for adapted populations. A negative heterosis effect could be observed for the desiccation tolerance because nearly all of the inbred lines had a higher tolerance to the desiccation stress than the hybrid strain. Improvement of the desiccation tolerance by breeding was only obtained when the adaptation process was included in the selection process, which was related to a higher phenotypic variance in the populations after adaptation. A total of eight selection and breeding steps were carried out. Without previous adaptation, the mean of the tolerated aw -value remained almost constant between 0.94 and 0.93. In contrast, when we adapted the DJs prior to the exposure to desiccation stress, the tolerable aw -values continuously dropped from 0.89 to 0.81. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Biocontrol; Desiccation tolerance; Genetic improvement; Heritability; Heterorhabditis bacteriophora; Heterosis; Selective breeding; Stress adaptation
1. Introduction The entomopathogenic nematode Heterorhabditis bacteriophora Poinar is a safe biological control agent (Ehlers, 2003), mainly used to control white grubs in turf (Ehlers and Peters, 1998) and weevil larvae in orna* Corresponding author. Fax: +49-4307-839834. E-mail address: [email protected] (R.-U. Ehlers).
1049-9644/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2004.03.009
mentals and strawberries (Cowles et al., in press; van Tol et al., in press). Like other rhabditid nematodes, H. bacteriophora forms developmentally arrested, nonfeeding third-stage dauer juveniles (DJs). The DJs are resistant to shear stress and can therefore be applied with conventional spray technology. For commercial use, H. bacteriophora is produced in monoxenic liquid culture in bioreactors together with their symbiotic bacterium Photorhabdus luminescens Thomas and
O. Strauch et al. / Biological Control 31 (2004) 218–226
Poinar (Ehlers, 2001). At the end of the production process, nematodes are separated from the spent medium and the bacteria by continuous centrifugation. For storage and transportation to the users, high densities of DJs are mixed with different clay minerals and packed in plastic bags. The survival in these formulations is limited to only a few weeks and is much reduced at room temperature (Grewal, 2000; Strauch et al., 2000). The poor shelf life of nematode products is the major limiting factor preventing a more wide-spread commercial exploitation of these biocontrol agents. Longevity of nematodes can be enhanced by the transfer of the DJs into a quiescent state, in which they use less energy and are more resistant to environmental extremes. The quiescence can be induced by moderate desiccation, which in the case of H. bacteriophora transfers this nematode into a weak anhydrobiotic state (Glazer, 2002). However, the desiccation tolerance of Heterorhabditis spp. is not well developed (Glazer, 1996; Liu and Glazer, 2000). Consequently, the availability of nematode strains with a higher desiccation tolerance would increase the ability for prolonged storage and enhance the quality of commercial nematode products. Moderate desiccation of nematodes induces the production of trehalose and glycerol that can replace water in biological membranes (Solomon et al., 1999; Womersley, 1990). The production of protective heat shock proteins was reported as well for the entomopathogenic nematode, Steinernema feltiae (Filipjev) (Solomon et al., 2000). They can stabilize the cell membranes in case of water loss and enhance the desiccation tolerance. These physiological adaptations can also be induced by transfer of nematodes into hypertonic non-ionic solutions (Charwat et al., 2002; Glazer and Salame, 2000). The dehydrating conditions in non-ionic solutions are described by the water activity (aw -value). Different responses of nematodes in hypertonic non-ionic or ionic solutions were discussed by Glazer and Salame (2000) and Piggott et al. (2002). Beside the desiccation stress in concentrated ionic solutions, toxic effect can occur by overtaxing the ionic regulation mechanisms. In this study, the non-ionic polymer polyethyleneglycol 600 was used to produce desiccation stress, which is comparable to conditions produced at lower relative humidity, but not to osmotic stress produced in ionic solutions. The objective of our investigation was to evaluate the genetic potential for an improvement of the desiccation tolerance of H. bacteriophora. There are several reasons to select this nematode species for our investigation. It has a world-wide distribution (Hominick, 2002) and its reproduction is through amphimictic as well as through automictic adults (Johnigk and Ehlers, 1999a,b) making it an excellent candidate for cross- and in-breeding. The possibility to produce offspring alternatively by self- or cross-fertilization is a prerequisite for a breeding program utilizing inbred lines.
219
The desiccation tolerance is a quantitative trait. The phenotypes are influenced by several genes and environmental factors. In such cases, the breeding success depends on the proportion of the genetically caused variance of the phenotypes, which is called the heritability (h2 ) of a trait (Falconer, 1984). The heritability of the desiccation tolerance has been investigated by Glazer et al. (1991) for H. bacteriophora strain HP88, originating from Utah, USA. For this strain, a low heritability of the desiccation tolerance (h2 ¼ 0:11) was reported. This investigation, however, did not consider the possibility to enhance the desiccation tolerance by adaptation to desiccation stress. Attempts to improve the desiccation tolerance should consider an adaptation phase prior to exposure to extreme desiccation stress as additional genes are involved in the selection process and this may consequently increase the success of a breeding program. In our investigation, the heritability and the progress during selective breeding were compared for the desiccation tolerance of adapted and nonadapted DJ of H. bacteriophora.
2. Materials and methods 2.1. Establishment of hybrid strains and inbred lines of H. bacteriophora The basis of the production of homozygous inbred lines was a hybrid strain (PS7), a pool of seven different isolates of H. bacteriophora from Europe and USA, following the method described by Johnigk et al. (2002). Prior to the breeding experiments, strain H06 originating from China and provided by Richou Han (Guangdong Entomological Institute, Guangzhou) was crossed with PS7 to further enlarge the genetic pool. For each strain (PS7 or H06), 12 Galleria mellonella (L.) larvae were treated with 50 DJs/insect. After 6 days at 25 °C, males and premature females were collected from the insect cadaver. Each of 50 males of one nematode strain was cultured together on agar plates with one premature female of the other strain (according to Iraki et al., 2000). The new DJ generation that had developed 14 days later, were pooled and 10,000 DJs were mixed with the three-fold number from the PS7 strain, in order to adjust the proportion of the H06 alleles in the final population to 1:8. With this hybrid strain, 96 G. mellonella were treated with 50 DJs/insect. The emerging DJs from the insect cadavers were the foundation strain PS8 used for selective breeding. 2.2. Separation of active and inactive dauer juveniles After exposure to the desiccation stress, live nematodes were separated from dead individuals using a sieving method. The bottom of 2 ml Eppendorf tubes
220
O. Strauch et al. / Biological Control 31 (2004) 218–226
were cut and replaced by 50-lm mesh sieves. On top of the sieve, a 5-mm cotton wool plug was placed. The cotton wool prevented immobile nematodes from passing through the sieve. The sieves were put into tubes of 8-mm diameter, filled with Ringer’s solution. Nematodes were pipetted on top of the cotton wool and the tubes were left at 25 °C. After 3 h, 98% (2%) of the active nematodes had accumulated on the bottom of the test tube. This method was used for batches up to 500 individuals for the optimization of the adaptation phase (see Section 2.3). For samples of several thousand nematodes, the separation method was unreliable as DJs became inactivated after application on the sieve probably due to the lack of oxygen and clumping of the DJs on top of the cotton wool layer preventing migration. Therefore, a water trap was developed for the separation of dead and alive nematodes. DJs were pipetted on a filter paper (18 18 mm), which was then placed on another filter paper layer to remove excess water. The nematodes accumulated in the middle of the filter paper which was placed on an aluminum platform (18 18 4 mm) into a petri dish of 3.5-cm diameter filled with 2 ml Ringer’s solution. Living DJs moved around randomly and after some time ended up in the Ringer’s solution where they were trapped. After 24 h at 25 °C, 97–100% of the DJs were found on the bottom of the petri dish when 500 100 DJs/trap were used (n ¼ 20) and 95–99% when 5000–10,000 DJs/trap were used (n ¼ 8). 2.3. Optimization of the adaptation phase To adapt nematodes to desiccation conditions, DJs (PS7) were transferred into a hygroscopic solution of polyethyleneglycol 600 (PEG: HOCH2 –CH2 –(O–CH2 – CH2 )ðn1Þ –OH). PEG 600 is a clear, viscose, and nontoxic, and non-ionic liquid (Beyer and Walter, 1981), which can be used to produce solutions with a wide range of different water activities (aw -values) by mixing with water. The water activity is defined as the relative proportion of unbound water in a sample. It was measured by determination of the relative humidity above the PEG suspension at a defined temperature (Aqua Lab CX-2, Decagon Devices, Pullman, WA). As the aw of a suspension is lowered, the greater amount of water is removed from the nematodes. To determine the most effective intensity and duration for the adaptation phase, dose–effect relationships were established. DJ batches of 1000 ml1 were exposed to aw -values of 0.995 (Ringer’s solution), 0.98, 0.97, and 0.96 for 72, 96, and 120 h at 25 °C. After the adaptation phase, the DJs were exposed to lower water activities (10 different aw -values between 0.96 and 0.75). DJ samples of 400 ll were transferred onto 10-lm mesh sieves and the suspension was removed by vacuum suction. The DJs were washed off the sieves with 1 ml of a PEG so-
lution with a lower aw -value. Samples were then transferred to 24-cell-well dishes and kept at 25 °C. After 24 h, the PEG solutions were replaced by tap water. Surviving DJs were determined as described above after a rehydration phase of 24 h at 25 °C. All treatments were carried out with one nematode batch at the same time. 2.4. Determination of the desiccation tolerance of inbred lines The desiccation tolerance of the inbred lines was determined for adapted and non-adapted DJs of the PS7 strain. The inbred lines were propagated in G. mellonella larvae prior to the experiments. Forty-eight insect larvae were infected with 20 DJs each and after 14 days the emerging DJs were pooled into one population. This was done to reduce the effect of batch to batch variability. During adaptation, the nematodes were kept for 72 h at 25 °C in a PEG solution with a water activity of 0.96. After adaptation, batches of 500 100 DJs were exposed to aw -values of 1, 0.94, 0.90, 0.84, or 0.76 for 24 h at 25 °C. Non-adapted DJs were exposed to aw values of 1, 0.95, 0.93, 0.91, or 0.86. Rehydration was in Ringer’s solution for 24 h at 25 °C and the determination of the percentage of active DJs was carried out as described above. 2.5. Selection process Genetic selection for desiccation tolerance was carried out with adapted and non-adapted DJ starting with the PS8 strain. During adaptation, the nematodes were kept for 72 h at 25 °C in a PEG solution of aw ¼ 0.96. After adaptation, batches of 10,000 3000 DJs were exposed for 24 h at 25 °C to 10 different aw -values between 1 and 0.7. Non-adapted DJs were exposed to aw -values between 1 and 0.88. Thereafter, the DJs were re-hydrated in Ringer’s solution for another 24 h at the same temperature. The determination of the percentage of active DJs was carried out as described above. The DJs from the treatment with a water activity that gave 5–10% survival (about 1000 individuals) were used for production of new progeny in last instar G. mellonella. Up to 50 insect larvae were infected with 20 DJs each in moist sand (10% water w/w) at 25 °C, and after about 14 days the emerging offspring from the cadavers were pooled and used for the next selection step carried out under the same conditions. This was done to reduce the effect of batch to batch variability. In total, eight selection steps were carried out. 2.6. Evaluation of data To calculate a mean desiccation tolerance and its variance, a cumulative normal distribution (Fig. 1) was fitted to the original data (aw -value and related per-
O. Strauch et al. / Biological Control 31 (2004) 218–226
221
Fig. 1. Data obtained after the first selection step of the desiccation stress adapted population of the H. bacteriophora hybrid strain PS8. Percentage active nematodes after exposure to different water activity values (aw ) and cumulative normal distribution, used to calculate the median of the tolerated aw -values. The selection pressure of 10% represents those individuals with the highest tolerance to desiccation stress, which were used for the consecutive breeding step. The expected selection response (R) is the product of the heritability h2 (0.46) and xs xb .
centage of active DJs). The fitting was carried out by minimizing the v2 for testing the original data versus the theoretical normal distribution. The regression coefficient between the original data and the fitted normal distribution was always higher than 0.9. The mean and standard deviation of the fitted normal distribution were used as an estimation of the median and standard deviation of the desiccation tolerance in the examined population. To detect significant differences between treatments used to define the optimal adaptation procedure and to compare the inbred lines, the Student’s t test was used considering unequal variances. Since the variance of the desiccation tolerance was not independent from the mean value, ANOVA could not be applied. The estimation of the heritability (h2 ¼ r2g =ðr2g þ 2 re Þ; r2g is the genetically caused variance, r2e is the environmentally caused variance) was carried out as described before (Johnigk et al., 2002). It can be assumed that the inbred lines originated from the strain PS7 were homozygous (Johnigk et al., 2002). Therefore, the observed variability of the desiccation tolerance in one line is a result of environmentally caused variability (r2e ). In contrast, differences in the mean tolerance between the inbred lines can be assumed as genetically caused (r2g ). The variability within and between the inbred lines was calculated based on the mean of the standard deviation in each line, respectively, the standard deviation of the mean values. To monitor the progress in breeding success, the obtained mean tolerated water activity was compared
with the expected success, which is defined as the reduction of the tolerated water activity in the offspring of the selected population. This success depends on the heritability, the variability of the trait, and the selection pressure. The variability of the trait is represented by the standard deviation in the basic population. The selection pressure is defined by the proportion of the population selected for further breeding (Fig. 1). The standard deviation of the trait and the proportion of the selected population define the difference between the median of the desiccation tolerance in the basic (xb ) and in the selected population (xs ). The median instead of the mean value was used here, since the desiccation tolerance in the selected population is not normally distributed. The difference xb xs represents the maximal possible selection success in the next generation, which is restricted by the heritability. It follows that the expected selection response (R) can be calculated as follows: R ¼ h2 ðxs xb Þ (Falconer, 1984). This value was compared with the obtained success.
3. Results 3.1. Influence of the adaptation phase on the desiccation tolerance The mean tolerated water activity could be lowered significantly by decreasing aw -values during the adaptation phase. The lowest mean tolerated aw -value (0.85) was achieved with an adaptation phase of 72 h at an aw -
222
O. Strauch et al. / Biological Control 31 (2004) 218–226
value of 0.96 (Fig. 2). A significant difference of the desiccation tolerance after an adaptation phase at aw values of 0.97 and 0.96 was not detected (Fig. 2). Therefore, a further increase in tolerance can probably not be achieved by lowering the aw beyond 0.96 during the adaptation phase. From our data, we cannot conclude whether a shorter exposition to an aw -value of 0.96 can increase the tolerance against lower water activities. Prolonging the adaptation phase decreased the tolerance to desiccation stress. The weakest desiccation tolerance was obtained after exposure to Ringer’s solution (aw ¼ 0.995). The variability of the desiccation tolerance
increased with decreasing aw -values during adaptation (Fig. 3). 3.2. Heritability of desiccation tolerance The mean tolerated aw -values ranged from 0.96 to 0.91 (PS7 ¼ 0.94) for non-adapted nematode inbred lines (Fig. 4A) and from 0.86 to 0.77 (PS7 ¼ 0.86) for adapted populations (Fig. 4B). The correlation coefficient for the tolerated water activity between the adapted and nonadapted populations was 0.18. The calculated heritability for the desiccation tolerance of non-adapted
Fig. 2. Influence of the water activity (aw ) and duration of exposure to desiccation stress during the adaptation phase on the mean tolerated water activity measured during the following exposure to different polyethyleneglycol (PEG 600) suspensions with variable aw -values. The tolerated mean aw -values increase significantly with increasing aw -values during the adaptation period until a value of 0.97. Prolonging the adaptation phase can significantly reduce the desiccation tolerance. Different letters at data points indicate significant differences (Student’s t test considering unequal variances, a 6 0:05).
Fig. 3. Relation of standard deviation of the tolerated aw -values and the intensity of the desiccation stress and duration of the adaptation period. The variability within the H. bacteriophora population increases with increasing desiccation stress during the adaptation phase. The duration of the exposure has no significant influence on the variability.
O. Strauch et al. / Biological Control 31 (2004) 218–226
223
Fig. 4. Mean tolerated water activity (aw ) of inbred lines and the foundation strain PS7. Error bars on the columns indicate standard deviation. The deviation is low for non-adapted H. bacteriophora inbred line populations (A) and higher in stress adapted populations (B). Different letters above the bars indicate significant differences between the mean values (Student’s t test considering unequal variances, a 6 0:05). The heritability h2 ¼ r2g =ðr2g þ r2e Þ, where r2g is the genetically caused variance and r2e is the environmentally caused variance.
H. bacteriophora was 0.46 (95% confidence limit: 0.35– 0.58) and 0.48 (95% confidence limit: 0.30–0.69) for the tolerance of adapted populations. Thus, the adaptation process had no influence on the heritability of the desiccation tolerance. Nearly all of the inbred lines had a higher tolerance to desiccation stress than the hybrid strain PS7 from which inbred lines had been produced (Figs. 4A and B). 3.3. Improvement of desiccation tolerance Whereas the desiccation tolerance of non-adapted nematodes could not be improved by selective breeding, the tolerance of adapted H. bacteriophora was responsive to this approach (Fig. 5). A total of eight selection
and breeding steps were carried out. Without previous adaptation, the mean of the tolerated aw -value remained almost constant between 0.94 and 0.93. In contrast, when adapting DJs prior to the exposure to desiccation stress, a breeding success was achieved. Tolerated aw values continuously dropped from 0.89 to 0.81 in the offspring of the selected DJs after the first three selection step. However, during the following steps, instead of an increase in desiccation tolerance, the populations became less resistant or the tolerance remained almost constant at aw between 0.82 and 0.81. The obtained selection success was less than the expected tolerance (Fig. 5). In most cases, however, the expected values lie inside the 95% confidence intervals of the obtained values. A significant difference between the
224
O. Strauch et al. / Biological Control 31 (2004) 218–226
Fig. 5. Recorded and expected mean tolerated water activity (aw ) of adapted and non-adapted populations of H. bacteriophora at subsequent selection steps. Error bars represent the 95% confidence interval.
obtained and expected success occurred after the fourth selection step for adapted populations when no further success was recorded.
4. Discussion This is the first reported attempt to improve the desiccation tolerance of an entomopathogenic nematode through genetic selection. The study has shown that selective breeding is a feasible approach to increase the tolerance of the nematodes towards desiccation stress only if DJs are stress-adapted prior to exposure to desiccation conditions. Adaptation resulted in an increased variability of the desiccation tolerance within a population (Fig. 3). The variability increased with the intensity of the stress during the adaptation phase. Is this an effect of the genetic variability in the genes, which have an influence on the adaptation process or the result of the variable response of the different phenotypes to the environmental conditions? Is the increasing variability genetically or environmentally caused or both? The genetically caused proportion (h2 ) on the phenotypic variation of the desiccation tolerance was not different in the adapted and the non-adapted populations. We can conclude that the increased variability was not only genetically caused, otherwise the heritability of the desiccation tolerance should be higher for the adapted populations. Three factors influence the breeding success, the heritability, the selection pressure, and the variability within the population. As the first two are almost the same for adapted and non-adapted populations, the breeding success was determined mainly by the high variability of the adapted population. The most conspicuous result obtained during the evaluation of the tolerance of the inbred lines is that nearly all of the inbred lines were more tolerant than the hybrid strain PS7, from which inbred lines had been
produced (Fig. 4). Similar results were obtained by Glazer et al. (1991) who showed that the mean desiccation tolerance of most of the inbred lines produced from HP88 was higher than in the original population. This effect might represent a heterosis effect. Heterosis effects are usually described as positive effects occurring in the progeny of crosses of homozygous individuals carrying different alleles. In our case, the heterosis effect reduced the desiccation tolerance of the hybrid. The genetic background (dominance and epistasis) of so called negative or positive heterosis effects are not necessarily different (Brewbaker, 1967; Falconer, 1984; Fischer, 1978). So far we can only assume that genes involved in the tolerance to desiccation stress might not be working synergistically and that negative effects are overcome through selection of inbred lines. Whether we were able to further improve the desiccation tolerance by combining different inbred lines and exclude those with antagonistic effects would be worth investigating, particularly as the mean tolerated water activity of the most successful inbred line D4 (0.77) is much below the tolerance obtained through selection starting from the hybrid strain (0.81 in the third selection step). Including the adaptation to desiccation stress in the breeding program reduced the mean tolerated aw -value from 0.89 to 0.81. These observations fit well with the expected breeding success for adapted and non-adapted populations (Fig. 5). However, expected values were always a little lower than the obtained values for the mean tolerated aw . For the calculation of the expected selection response R the estimated values of h2 was used and we assumed that h2 remained constant during the breeding process. This assumption is wrong in the case of a successful selection process. In this case, the genetic variance in the populations is reduced, whereas the influence of the environmentally caused variability remains constant. Consequently, h2 will decrease to zero at the end of the breeding process (Falconer, 1984). Since the decline of h2 could not previously be
O. Strauch et al. / Biological Control 31 (2004) 218–226
calculated, it could not be included in the calculation for the expected success. Therefore, the calculated R is an overestimation from the second selection step onwards. Another reason for an overestimation of R could be the fact that the selected populations produce two generations inside the insect before the DJs emerged. Thus, the offspring used for the next selection step was a result of self- and cross-fertilization (Johnigk and Ehlers, 1999a,b) without a selection pressure. Finally the heritability was estimated by using PS7 and the selection process was carried out with PS8. PS8 might have a higher heritability due to a enhanced genetic variability, but this is not proven so far. The low desiccation tolerance of H. bacteriophora and resulting short shelf-life of nematode products are the major factors preventing a more widespread use of nematodes in biological control (Georgis, 2002). In order to overcome this situation, other approaches than selective breeding might be feasible. Genes have been characterized also from nematodes which can increase desiccation tolerance (Browne et al., 2002; Gal et al., 2003; Solomon et al., 2000). Genetic transformation of nematodes might be a more promising way to reach the goal. Cross-breeding attempts should include isolates of H. bacteriophora from relatively warm and dry regions like the Middle East (Glazer et al., 1993; Iraki et al., 2000) or Turkey (Susurluk et al., 2001). Mutagenesis can also be used to improve desiccation tolerance (Burnell, 2002). Other beneficial traits need to be characterized and improved inbred lines or products of selective breeding should be crossed into commercial strains already selected for enhanced performance in liquid culture (Johnigk et al., 2002). Such programs would have the advantage of yielding methods to monitor the genetic stability of commercially used populations and thus could contribute to improve the quality and shelflife of nematode-based products in the future.
Acknowledgments We gratefully acknowledge the financial support of the Deutsche Forschungsgemeinschaft (Project Eh 116/ 4-1) and thank Manfred H€ uhn (Institute for Plant Production and Plant Breeding, University Kiel) for his comments on the results.
References Beyer, H., Walter, W., 1981. Lehrbuch der organischen Chemie. Hirzel Verlag, Stuttgart. Brewbaker, J.L., 1967. Angewandte Genetik. Gustav Fischer Verlag, Jena. Browne, J., Tunnacliffe, A., Burnell, A., 2002. Plant desiccation gene found in nematode. Nature 416, 38.
225
Burnell, A., 2002. Genetics and genetic improvement. In: Gaugler, R. (Ed.), Entomopathogenic Nematology. CABI Publishing, Oxon, UK, pp. 241–263. Charwat, S.M., Fischer, J.M., Wyss, U., 2002. The effect of osmotic stress on desiccation survival and water content of four nematode species. Nematology 4, 89–97. Cowles, R.S., Polavarapu, S., Ehlers, R.-U., Williams, R.N., in press. Management of insect pests in small fruit crops with nematodes. In: Grewal, P., Ehlers, R.-U., Shapiro-Ilan, D. (Eds.), Nematodes as Biological Control Agents. CABI Publishing, Oxon, UK. Ehlers, R.-U., 2001. Mass production of entomopathogenic nematodes for plant protection. Appl. Microbiol. Biotechnol. 56, 623–633. Ehlers, R.-U., 2003. Biocontrol nematodes. In: Hokkanen, H.M.T., Hajek, A.E. (Eds.), Environmental Impacts of Microbial Insecticides. Kluwer Academic Publishers, Dordrecht, NL, pp. 177–220. Ehlers, R.-U., Peters, A., 1998. Bek€ampfung von Engerlingen auf Sportrasen. Rasen/Turf/Gazon 29, 60–67. Falconer, D.S., 1984. Quantitative Genetics. Hirzel Verlag, Stuttgart. Fischer, H.E., 1978. Heterosis. Gustav Fischer Verlag, Jena. Gal, T.Z., Glazer, I., Koltai, H., 2003. Differential gene expression during desiccation stress in the insect-killing nematode Steinernema feltiae IS-6. J. Parasitol. 89, 761–766. Georgis, R., 2002. The Biosys experiment: an insider’s perspective. In: Gaugler, R. (Ed.), Entomopathogenic Nematology. CABI Publishing, Oxon, UK, pp. 357–372. Glazer, I., 1996. Survival mechanisms of entomopathogenic nematodes. Biocontrol Sci. Technol. 6, 373–378. Glazer, I., 2002. Survival biology. In: Gaugler, R. (Ed.), Entomopathogenic Nematology. CABI Publishing, Oxon, UK, pp. 169–187. Glazer, I., Salame, L., 2000. Osmotic survival of the entomopathogenic nematode Steinernema carpocapse. Biol. Control 18, 251–257. Glazer, I., Gaugler, R., Segal, D., 1991. Genetics of the entomopathogenic nematode Heterorhabditis bacteriophora (strain HP88): the diversity of beneficial traits. J. Nematol. 23, 324–333. Glazer, I., Liran, N., Poinar, G.O., Smits, P.H., 1993. Identification and biological activity of newly isolated heterorhabditid populations from Israel. Fund. Appl. Nematol. 16, 467–472. Grewal, P.S., 2000. Enhanced ambient storage stability of an entomopathogenic nematode through anhydrobiosis. Pest Manag. Sci. 56, 401–406. Hominick, W.M., 2002. Biogeography. In: Gaugler, R. (Ed.), Entomopathogenic Nematology. CABI Publishing, Oxon, UK, pp. 115– 143. Iraki, N., Salah, N., Sansour, M.A., Segal, D., Glazer, I., Johnigk, S.A., Hussein, M.A., Ehlers, R.-U., 2000. Isolation and characterization of two entomopathogenic nematode strains, Heterorhabditis indica (Nematoda, Rhabditida), from the West Bank, Palestinian Territories. J. Appl. Entomol. 124, 375–380. Johnigk, S.-A., Ehlers, R.-U., 1999a. Juvenile development and life cycle of Heterorhabditis bacteriophora and H. indica (Nematoda: Heterorhabditidae). Nematology 1, 251–260. Johnigk, S.-A., Ehlers, R.-U., 1999b. Endotokia matricida in hermaphrodites of Heterorhabditis spp. and the effect of the food supply. Nematology 1, 717–726. Johnigk, S.-A., Hollmer, S., Strauch, O., Wyss, U., Ehlers, R.-U., 2002. Heritability of the liquid culture mass production potential of the entomopathogenic nematode Heterorhabditis bacteriophora. Biocontrol Sci. Technol. 12, 267–276. Liu, Q.Z., Glazer, I., 2000. Desiccation survival of entomopathogenic nematodes of the genus Heterorhabditis. Phytoparasitica 28, 331– 340. Piggott, S.J., Liu, Q.Z., Glazer, I., Wright, D.J., 2002. Does osmoregulatory behaviour in entomopathogenic nematodes predispose desiccation tolerance? Nematology 4, 483–487. Solomon, A., Paperna, I., Glazer, I., 1999. Desiccation survival of the entomopathogenic nematode Steinernema feltiae: induction of anhydrobiosis. Nematology 1, 61–68.
226
O. Strauch et al. / Biological Control 31 (2004) 218–226
Solomon, A., Salomon, R., Paperna, I., Glazer, I., 2000. Desiccation stress of entomopathogenic nematodes induces the accumulation of a novel heat-stable protein. Parasitology 121, 409–416. Strauch, O., Niemann, I., Neumann, A., Schmidt, A.J., Peters, A., Ehlers, R.-U., 2000. Storage and formulation of the entomopathogenic nematodes Heterorhabditis indica and H. bacteriophora. BioControl 45, 483–500. Susurluk, A., Dix, I., Stackebrandt, E., Strauch, O., Wyss, U., Ehlers, R.-U., 2001. Identification and ecological characterisation of three
entomopathogenic nematode–bacterium complexes from Turkey. Nematology 3, 833–841. van Tol, R., Mannion, C., Raupp, M., in press. Nursery and tree applications. In: Grewal, P., Ehlers, R.-U., Shapiro-Ilan, D. (Eds.), Nematodes as Biological Control Agents. CABI Publishing, Oxon, UK. Womersley, C.Z., 1990. Dehydration survival and anhydrobiotic potential. In: Gaugler, R., Kaya, H.K. (Eds.), Entomopathogenic Nematodes in Biological Control. CRC Press, Boca Raton, pp. 117–138.
Genetic improvement of the desiccation tolerance of the entomopathogenic nematode Heterorhabditis bacteriophora through selective breeding O. Strauch, J. Oestergaard, S. Hollmer, and R.-U. Ehlers* Department for Biotechnology and Biological Control, Institute for Phytopathology, Christian-Albrechts-University Kiel, Raisdorf 24223, Germany Received 6 January 2004; accepted 12 March 2004 Available online 6 May 2004
Abstract The commercial exploitation of the entomopathogenic nematode Heterorhabditis bacteriophora in biological control is limited due to its relatively short shelf life, that is related to its low tolerance of environmental extremes such as desiccation. Desiccation is a result of evaporation at low humidity or hypertonic osmotic conditions. For storage and transportation infective dauer juveniles (DJs) are mixed with clay minerals. To maintain DJ quality their metabolism is reduced by transfer of the DJs into a quiescent state, which is induced by moderate desiccation. If the nematodes were more resistant to desiccation stress, they could be further desiccated to prolong DJ survival and shelf life of nematode-based products. The reduction of the nematode metabolism and the survival in the quiescent stage could be enhanced by an increased resistance to desiccation stress. The objective of this investigation was to determine the genetic variability of the desiccation tolerance of H. bacteriophora and to exploit this variability for an enhancement of desiccation tolerance by breeding. A hybrid strain resulting from crosses of eight H. bacteriophora isolates from different geographic origins was used in our investigation. The desiccation stress was adjusted by hygroscopic polyethyleneglycol (PEG 600) solutions. By lowering the water activity (aw -value) of this solution, the removal of water from the DJs is enhanced. The influence of an adaptation phase on the desiccation tolerance was investigated. The lowest mean tolerated aw -value (0.85) was achieved with an adaptation phase of 72 h at an aw -value of 0.96. The variance of the desiccation tolerance increased with the reduction of the aw -value during adaptation. The heritability of the trait, determined by using homozygous inbred lines, was 0.46 for non-adapted and 0.48 for adapted populations. A negative heterosis effect could be observed for the desiccation tolerance because nearly all of the inbred lines had a higher tolerance to the desiccation stress than the hybrid strain. Improvement of the desiccation tolerance by breeding was only obtained when the adaptation process was included in the selection process, which was related to a higher phenotypic variance in the populations after adaptation. A total of eight selection and breeding steps were carried out. Without previous adaptation, the mean of the tolerated aw -value remained almost constant between 0.94 and 0.93. In contrast, when we adapted the DJs prior to the exposure to desiccation stress, the tolerable aw -values continuously dropped from 0.89 to 0.81. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Biocontrol; Desiccation tolerance; Genetic improvement; Heritability; Heterorhabditis bacteriophora; Heterosis; Selective breeding; Stress adaptation
1. Introduction The entomopathogenic nematode Heterorhabditis bacteriophora Poinar is a safe biological control agent (Ehlers, 2003), mainly used to control white grubs in turf (Ehlers and Peters, 1998) and weevil larvae in orna* Corresponding author. Fax: +49-4307-839834. E-mail address: [email protected] (R.-U. Ehlers).
1049-9644/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2004.03.009
mentals and strawberries (Cowles et al., in press; van Tol et al., in press). Like other rhabditid nematodes, H. bacteriophora forms developmentally arrested, nonfeeding third-stage dauer juveniles (DJs). The DJs are resistant to shear stress and can therefore be applied with conventional spray technology. For commercial use, H. bacteriophora is produced in monoxenic liquid culture in bioreactors together with their symbiotic bacterium Photorhabdus luminescens Thomas and
O. Strauch et al. / Biological Control 31 (2004) 218–226
Poinar (Ehlers, 2001). At the end of the production process, nematodes are separated from the spent medium and the bacteria by continuous centrifugation. For storage and transportation to the users, high densities of DJs are mixed with different clay minerals and packed in plastic bags. The survival in these formulations is limited to only a few weeks and is much reduced at room temperature (Grewal, 2000; Strauch et al., 2000). The poor shelf life of nematode products is the major limiting factor preventing a more wide-spread commercial exploitation of these biocontrol agents. Longevity of nematodes can be enhanced by the transfer of the DJs into a quiescent state, in which they use less energy and are more resistant to environmental extremes. The quiescence can be induced by moderate desiccation, which in the case of H. bacteriophora transfers this nematode into a weak anhydrobiotic state (Glazer, 2002). However, the desiccation tolerance of Heterorhabditis spp. is not well developed (Glazer, 1996; Liu and Glazer, 2000). Consequently, the availability of nematode strains with a higher desiccation tolerance would increase the ability for prolonged storage and enhance the quality of commercial nematode products. Moderate desiccation of nematodes induces the production of trehalose and glycerol that can replace water in biological membranes (Solomon et al., 1999; Womersley, 1990). The production of protective heat shock proteins was reported as well for the entomopathogenic nematode, Steinernema feltiae (Filipjev) (Solomon et al., 2000). They can stabilize the cell membranes in case of water loss and enhance the desiccation tolerance. These physiological adaptations can also be induced by transfer of nematodes into hypertonic non-ionic solutions (Charwat et al., 2002; Glazer and Salame, 2000). The dehydrating conditions in non-ionic solutions are described by the water activity (aw -value). Different responses of nematodes in hypertonic non-ionic or ionic solutions were discussed by Glazer and Salame (2000) and Piggott et al. (2002). Beside the desiccation stress in concentrated ionic solutions, toxic effect can occur by overtaxing the ionic regulation mechanisms. In this study, the non-ionic polymer polyethyleneglycol 600 was used to produce desiccation stress, which is comparable to conditions produced at lower relative humidity, but not to osmotic stress produced in ionic solutions. The objective of our investigation was to evaluate the genetic potential for an improvement of the desiccation tolerance of H. bacteriophora. There are several reasons to select this nematode species for our investigation. It has a world-wide distribution (Hominick, 2002) and its reproduction is through amphimictic as well as through automictic adults (Johnigk and Ehlers, 1999a,b) making it an excellent candidate for cross- and in-breeding. The possibility to produce offspring alternatively by self- or cross-fertilization is a prerequisite for a breeding program utilizing inbred lines.
219
The desiccation tolerance is a quantitative trait. The phenotypes are influenced by several genes and environmental factors. In such cases, the breeding success depends on the proportion of the genetically caused variance of the phenotypes, which is called the heritability (h2 ) of a trait (Falconer, 1984). The heritability of the desiccation tolerance has been investigated by Glazer et al. (1991) for H. bacteriophora strain HP88, originating from Utah, USA. For this strain, a low heritability of the desiccation tolerance (h2 ¼ 0:11) was reported. This investigation, however, did not consider the possibility to enhance the desiccation tolerance by adaptation to desiccation stress. Attempts to improve the desiccation tolerance should consider an adaptation phase prior to exposure to extreme desiccation stress as additional genes are involved in the selection process and this may consequently increase the success of a breeding program. In our investigation, the heritability and the progress during selective breeding were compared for the desiccation tolerance of adapted and nonadapted DJ of H. bacteriophora.
2. Materials and methods 2.1. Establishment of hybrid strains and inbred lines of H. bacteriophora The basis of the production of homozygous inbred lines was a hybrid strain (PS7), a pool of seven different isolates of H. bacteriophora from Europe and USA, following the method described by Johnigk et al. (2002). Prior to the breeding experiments, strain H06 originating from China and provided by Richou Han (Guangdong Entomological Institute, Guangzhou) was crossed with PS7 to further enlarge the genetic pool. For each strain (PS7 or H06), 12 Galleria mellonella (L.) larvae were treated with 50 DJs/insect. After 6 days at 25 °C, males and premature females were collected from the insect cadaver. Each of 50 males of one nematode strain was cultured together on agar plates with one premature female of the other strain (according to Iraki et al., 2000). The new DJ generation that had developed 14 days later, were pooled and 10,000 DJs were mixed with the three-fold number from the PS7 strain, in order to adjust the proportion of the H06 alleles in the final population to 1:8. With this hybrid strain, 96 G. mellonella were treated with 50 DJs/insect. The emerging DJs from the insect cadavers were the foundation strain PS8 used for selective breeding. 2.2. Separation of active and inactive dauer juveniles After exposure to the desiccation stress, live nematodes were separated from dead individuals using a sieving method. The bottom of 2 ml Eppendorf tubes
220
O. Strauch et al. / Biological Control 31 (2004) 218–226
were cut and replaced by 50-lm mesh sieves. On top of the sieve, a 5-mm cotton wool plug was placed. The cotton wool prevented immobile nematodes from passing through the sieve. The sieves were put into tubes of 8-mm diameter, filled with Ringer’s solution. Nematodes were pipetted on top of the cotton wool and the tubes were left at 25 °C. After 3 h, 98% (2%) of the active nematodes had accumulated on the bottom of the test tube. This method was used for batches up to 500 individuals for the optimization of the adaptation phase (see Section 2.3). For samples of several thousand nematodes, the separation method was unreliable as DJs became inactivated after application on the sieve probably due to the lack of oxygen and clumping of the DJs on top of the cotton wool layer preventing migration. Therefore, a water trap was developed for the separation of dead and alive nematodes. DJs were pipetted on a filter paper (18 18 mm), which was then placed on another filter paper layer to remove excess water. The nematodes accumulated in the middle of the filter paper which was placed on an aluminum platform (18 18 4 mm) into a petri dish of 3.5-cm diameter filled with 2 ml Ringer’s solution. Living DJs moved around randomly and after some time ended up in the Ringer’s solution where they were trapped. After 24 h at 25 °C, 97–100% of the DJs were found on the bottom of the petri dish when 500 100 DJs/trap were used (n ¼ 20) and 95–99% when 5000–10,000 DJs/trap were used (n ¼ 8). 2.3. Optimization of the adaptation phase To adapt nematodes to desiccation conditions, DJs (PS7) were transferred into a hygroscopic solution of polyethyleneglycol 600 (PEG: HOCH2 –CH2 –(O–CH2 – CH2 )ðn1Þ –OH). PEG 600 is a clear, viscose, and nontoxic, and non-ionic liquid (Beyer and Walter, 1981), which can be used to produce solutions with a wide range of different water activities (aw -values) by mixing with water. The water activity is defined as the relative proportion of unbound water in a sample. It was measured by determination of the relative humidity above the PEG suspension at a defined temperature (Aqua Lab CX-2, Decagon Devices, Pullman, WA). As the aw of a suspension is lowered, the greater amount of water is removed from the nematodes. To determine the most effective intensity and duration for the adaptation phase, dose–effect relationships were established. DJ batches of 1000 ml1 were exposed to aw -values of 0.995 (Ringer’s solution), 0.98, 0.97, and 0.96 for 72, 96, and 120 h at 25 °C. After the adaptation phase, the DJs were exposed to lower water activities (10 different aw -values between 0.96 and 0.75). DJ samples of 400 ll were transferred onto 10-lm mesh sieves and the suspension was removed by vacuum suction. The DJs were washed off the sieves with 1 ml of a PEG so-
lution with a lower aw -value. Samples were then transferred to 24-cell-well dishes and kept at 25 °C. After 24 h, the PEG solutions were replaced by tap water. Surviving DJs were determined as described above after a rehydration phase of 24 h at 25 °C. All treatments were carried out with one nematode batch at the same time. 2.4. Determination of the desiccation tolerance of inbred lines The desiccation tolerance of the inbred lines was determined for adapted and non-adapted DJs of the PS7 strain. The inbred lines were propagated in G. mellonella larvae prior to the experiments. Forty-eight insect larvae were infected with 20 DJs each and after 14 days the emerging DJs were pooled into one population. This was done to reduce the effect of batch to batch variability. During adaptation, the nematodes were kept for 72 h at 25 °C in a PEG solution with a water activity of 0.96. After adaptation, batches of 500 100 DJs were exposed to aw -values of 1, 0.94, 0.90, 0.84, or 0.76 for 24 h at 25 °C. Non-adapted DJs were exposed to aw values of 1, 0.95, 0.93, 0.91, or 0.86. Rehydration was in Ringer’s solution for 24 h at 25 °C and the determination of the percentage of active DJs was carried out as described above. 2.5. Selection process Genetic selection for desiccation tolerance was carried out with adapted and non-adapted DJ starting with the PS8 strain. During adaptation, the nematodes were kept for 72 h at 25 °C in a PEG solution of aw ¼ 0.96. After adaptation, batches of 10,000 3000 DJs were exposed for 24 h at 25 °C to 10 different aw -values between 1 and 0.7. Non-adapted DJs were exposed to aw -values between 1 and 0.88. Thereafter, the DJs were re-hydrated in Ringer’s solution for another 24 h at the same temperature. The determination of the percentage of active DJs was carried out as described above. The DJs from the treatment with a water activity that gave 5–10% survival (about 1000 individuals) were used for production of new progeny in last instar G. mellonella. Up to 50 insect larvae were infected with 20 DJs each in moist sand (10% water w/w) at 25 °C, and after about 14 days the emerging offspring from the cadavers were pooled and used for the next selection step carried out under the same conditions. This was done to reduce the effect of batch to batch variability. In total, eight selection steps were carried out. 2.6. Evaluation of data To calculate a mean desiccation tolerance and its variance, a cumulative normal distribution (Fig. 1) was fitted to the original data (aw -value and related per-
O. Strauch et al. / Biological Control 31 (2004) 218–226
221
Fig. 1. Data obtained after the first selection step of the desiccation stress adapted population of the H. bacteriophora hybrid strain PS8. Percentage active nematodes after exposure to different water activity values (aw ) and cumulative normal distribution, used to calculate the median of the tolerated aw -values. The selection pressure of 10% represents those individuals with the highest tolerance to desiccation stress, which were used for the consecutive breeding step. The expected selection response (R) is the product of the heritability h2 (0.46) and xs xb .
centage of active DJs). The fitting was carried out by minimizing the v2 for testing the original data versus the theoretical normal distribution. The regression coefficient between the original data and the fitted normal distribution was always higher than 0.9. The mean and standard deviation of the fitted normal distribution were used as an estimation of the median and standard deviation of the desiccation tolerance in the examined population. To detect significant differences between treatments used to define the optimal adaptation procedure and to compare the inbred lines, the Student’s t test was used considering unequal variances. Since the variance of the desiccation tolerance was not independent from the mean value, ANOVA could not be applied. The estimation of the heritability (h2 ¼ r2g =ðr2g þ 2 re Þ; r2g is the genetically caused variance, r2e is the environmentally caused variance) was carried out as described before (Johnigk et al., 2002). It can be assumed that the inbred lines originated from the strain PS7 were homozygous (Johnigk et al., 2002). Therefore, the observed variability of the desiccation tolerance in one line is a result of environmentally caused variability (r2e ). In contrast, differences in the mean tolerance between the inbred lines can be assumed as genetically caused (r2g ). The variability within and between the inbred lines was calculated based on the mean of the standard deviation in each line, respectively, the standard deviation of the mean values. To monitor the progress in breeding success, the obtained mean tolerated water activity was compared
with the expected success, which is defined as the reduction of the tolerated water activity in the offspring of the selected population. This success depends on the heritability, the variability of the trait, and the selection pressure. The variability of the trait is represented by the standard deviation in the basic population. The selection pressure is defined by the proportion of the population selected for further breeding (Fig. 1). The standard deviation of the trait and the proportion of the selected population define the difference between the median of the desiccation tolerance in the basic (xb ) and in the selected population (xs ). The median instead of the mean value was used here, since the desiccation tolerance in the selected population is not normally distributed. The difference xb xs represents the maximal possible selection success in the next generation, which is restricted by the heritability. It follows that the expected selection response (R) can be calculated as follows: R ¼ h2 ðxs xb Þ (Falconer, 1984). This value was compared with the obtained success.
3. Results 3.1. Influence of the adaptation phase on the desiccation tolerance The mean tolerated water activity could be lowered significantly by decreasing aw -values during the adaptation phase. The lowest mean tolerated aw -value (0.85) was achieved with an adaptation phase of 72 h at an aw -
222
O. Strauch et al. / Biological Control 31 (2004) 218–226
value of 0.96 (Fig. 2). A significant difference of the desiccation tolerance after an adaptation phase at aw values of 0.97 and 0.96 was not detected (Fig. 2). Therefore, a further increase in tolerance can probably not be achieved by lowering the aw beyond 0.96 during the adaptation phase. From our data, we cannot conclude whether a shorter exposition to an aw -value of 0.96 can increase the tolerance against lower water activities. Prolonging the adaptation phase decreased the tolerance to desiccation stress. The weakest desiccation tolerance was obtained after exposure to Ringer’s solution (aw ¼ 0.995). The variability of the desiccation tolerance
increased with decreasing aw -values during adaptation (Fig. 3). 3.2. Heritability of desiccation tolerance The mean tolerated aw -values ranged from 0.96 to 0.91 (PS7 ¼ 0.94) for non-adapted nematode inbred lines (Fig. 4A) and from 0.86 to 0.77 (PS7 ¼ 0.86) for adapted populations (Fig. 4B). The correlation coefficient for the tolerated water activity between the adapted and nonadapted populations was 0.18. The calculated heritability for the desiccation tolerance of non-adapted
Fig. 2. Influence of the water activity (aw ) and duration of exposure to desiccation stress during the adaptation phase on the mean tolerated water activity measured during the following exposure to different polyethyleneglycol (PEG 600) suspensions with variable aw -values. The tolerated mean aw -values increase significantly with increasing aw -values during the adaptation period until a value of 0.97. Prolonging the adaptation phase can significantly reduce the desiccation tolerance. Different letters at data points indicate significant differences (Student’s t test considering unequal variances, a 6 0:05).
Fig. 3. Relation of standard deviation of the tolerated aw -values and the intensity of the desiccation stress and duration of the adaptation period. The variability within the H. bacteriophora population increases with increasing desiccation stress during the adaptation phase. The duration of the exposure has no significant influence on the variability.
O. Strauch et al. / Biological Control 31 (2004) 218–226
223
Fig. 4. Mean tolerated water activity (aw ) of inbred lines and the foundation strain PS7. Error bars on the columns indicate standard deviation. The deviation is low for non-adapted H. bacteriophora inbred line populations (A) and higher in stress adapted populations (B). Different letters above the bars indicate significant differences between the mean values (Student’s t test considering unequal variances, a 6 0:05). The heritability h2 ¼ r2g =ðr2g þ r2e Þ, where r2g is the genetically caused variance and r2e is the environmentally caused variance.
H. bacteriophora was 0.46 (95% confidence limit: 0.35– 0.58) and 0.48 (95% confidence limit: 0.30–0.69) for the tolerance of adapted populations. Thus, the adaptation process had no influence on the heritability of the desiccation tolerance. Nearly all of the inbred lines had a higher tolerance to desiccation stress than the hybrid strain PS7 from which inbred lines had been produced (Figs. 4A and B). 3.3. Improvement of desiccation tolerance Whereas the desiccation tolerance of non-adapted nematodes could not be improved by selective breeding, the tolerance of adapted H. bacteriophora was responsive to this approach (Fig. 5). A total of eight selection
and breeding steps were carried out. Without previous adaptation, the mean of the tolerated aw -value remained almost constant between 0.94 and 0.93. In contrast, when adapting DJs prior to the exposure to desiccation stress, a breeding success was achieved. Tolerated aw values continuously dropped from 0.89 to 0.81 in the offspring of the selected DJs after the first three selection step. However, during the following steps, instead of an increase in desiccation tolerance, the populations became less resistant or the tolerance remained almost constant at aw between 0.82 and 0.81. The obtained selection success was less than the expected tolerance (Fig. 5). In most cases, however, the expected values lie inside the 95% confidence intervals of the obtained values. A significant difference between the
224
O. Strauch et al. / Biological Control 31 (2004) 218–226
Fig. 5. Recorded and expected mean tolerated water activity (aw ) of adapted and non-adapted populations of H. bacteriophora at subsequent selection steps. Error bars represent the 95% confidence interval.
obtained and expected success occurred after the fourth selection step for adapted populations when no further success was recorded.
4. Discussion This is the first reported attempt to improve the desiccation tolerance of an entomopathogenic nematode through genetic selection. The study has shown that selective breeding is a feasible approach to increase the tolerance of the nematodes towards desiccation stress only if DJs are stress-adapted prior to exposure to desiccation conditions. Adaptation resulted in an increased variability of the desiccation tolerance within a population (Fig. 3). The variability increased with the intensity of the stress during the adaptation phase. Is this an effect of the genetic variability in the genes, which have an influence on the adaptation process or the result of the variable response of the different phenotypes to the environmental conditions? Is the increasing variability genetically or environmentally caused or both? The genetically caused proportion (h2 ) on the phenotypic variation of the desiccation tolerance was not different in the adapted and the non-adapted populations. We can conclude that the increased variability was not only genetically caused, otherwise the heritability of the desiccation tolerance should be higher for the adapted populations. Three factors influence the breeding success, the heritability, the selection pressure, and the variability within the population. As the first two are almost the same for adapted and non-adapted populations, the breeding success was determined mainly by the high variability of the adapted population. The most conspicuous result obtained during the evaluation of the tolerance of the inbred lines is that nearly all of the inbred lines were more tolerant than the hybrid strain PS7, from which inbred lines had been
produced (Fig. 4). Similar results were obtained by Glazer et al. (1991) who showed that the mean desiccation tolerance of most of the inbred lines produced from HP88 was higher than in the original population. This effect might represent a heterosis effect. Heterosis effects are usually described as positive effects occurring in the progeny of crosses of homozygous individuals carrying different alleles. In our case, the heterosis effect reduced the desiccation tolerance of the hybrid. The genetic background (dominance and epistasis) of so called negative or positive heterosis effects are not necessarily different (Brewbaker, 1967; Falconer, 1984; Fischer, 1978). So far we can only assume that genes involved in the tolerance to desiccation stress might not be working synergistically and that negative effects are overcome through selection of inbred lines. Whether we were able to further improve the desiccation tolerance by combining different inbred lines and exclude those with antagonistic effects would be worth investigating, particularly as the mean tolerated water activity of the most successful inbred line D4 (0.77) is much below the tolerance obtained through selection starting from the hybrid strain (0.81 in the third selection step). Including the adaptation to desiccation stress in the breeding program reduced the mean tolerated aw -value from 0.89 to 0.81. These observations fit well with the expected breeding success for adapted and non-adapted populations (Fig. 5). However, expected values were always a little lower than the obtained values for the mean tolerated aw . For the calculation of the expected selection response R the estimated values of h2 was used and we assumed that h2 remained constant during the breeding process. This assumption is wrong in the case of a successful selection process. In this case, the genetic variance in the populations is reduced, whereas the influence of the environmentally caused variability remains constant. Consequently, h2 will decrease to zero at the end of the breeding process (Falconer, 1984). Since the decline of h2 could not previously be
O. Strauch et al. / Biological Control 31 (2004) 218–226
calculated, it could not be included in the calculation for the expected success. Therefore, the calculated R is an overestimation from the second selection step onwards. Another reason for an overestimation of R could be the fact that the selected populations produce two generations inside the insect before the DJs emerged. Thus, the offspring used for the next selection step was a result of self- and cross-fertilization (Johnigk and Ehlers, 1999a,b) without a selection pressure. Finally the heritability was estimated by using PS7 and the selection process was carried out with PS8. PS8 might have a higher heritability due to a enhanced genetic variability, but this is not proven so far. The low desiccation tolerance of H. bacteriophora and resulting short shelf-life of nematode products are the major factors preventing a more widespread use of nematodes in biological control (Georgis, 2002). In order to overcome this situation, other approaches than selective breeding might be feasible. Genes have been characterized also from nematodes which can increase desiccation tolerance (Browne et al., 2002; Gal et al., 2003; Solomon et al., 2000). Genetic transformation of nematodes might be a more promising way to reach the goal. Cross-breeding attempts should include isolates of H. bacteriophora from relatively warm and dry regions like the Middle East (Glazer et al., 1993; Iraki et al., 2000) or Turkey (Susurluk et al., 2001). Mutagenesis can also be used to improve desiccation tolerance (Burnell, 2002). Other beneficial traits need to be characterized and improved inbred lines or products of selective breeding should be crossed into commercial strains already selected for enhanced performance in liquid culture (Johnigk et al., 2002). Such programs would have the advantage of yielding methods to monitor the genetic stability of commercially used populations and thus could contribute to improve the quality and shelflife of nematode-based products in the future.
Acknowledgments We gratefully acknowledge the financial support of the Deutsche Forschungsgemeinschaft (Project Eh 116/ 4-1) and thank Manfred H€ uhn (Institute for Plant Production and Plant Breeding, University Kiel) for his comments on the results.
References Beyer, H., Walter, W., 1981. Lehrbuch der organischen Chemie. Hirzel Verlag, Stuttgart. Brewbaker, J.L., 1967. Angewandte Genetik. Gustav Fischer Verlag, Jena. Browne, J., Tunnacliffe, A., Burnell, A., 2002. Plant desiccation gene found in nematode. Nature 416, 38.
225
Burnell, A., 2002. Genetics and genetic improvement. In: Gaugler, R. (Ed.), Entomopathogenic Nematology. CABI Publishing, Oxon, UK, pp. 241–263. Charwat, S.M., Fischer, J.M., Wyss, U., 2002. The effect of osmotic stress on desiccation survival and water content of four nematode species. Nematology 4, 89–97. Cowles, R.S., Polavarapu, S., Ehlers, R.-U., Williams, R.N., in press. Management of insect pests in small fruit crops with nematodes. In: Grewal, P., Ehlers, R.-U., Shapiro-Ilan, D. (Eds.), Nematodes as Biological Control Agents. CABI Publishing, Oxon, UK. Ehlers, R.-U., 2001. Mass production of entomopathogenic nematodes for plant protection. Appl. Microbiol. Biotechnol. 56, 623–633. Ehlers, R.-U., 2003. Biocontrol nematodes. In: Hokkanen, H.M.T., Hajek, A.E. (Eds.), Environmental Impacts of Microbial Insecticides. Kluwer Academic Publishers, Dordrecht, NL, pp. 177–220. Ehlers, R.-U., Peters, A., 1998. Bek€ampfung von Engerlingen auf Sportrasen. Rasen/Turf/Gazon 29, 60–67. Falconer, D.S., 1984. Quantitative Genetics. Hirzel Verlag, Stuttgart. Fischer, H.E., 1978. Heterosis. Gustav Fischer Verlag, Jena. Gal, T.Z., Glazer, I., Koltai, H., 2003. Differential gene expression during desiccation stress in the insect-killing nematode Steinernema feltiae IS-6. J. Parasitol. 89, 761–766. Georgis, R., 2002. The Biosys experiment: an insider’s perspective. In: Gaugler, R. (Ed.), Entomopathogenic Nematology. CABI Publishing, Oxon, UK, pp. 357–372. Glazer, I., 1996. Survival mechanisms of entomopathogenic nematodes. Biocontrol Sci. Technol. 6, 373–378. Glazer, I., 2002. Survival biology. In: Gaugler, R. (Ed.), Entomopathogenic Nematology. CABI Publishing, Oxon, UK, pp. 169–187. Glazer, I., Salame, L., 2000. Osmotic survival of the entomopathogenic nematode Steinernema carpocapse. Biol. Control 18, 251–257. Glazer, I., Gaugler, R., Segal, D., 1991. Genetics of the entomopathogenic nematode Heterorhabditis bacteriophora (strain HP88): the diversity of beneficial traits. J. Nematol. 23, 324–333. Glazer, I., Liran, N., Poinar, G.O., Smits, P.H., 1993. Identification and biological activity of newly isolated heterorhabditid populations from Israel. Fund. Appl. Nematol. 16, 467–472. Grewal, P.S., 2000. Enhanced ambient storage stability of an entomopathogenic nematode through anhydrobiosis. Pest Manag. Sci. 56, 401–406. Hominick, W.M., 2002. Biogeography. In: Gaugler, R. (Ed.), Entomopathogenic Nematology. CABI Publishing, Oxon, UK, pp. 115– 143. Iraki, N., Salah, N., Sansour, M.A., Segal, D., Glazer, I., Johnigk, S.A., Hussein, M.A., Ehlers, R.-U., 2000. Isolation and characterization of two entomopathogenic nematode strains, Heterorhabditis indica (Nematoda, Rhabditida), from the West Bank, Palestinian Territories. J. Appl. Entomol. 124, 375–380. Johnigk, S.-A., Ehlers, R.-U., 1999a. Juvenile development and life cycle of Heterorhabditis bacteriophora and H. indica (Nematoda: Heterorhabditidae). Nematology 1, 251–260. Johnigk, S.-A., Ehlers, R.-U., 1999b. Endotokia matricida in hermaphrodites of Heterorhabditis spp. and the effect of the food supply. Nematology 1, 717–726. Johnigk, S.-A., Hollmer, S., Strauch, O., Wyss, U., Ehlers, R.-U., 2002. Heritability of the liquid culture mass production potential of the entomopathogenic nematode Heterorhabditis bacteriophora. Biocontrol Sci. Technol. 12, 267–276. Liu, Q.Z., Glazer, I., 2000. Desiccation survival of entomopathogenic nematodes of the genus Heterorhabditis. Phytoparasitica 28, 331– 340. Piggott, S.J., Liu, Q.Z., Glazer, I., Wright, D.J., 2002. Does osmoregulatory behaviour in entomopathogenic nematodes predispose desiccation tolerance? Nematology 4, 483–487. Solomon, A., Paperna, I., Glazer, I., 1999. Desiccation survival of the entomopathogenic nematode Steinernema feltiae: induction of anhydrobiosis. Nematology 1, 61–68.
226
O. Strauch et al. / Biological Control 31 (2004) 218–226
Solomon, A., Salomon, R., Paperna, I., Glazer, I., 2000. Desiccation stress of entomopathogenic nematodes induces the accumulation of a novel heat-stable protein. Parasitology 121, 409–416. Strauch, O., Niemann, I., Neumann, A., Schmidt, A.J., Peters, A., Ehlers, R.-U., 2000. Storage and formulation of the entomopathogenic nematodes Heterorhabditis indica and H. bacteriophora. BioControl 45, 483–500. Susurluk, A., Dix, I., Stackebrandt, E., Strauch, O., Wyss, U., Ehlers, R.-U., 2001. Identification and ecological characterisation of three
entomopathogenic nematode–bacterium complexes from Turkey. Nematology 3, 833–841. van Tol, R., Mannion, C., Raupp, M., in press. Nursery and tree applications. In: Grewal, P., Ehlers, R.-U., Shapiro-Ilan, D. (Eds.), Nematodes as Biological Control Agents. CABI Publishing, Oxon, UK. Womersley, C.Z., 1990. Dehydration survival and anhydrobiotic potential. In: Gaugler, R., Kaya, H.K. (Eds.), Entomopathogenic Nematodes in Biological Control. CRC Press, Boca Raton, pp. 117–138.