Introduction

Entomopathogenic nematodes (EPN) of the genus Heterorhabditis Poinar 1975 (Rhabditidomorpha: Heterorhabditidae) are symbiotically associated with the bacterium Photorhabdus luminescens Thomas and Poinar 1979. They are comparatively safe biocontrol agents (Ehlers 2003) used to control insect pests in cryptic environments (Grewal et al. 2005). Recently, the potential of H. bacteriophora to control larvae of the invasive pest Western Corn Rootworm (Diabrotica virgifera virgifera) has been reported (Toepfer et al. 2008). EPN are produced in liquid culture in industry-scale bioreactors (Ehlers 2001) to provide nematode-based biocontrol products for integrated pest management at reasonable cost. H. bacteriophora Poinar 1975, like many other rhaditid nematodes, form developmentally arrested, non-feeding, third-stage dauer juveniles (DJs) that can be stored for some time (Strauch et al. 2000). DJs are resistant to shear stress and can therefore be applied with conventional spraying technology (Ehlers and Shapiro-Ilan 2005), providing the temperature during agitation is not too high (Łączyński et al. 2006). Despite these properties making them excellent biological control agents, Heterorhabditis spp. also possess some handicaps, like their short shelf-life, which limits a more widespread use.

In order to preserve product quality, EPN are usually stored at temperature <10°C to reduce their metabolism. During transportation, however, they are often exposed to higher temperature and then their longevity is further reduced (Strauch et al. 2000). Another possibility to reduce their metabolism is the induction of quiescence by exposure to limited desiccation conditions. However, the potential of surviving desiccation stress within heterorhabditids is limited (Glazer 2002; Grewal et al. 2006). To overcome these limitations selective breeding for improved tolerance to extreme environmental conditions, like heat and desiccation was proposed by Gaugler (1986).The heritability of the traits heat and desiccation tolerance is relatively high in H. bacteriophora (Glazer et al. 1991; Strauch et al. 2004; Ehlers et al. 2005), making selective breeding a feasible approach for genetic improvement of these traits. The approach to genetic improvement includes hybridization to obtain a larger gene-pool and subsequent genetic selection. Improvement of desiccation tolerance was already obtained by Strauch et al. (2004) and of heat tolerance by Ehlers et al. (2005) through genetic selection. Mukuka et al. (2010a, b) recorded similar tolerance for heat and desiccation stress just by screening among natural populations of H. bacteriophora. The three most tolerant strains were then crossed by using only those individuals of a population for crosses, which had tolerated the highest stress conditions. These resulting hybrids provided additional progress in stress tolerance (Mukuka et al. 2010c). The maximum mean tolerated temperature (survival of 50% of the DJ population after heat treatment) reached 42°C, and the desiccation tolerance decreased to a minimum mean tolerated water activity (a w) of 0.65 (Mukuka et al. 2010c).

However, genetic improvement remains controversial because many researchers think laboratory selection programmes reduce genetic variability and integrity, introducing correlated deleterious effects (Roush 1979, Bedding et al. 1993). Reduction in environmental tolerance (heat, desiccation and UV) and reproductive potential as a result of subsequent reproduction under laboratory conditions have been reported (Shapiro et al. 1996; Wang and Grewal 2002). Phenotypic trait changes may also arise from non-genetic factors such as poor nutrition and disease. Some bottlenecks during the selection regime might be reduced by selecting a large population of survivors at each cycle of selection (Hopper et al. 1993). However, regardless of the approach, trait stability is an important factor, should genetically improved strains be used commercially. The inadvertent transfer, by genetic crosses, of genetic material other than the desired trait itself could compromise pathogenicity, reproduction potential or other beneficial traits of the resultant hybrids (Segal and Glazer 2000).

The pathogenicity (Han and Ehlers 2000) and reproductive potential (Grewal et al. 1994) have been well elaborated and documented in Heterorhabditis spp. Virulence defined as “disease producing power” (Tanada and Kaya 1993) is caused by the symbiotic bacteria P. luminescens that enters the insect passively within the nematodes gut (Han and Ehlers 2000). The ability of DJs to penetrate into the host is thus an essential step defining virulence (Dowds and Peters 2002). The portal of entry for heterorhabditid DJs is through natural openings or directly through the cuticle (Koppenhöfer et al. 2004).

The objective of this study was to compare the fitness of hybrid strains to the already available commercial hybrid strain EN 01. This was done by evaluating the virulence, host penetration and reproductive potential that could have been compromised after hybridization.

Materials and methods

Nematode strains

The H. bacteriophora hybrid strains (Table 1) had been produced within the study reported by (Mukuka et al. 2010c). The virulence and reproduction potential was compared with the hybrid strain EN 01, which is the commercially used strain of e-nema GmbH (Schwentinental, Germany). Before experimentation, the hybrids were cultured in vivo using last instar of Galleria mellonella (Lepidoptera, Pyralidae) as described by Kaya and Stock (1997). The nematodes were stored in Ringer’s solution at 10°C and used within 1 week after emergence.

Table 1 Description of hybrid strains, their mean tolerated temperature (°C) and/or water activity (a w) after adaptation or without prior adaptation before exposure to stress conditions according to Mukuka et al. (2010c) and mean ranking of fitness according to results presented in Table 2
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Virulence test

Virulence tests were conducted by assessment of the mortality caused by two DJs against one last instar of G. mellonella using a method described in details by Peters (2005). Two DJs were placed into a well with 16 mm diameter of a 24-cell-well plate (Iwaki 24 well, Asahi Techno Glass, Tokyo, Japan) containing 1 g of sterile sand (10% water w/w). Then one last instar G. mellonella was added and the plates were closed with Parafilm. After incubation for 72 h in the dark at 25°C, the number of dead insect was recorded as percentage of the total number of insects tested in each replicate (n = 24). The experiment was repeated five times with different batches of DJs for each hybrid.

Host penetration

Host penetration was also assessed with last instar G. mellonella. The experimental procedure was as described but 100 DJs/insect were applied. After 5 days, three G. mellonella cadavers were dissected from one batch and the total number of hermaphrodites, which had developed from invading DJs, was recorded. A total of three G. mellonella per hybrid strain was dissected from three independent replicates inoculated with different batches of DJs. The total number of hermaphrodites was recorded and divided by 100 to determine percent nematode penetration.

Reproductive potential

Reproductive potential was determined by infecting last instar of G. mellonella with 100 DJs/insect as described above. After 72 h at 25°C, five infected insect cadavers was picked randomly and transferred to a White trap (White 1927), placing individual cadavers in each trap. Three batches of nematodes (replicates) were evaluated giving a total of 15 White traps (individual cadavers) per nematode hybrid strain. The total number of DJs produced per G. mellonella was recorded 14 days after infection.

Ranking

Strains were put in order according to their performance in virulence, host penetration and reproduction (Table 2). The reproduction potential per invaded DJ was calculated by dividing the reproductive success (mean number of DJs harvested from one larva of G. mellonella) by the mean number of invaded DJs. The mean ranking of the strains was assessed by adding the individual ranking for virulence, penetration, reproduction and reproduction per DJ and then ranking the strains.

Table 2 Ranking of virulence (percentage dead insects after exposure to 2 DJs/insect for 72 h) host penetration (percentage of invaded DJs after exposure of 100 DJs/insect for 72 h) and mean reproductive potential (n = 15) of H. bacteriophora hybrid strains and a commercial strain (in italics)
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Statistical analysis

The mortality data were corrected for control mortality following the formula by Abbott (1925) and were arcsin-transformed. Treatment differences were detected through ANOVA (P ≤ 0.05) and differences between treatments were compared using Tukey’s HSD test. Correlation between virulence and DJ penetration was assessed using the Pearson’s correlation coefficient at a 5% confidence level.

Results

Virulence

The mean mortality of G. mellonella ranged between 8.6 and 75.7% (Fig. 1). Significant difference were recorded (F = 39.9; df = 11, 59; P ≤ 0.0001) with HH1 (75.7%) causing the highest mortality followed by DA2 (75.5%), HA1 (71.3%) and then the commercial strain EN01. The lowest mortality was recorded for DD2 (8.6%), followed by DA1 (12.1%) and then D3 (19.0%).

Fig. 1
figure 1

Mean Abbott-corrected mortality (%) of last instar G. mellonella after exposure to two DJs/insect for 72 h at 25°C to different H. bacteriophora hybrid strains and a commercial strain (EN01). Each column represents a mean of five replicates with different DJ batches of 24 insects per replicate. Different letters on the error bars indicate significant differences according to HSD test (P < 0.05)

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Host penetration

Penetration into G. mellonella was always less than 20%, however, significant differences were recorded (F = 4.08; df = 11, 107; P ≤ 0.0001) among the nematode strains. The highest penetration was recorded for HA1 (17.7%) followed by H3 (17.1%) and then DD2 (16.1%). The lowest number of penetrated DJs was recorded for the commercial strain EN01 (4.6%), although not significantly different from HH1 (6.2%), DA1 (7.1%), D3 (7.7%) and HD4 (9.6%) (Fig. 2). No significant correlation was assessed between virulence and DJ penetration (r = −0.06, P = 0.85).

Fig. 2
figure 2

Mean DJ penetration (%) of different H. bacteriophora hybrid strains and a commercial strain (EN01) in G. mellonella last instars after 5 days exposure to 100 DJs per insect at 25°C. Each bar represents a mean of three replicates with different DJ batches of three insect larvae per replicate. Different letters on the error bars indicate significant differences according to HSD test (P < 0.05)

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Reproductive potential

The number of DJs produced per G. mellonella ranged from 54,066 to 271,720 (Fig. 3). The highest in production of DJs was HA2 (271,720), followed by HA1 (233,546) and then HH2 (183,396). The lowest number of offspring was recorded for DD2 (54,066), followed by D3 (64,066) and then HD4 (102,066). The commercial strain EN01 produced 142,356 DJs and was only significantly different from the four hybrid strains HA2 (271,720), HA1 (233,546), D3 (64,066) and DD2 (54,066) (F = 20.918; df = 11, 179; P ≤ 0.0001).

Fig. 3
figure 3

Mean number of DJ offspring obtained with different H. bacteriophora hybrid strains and a commercial strain (EN01) from last instar G. mellonella larva after 72 h exposure with 100 DJs of H. bacteriophora per insect larvae and recorded 14 days after infection. Each bar represents a mean of three replicates of five insect larvae per replicate. Different letters on the error bars indicate significant differences according to HSD test (P < 0.05)

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Ranking

Ranking of the strain performance is summarized in Table 2. Ranking at position one for reproduction per invaded DJ is the commercial strain. The result of the mean ranking is presented in Table 1. When the additive ranking resulted in the same sum, the same ranking was given. The commercial strain is ranking on position 4. With exception of strain DA2, the result of crossing three strains with improved desiccation tolerance after adaptation to desiccation stress, ranking on position 3, almost all strains ranking lower than the commercial strain are those with enhanced desiccation tolerance.

Discussion

Only the heat-tolerant strains were superior or similar in fitness to the commercial strain EN 01. Heat tolerant hybrid strains caused relatively higher virulence and were superior in host penetration and reproductive potential compared to desiccation tolerant hybrid strains (Table 2). The only exception was strain DA2, which was high in virulence and in a middle position in the ranking of other fitness characters. The results thus provide indication for trade-off effects as a result of increased tolerance to desiccation stress. Similar observations are reported by Grewal et al. (2002), who reported positive correlations between heat, hypoxia and UV tolerance and DJ longevity, but a negative correlation for desiccation tolerance.

The breeding programme had produced crosses of strains, which were tolerant without a prior stress adaptation (HH for heat, and DD for desiccation tolerant hybrids) and those which had an increased tolerance after adaptation to stress (HA for heat and DA for desiccation tolerant hybrids). For hybrid strains produced for enhanced tolerance, those that included the adaptation to stress conditions were always superior in fitness compared to those which had not been exposed to adaptation in the selection for cross breeding (Tables 1, 2). This could possibly be a result of pleiotropy (Gaugler et al. 1990) for genes involved in reaction to stress adaptation. Comparing hybrid strains with one or two crosses in heat or desiccation tolerance with hybrids H3, D3, crossing the populations with enhanced tolerance including or excluding an adaptation prior to stress exposure and HD4, which crossed H3 and D3, a further reduction in fitness was noted only in H3, but not in D3 or the hybrid HD4, which included heat and desiccation tolerant strains.

The ability of DJs to penetrate into the host is an essential step in the pathogenesis of EPN. The higher the number of DJs the higher should be the virulence. Nevertheless, the probability of any DJ to successfully penetrate and establish in even highly susceptible host is low (Gaugler et al. 1990). Host penetration is generally low in H. bacteriophora. Our host penetration results are similar to Shapiro et al. (1996), who recorded a host penetration of hybrid strains of H. bacteriophora averaging 27% at 30°C and 4% at 25°C. Our results document the lowest penetration activity of 4.8% for the commercial strain; however, its virulence is not low ranking at position 4. No correlation was assessed for the results of host penetration and virulence in this study, which is surprising. The crosses had been produced on the bacterium P. luminescens originating from the female partner always; thus the hybrids carried different strains of the symbiotic bacterium. As the virulence of H. bacteriophora depends on the symbiotic bacterium (Han and Ehlers 2000), variable virulence of the different bacterial strains might have caused the mismatch between host penetration and virulence.

The reproduction potential of the commercial strain was ranking at position 7; however, if calculated as number of offspring per invaded DJ, it is on position 1. The commercial strain has long been produced in liquid culture. In liquid media, amphimixis is not possible as males cannot attach to females and thus no copulation occurs. This causes automatic selection of inbred lines with a high reproductive potential, why it is not surprising to find the commercial strain at position 1 for the reproduction potential per DJ.

The high fitness of the heat tolerant hybrid strains could be attributed to the maximised genetic variability during crosses (Mukuka et al. 2010c). The genetic crosses probably provided multiple alleles at a locus that removes strain-specific co-adapted gene complexes and/or epistatic combinations responsible for resisting changes in gene frequency due to artificial selection (White et al. 1970). In our hybrids, genetic drift did not occur because at each hybridisation step, although selection pressure was included (only the 10% most tolerant individuals were used for crosses), hundreds of parents for subsequent crossings were used. Normally, when a few parents are used, it is possible to introduce other alleles of independent genes that can bring in a positive or negative effect with the selected trait.

The hybrid HD4 has been further propagated including selection pressure for heat and desiccation and additional progress in tolerance was obtained, particularly in heat tolerance (Mukuka et al. 2010c). It will be interesting to investigate whether the genetic selection has similar effects like reported in this study. Important will also be, whether the beneficial traits can be stabilized in the population. The creation of inbred line of the hybrid lines through liquid culture production will possibly prevent beneficial traits of heat and desiccation tolerance to decline (Bai et al. 2005). From a practical standpoint, there is need to carry out the virulence tests on less susceptible insect as well.