Genetic Improvement

Genetics is a powerful means of enhancing the beneficial traits of biological control agents. This is exemplified by the enhancement of the tolerance of EPN Heterorhabditis bacteriophora towards environmental extremes. Genetics offers a powerful means of enhancing the desired traits in many species of crops and livestock. Genetic engineering shows early promise for the rational improvement of entomopathogenic nematodes. There are no longer any substantial methodological, regulatory, or implementation barriers to genetic engineering of nematodes. Genetic improvement endevours have included screening and isolation of populations exhibiting the desired traits. Successful genetic selection depends on the presence of genetic variation for the particular trait in the population (heritability,h2). To evaluate the genetic diversity for heat tolerance in the HP88 strain we have generated from it several inbred lines and compared their performance in a heat tolerance assay (survival of a 6 hours exposure to 37°C) .The heritability value in this population was very high (h2=0.98) suggesting that selection could be an effective approach for enhancing its heat tolerance (Glazer et al., 1991). Cross breeding is an effective means for combining several desired traits into a single superior strain. We therefore tested whether the trait of heat tolerance could be transferred by crossing from the IS 5 to the commercial HP 88 strain. Crosses were performed on petriplates between single hermaphrodites from the HP 88 strain and 4-6 IS 5 males (Shapiro et al., 1997). Insect parasitic nematodes possess many of the attributes of ideal biological control agents, but intolerance to extreme temperatures can restrict their use. Whether heat shock treatments could improve nematode survival and infectivity were examined at temperatures that normally inhabit their activity (35 and 40°C). Nematodes exposed to a sub lethal temp (35°C) for 3 h with a latency period of 1-2 h at 25°C killed insects at 35 and 40°C. Correlative evidence was obtained between increased thermo-tolerance and the synthesis of 70k Da heat shock proteins (hsps).These results provide the first evidence of hsp synthesis in the development of thermo-tolerance and biological activity in the non feeding developmentally arrested IJ nematodes .Transformation by microinjection was assessed. The transformation was found to be heritable and stable .No changes in fitness were observed in the transformed strain (Gaugler et al., 1997). Selection and hybridization have been employed in developing genetically improved strains. These approaches may fail if the available nematode population do not possess sufficient genetic diversity for the beneficial traits (Glazer et al., 1991). A lack of genetic diversity may be overcome by surveying natural population for the desired traits (Gaugler, 1987). Selection is employed to find genetically superior nematode species. Steinernematid species with increased host finding ability has been successfully selected. A heat tolerant strain of Heterorhabditis bacteriophora designation IS 5 was discovered in Negev desert, Israel in 1996 and was found superior to HP 88, a commercially available strain of H. bacteriophora. Stability of the heat tolerance trait and fitness of H. bacteriophora IS 5 was tested in the laboratory after multiple passages through the last instar G. mellonella larvae.

Hybridization is an alternative approach because of its relative simplicity and its speed in transforming beneficial genes. Hybridization is used for genetic improvement of heat tolerance in H. bacteriophora (Shapiro et al., 1997). Heat tolerant strain of H. bacteriophora IS 5 hybridize with commercially available strain of H. bacteriophora HP 88 and succeeded in developing progenies with heat tolerance trait. Genetic engineering has been touted as the vehicle that would drive biological insecticides to equal footing with chemicals, but the promise remains elusive. This disappointment has resulted in large part from regulatory constraints on microbial agents. Insecticidal nematodes, however, possess a unique niche in the regulatory environment. If transgenes are carefully selected, it should be possible to produce genetically enhanced nematodes without compromising environmental protection (Gaugler et al., 1997). Ordinarily, the idea of genetically modifying an organism as complex as Steinernematid and Heterorhabditid nematodes could not be seriously contemplated. Given the knowledge of and techniques for Caenorhabditis elegans (a model organism for three decades), an extraordinary opportunity exists to exploit the new technology, since these nematodes are within the same super family and closely related to C. elegans. Additionally, entomopathogenic nematodes are well suited for genetic studies due to their simplicity, transparency, ease of laboratory culture, short generation time, and small genome size (Gaugler & Hashmi, 1996). Furthermore, Heterorhabditis is a self-fertilizing hermaphrodite, thus mutations become homozygous automatically and pure lines can be maintained.

Hashmi et al., (1995) developed transformation methods and markers that have resulted in first genetically engineered insect parasite. They introduced an hsp 70 gene from Caenorhaditis elegans into H.bacteriophora ,increasing the copy number from one up to ten .The overexpression of HSP resulted in transgenic nematodes much better than un modified nematodes at surviving high temperature (Hashmi et al.,1998). Hashmi et al., (1997) analyzed the molecular organization of hsp70 genes in five species and isolates of heterorhabditis and one steinernematid species using pcr and RFLP. They found homology with hsp70 genes of C.elegans. In addition RFLPs with hsp 70 probe revealed different banding patterns for heterorhabditis species and isolates. Fitness of a genetically improved S.carpocapsae was evaluated before use commercially (Gaugler et al., 1990). The control potential of a strain (ScP) of Steinernema feltiae [S. bibionis] was evaluated for the management of L. mali.(Grewal et al.,1993). The feasibility of genetic selection as a means of enhancing resistance of Heterorhabditis bacteriophora strain HP88 to fenamiphos, oxamyl and avermectin was examined. Estimates of heritability (h2) of resistance to the 3 nematicides were evaluated(Glazer et al., 1997). The efficacy for control of Popillia japonica in blue-grass turf in New Jersey in 1991 of a strain of Steinernema carpocapsae, selectively bred in the laboratory for improved host-finding, was not enhanced.(Gaugler et al., 1994).

A method of selecting a strain of Steinernema feltiae that was effective against Lycoriella solani [L. mali] in mushroom was described in detail by Tomalak (1994) The efficacy of this strain was evaluated and compared with the efficacy of 2 unselected strains. The pattern of emergence of infective juveniles of S. glaseri was altered when selection was used to test for a genetic component (Stuart et al., 1996). Emergence pattern was found to have a genetic component and any selection effort should be with species adapted for the target host. In H.bacteriophora selection for heat tolerance proved to be effective but there was deterioration in reproductive potential (Segal et al.,1999). The success in genetic transformation of EPN opens the way for generating transgenic nematodes carrying genes conferring resistance to various environmental extremes, most notably heat shock genes. Genetic selection for enhanced penetration activity and virulence was carried out (Sulistyanto et al., 1999) with Tipula oleracea and Phyllopertha horticola successfully.

Abstracts
Hashmi-S; Hashmi-G; Gaugler-R. 1995. Genetic transformation of an entomopathogenic nematode by microinjection. Journal-of-Invertebrate-Pathology, 66:3, 293-296; 22 ref.
The first successful transformation of an entomopathogenic nematode is reported. Foreign genes were introduced in Heterorhabditis bacteriophora HP88 by microinjection using vectors carrying the Caenorhabditis elegans genes coding for the roller phenotype and 16-kDa heat shock protein (hsp16) gene. A translational fusion made by inserting lacZ in frame into hsp16 was expressed in the body musculature, hypodermis, and pharyngeal muscles. The transcription of the hsp16/lacZ transgenes resulted in the rapid synthesis of detectable levels of beta-galactosidase. This research opens new avenues for genetic modification of entomopathogenic nematodes.

Glazer-I; Salame-L; Segal-D. 1997. Genetic enhancement of nematicide resistance in entomopathogenic nematodes. Biocontrol-Science-and-Technology, 7:4, 499-512; 41 ref.
The feasibility of genetic selection as a means of enhancing resistance of Heterorhabditis bacteriophora strain HP88 to fenamiphos, oxamyl and avermectin was examined. Estimates of heritability (h2) of resistance to the 3 nematicides were obtained from analysis of inbred lines derived from the base population. The heritability estimate for fenamiphos was h2=0.31, for oxamyl h2=0.71 and for avermectin h2=0.46. Five rounds of selection were performed. Thereafter, each line was divided into 2: for one subline selection continued for 6 additional rounds. The other subline was reared without selection for the 6 additional rounds. After the eleventh round, resistance to the nematicides was examined as were several traits relevant to biocontrol efficacy including virulence, heat tolerance and reproduction potential. Selection resulted in an 8 to 9-fold increase in resistance to fenamiphos and avermectin and a 70-fold increase in resistance to oxamyl. The enhanced resistance oxamyl and avermectin, and to a lesser extent to fenamiphos, was stable and continued after selection was relaxed. No deterioration in traits relevant to biocontrol efficacy was observed in the selected lines as compared with the base population. The selected lines displayed enhanced cross-resistance towards some, but not all, of the nematicides tested. These results demonstrated that genetic selection can be used to enhance resistance of entomopathogenic nematodes to certain environmental stresses. The selected lines will be useful bioinsecticides in the context of integrated pest management.

Gaugler-R; Campbell-JF; McGuire-TR. 1990. Fitness of a genetically improved entomopathogenic nematode. Journal-of-Invertebrate-Pathology, 56:1, 106-116.
A strain of Steinernema carpocapsae [Neoaplectana carpocapsae] (G-13) selectively bred for improved host-finding was compared to 2 wild-type strains, the commercially available All strain and the Foundation strain from which the G-13 strain was derived, for changes in fitness. Selection did not affect pathogenicity to Galleria mellonella, mobility, sex ratio or morphology. However, the G-13 strain showed a gain of fitness with regard to host penetration and reproductive potential and a loss of fitness for storage stability. Acquiring enhanced host-finding abilities does not appear to be correlated with a serious reduction in overall fitness and the potential of the selected G-13 strain for inundative biological control appears unlikely to be impaired. The possible significance of the 3 correlated responses to selection is discussed.

Grewal-PS; Tomalak-M; Keil-CBO;Gaugler-R. 1993. Evaluation of a genetically selected strain of Steinernema feltiae against the mushroom sciarid Lycoriella mali. Annals-of-Applied-Biology, 123:3, 695-702; 29 ref.
The control potential of a strain (ScP) of Steinernema feltiae [S. bibionis] was evaluated for the management of L. mali. Trials were conducted at 2 commercial mushroom (Agaricus bisporus) farms in Pennsylvania with large and small infestations of the pest. The efficacy of the ScP strain was compared with the SN strain of S. feltiae and the chitin synthesis inhibitor, diflubenzuron. At low densities of L. mali, the strains did not differ in efficacy and caused 85-94% reduction in pest populations. At high pest densities with a mixed infestation of Megaselia halterata, the ScP strain caused reduction of 56-83% in populations of L. mali whereas the SN strain caused reductions of 51-73%. Two doses of the improved strain (0.5X106 and 1.0X106 infective juveniles per m2 cropping area) did not differ in efficacy from diflubenzuron which caused 80% reduction in populations of L. mali. The lower dose (0.5X106) of the SN strain was less effective than diflubenzuron. The ScP strain had a major advantage over the SN strain in persistence in casing material. Nematodes had no significant effect on mushroom yields.

Gaugler-R; Glazer-I; Campbell-JF; Liran-N. 1994. Laboratory and field evaluation of an entomopathogenic nematode genetically selected for improved host-finding. Journal-of-Invertebrate-Pathology, 63:1, 68-73; 29 ref.
The efficacy for control of Popillia japonica in blue-grass turf in New Jersey in 1991 of a strain of S. carpocapsae, selectively bred in the laboratory for improved host-finding, was not enhanced. Larval populations were reduced by selected (S20) and wild type (All) strains by 25-30%, compared with 70% for a chemical insecticide. Laboratory tests conducted in soil-filled pots against Maladera matrida gave similar results. The selected S20 strain was, nevertheless, better than the All and HP88 strains at locating larvae. Aggregation of S20 nematodes, which are known to have enhanced chemosensitivity to carbon dioxide, was observed at or near the spiracles; these are covered with sieve plates however, and are not a portal of nematode entry. In spite of selection for greater response to host cues, S20 persisted in nictating, even when a host was present. Low host finding ability is said to be responsible for the unfavorable field results obtained with S. carpocapsae in the control of scarab larvae. S. carpocapsae was poorly adapted to cause infection. It is concluded that poor adaptation cannot be overcome by genetic improvement and that future selection efforts for biological control with nematodes should be with species adapted for the target host.

Stuart-RJ; Lewis-EE; Gaugler-R. 1996. Selection alters the pattern of emergence from the host cadaver in the entomopathogenic nematode, Steinernema glaseri. Parasitology, 113:2, 183-189; 24 ref.
The authors used selection to test for a genetic component to the pattern of emergence of infective juveniles from the host (Galleria mellonella) cadaver in S. glaseri, and whether other traits would respond to selection on this major and complex life-history character. They selected for early ('fast') and late ('slow') emerging lines by perpetuating nematodes that emerged on the 1st and after the 7th day of emergence, respectively. After 12 cycles of selection, the pattern for the slow line but not the fast line differed significantly from the base population. Cumulative emergence for the slow line was less than the base population from Days 4 to 14 of the 18-day emergence period. The maximum difference occurred on the 4th day when 72.6% of emergence was complete for the base population but only 55.4% for the slow line. Decreases in infective juvenile size over the emergence period were consistent with the change in emergence pattern, but variation in sex ratios was not. No differences in infectivity was found. These results indicate that the emergence pattern has a genetic component, and that genetic variability for this trait occurs in natural populations. Furthermore, the asymmetric response to selection suggests that the authors' field population (from New Jersey, USA) is under strong selection for a highly skewed early emergence.

Hashmi-G; Hashmi-S; Selvan-S; Grewal-PS; Gaugler-R. 1997. Polymorphism in heat shock protein gene (hsp70) in entomopathogenic nematodes (Rhabditida). Journal-of-Thermal-Biology, 22:2, 143-149.
To assess variation among species of entomopathogenic nematodes for thermotolerance, a search was initiated for the molecular organization of heat-inducible hsp70 genes in these nematodes. Five Heterorhabditis species/isolates with different temperature optima for survival and one warm-adapted species of Steinernema were tested. PCR and RFLP analyses of hsp70 in Heterorhabditis species and S. scapterisci demonstrated a putative homology with the Caenorhabditis elegans hsp70 A gene, thus indicating an evolutionary conserved nature among different nematode species. RFLPs with the hsp70 A gene probe revealed different banding patterns for Heterorhabditis species and isolates. This is the first report on the identification of any hsp70 gene in entomopathogenic nematodes. This observation establishes a unique correlation between geographical distribution and polymorphisms for hsp70 A gene in these nematodes.

Hashmi-S; Gaugler-R; Abad-P(ed.); Burnell-A (ed.); Laumond-C (ed.); Boemare-N (ed.); Coudert-F. 1997. Genetic improvement of entomopathogenic nematodes. Entomopathogenic nematodes: genetic and molecular biology of entomopathogenic nematodes. Proceedings held at the Centre de Recherches Agronomiques de Provence-Alpes-Cote d'Azur Institut National de la Recherche Agronomique, Antibes, France, 1 to 5 April, 1997. 1999, 143-153; 44 ref.
Genetic engineering shows early promise for the rational improvement of entomopathogenic nematodes. There are no longer any substantial methodological, regulatory, or implementation barriers to genetic engineering of nematodes. Efficient new transformation methods have been developed. Markers are available to study gene expression without harm to the nematode. Transgenic strains have been generated, notably a strain of H. bacteriophora possessing enhanced thermo-tolerance. This strain has been field released, joining BT and baculoviruses as engineered insect natural enemies that have been released into the environment. The keys to receiving regulatory approval for the release included (1) engineering a commercial rather than ecological advantage into the nematode, and (2) over expressing an existing gene product rather than introducing a truly novel gene.

Segal-D; Shapiro-DI; Glazer-I; Abad-P (ed.); Burnell-A (ed.); Laumond-C (ed.); Boemare-N (ed.); Coudert-F. 1999. Genetic approaches for improvement of heat tolerance in entomopathogenic nematodes. Entomopathogenic nematodes: genetic and molecular biology of entomopathogenic nematodes. Proceedings held at the Centre de Recherches Agronomiques de Provence-Alpes-Cote d'Azur Institut National de la Recherche Agronomique, Antibes, France, 1 to 5 April, 1997. 1999, 103-112; 34 ref.
Some of the approaches for enhancing tolerance of Heterorhabditis bacteriophora to environmental extremes, in particular heat are reviewed. Selection for heat tolerance proved to be effective, but was associated in deterioration of reproductive potential. On the other hand selection for resistance to nematicides was very effective, lasted when the selection pressure was removed, and did not compromise other parameters of biocontrol efficacy. Screening for natural isolates resulted in the identification of a heat tolerant strain IS-5. Using genetic markers and cross hybridization it is demonstrated that the trait was dominant and transferable to the commercial strain HP88 without concomitant reduction in bio-control efficacy. Mutagenesis is useful for generating both mutants displaying desired beneficial traits and marker mutations. The utility of the latter is demonstrated. Finally, genetic engineering is a most promising tool for enhancing beneficial traits in entomopathogenic nematodes (EPN). The success in genetic transformation of EPN opens the way for generating transgenic nematodes carrying genes conferring resistance to various environmental extremes, most notably heat shock genes.

Grewal-PS; Tomalak-M; Keil-CBO; Gaugler-R. 1993. Evaluation of a genetically selected strain of Steinernema feltiae against the mushroom sciarid Lycoriella mali. Annals-of-Applied-Biology 123: 3, 695-702.
The control potential of a strain (ScP) of Steinernema feltiae [S. bibionis] was evaluated for the management of L. mali. Trials were conducted at 2 commercial mushroom (Agaricus bisporus) farms in Pennsylvania with large and small infestations of the pest. The efficacy of the ScP strain was compared with the SN strain of S. feltiaeand the chitin synthesis inhibitor, diflubenzuron. At low densities of L. mali, the strains did not differ in efficacy and caused 85-94% reduction in pest populations. At high pest densities with a mixed infestation of Megaselia halterata, the ScP strain caused reduction of 56-83% in populations of L. mali whereas the SN strain caused reductions of 51-73%. Two doses of the improved strain (0.5X106 and 1.0X106 infective juveniles per m2 cropping area) did not differ in efficacy from diflubenzuron which caused 80% reduction in populations of L. mali. The lower dose (0.5X106) of the SN strain was less effective than diflubenzuron. The ScP strain had a major advantage over the SN strain in persistence in casing material. Nematodes had no significant effect on mushroom yields.

Tomalak-M. 1994. Selective breeding of Steinernema feltiae (Filipjev) (Nematoda: Steinernematidae) for improved efficacy in control of a mushroom fly, Lycoriella solani Winnertz (Diptera:Sciaridae). Biocontrol-Science and Technology, 4:2, 187-198.
A method of selecting a strain of Steinernema feltiae that was effective against Lycoriella solani [L. mali] is described in detail. The efficacy of this strain was evaluated and compared with the efficacy of 2 unselected strains. During selection, preference was given to nematodes with the greatest ability to: search effectively for larvae of the target insect in their natural habitat; infect them shortly after application; and to reproduce in the haemocoel of the target insect. After 34 rounds of selection, a 4-fold improvement was achieved in nematode ability to find and parasitize 3rd- and 4th-instar larvae of L. mali in the mushroom substrate. Mortality of the pest 24 hours after application of the nematode was 22.5%, however, following selection, mortality of L. mali was 92.5%. A further experiment in a mixed age mushroom house established that the selected nematode strain was significantly better than both unselected strains, causing a 91.1-92.7% reduction in emergence of adults of L. mali from the mushroom substrate. No difference was observed between the efficacy of the selected nematodes applied at 1 X 106 and 3 X 106 infective juveniles/m2, while the unselected strains performed significantly better at the greater concn. All 3 strains were persistent in the mushroom casing, apparently due to recycling in the insect host.

Tomalak-M; Simoes-N (ed.); Boemare-N (ed.); Ehlers-R-U. 1998. Selection of Steinernema feltiae for enhanced virulence against Lycoriella solani. Entomopathogenic nematodes. Pathogenicity of entomopathogenic nematodes versus insect defence mechanisms: impact on selection of virulent strains. Proceedings held at Universidade dos Acres, Ponta Delgada, Acores, Portugal, 17 to 20 March 1996. 1998, 243-255; 23 ref.
Preliminary study revealed that among steinernematid and heterorhabditid nematodes only Steinernema feltiae could readily infect larvae of sciarid flies [Lycoriella mali]. The pests control efficacy in a mushroom casing was, however, significantly lower then that recorded in a peat-moss substrate used in greenhouse cultures. A series of laboratory experiments showed that the nematode host-finding and infectivity in the mushroom substrate could be strongly affected by a number of environmental factors, including the presence of growing mycelium and high moisture of the mushroom casing. To improve the parasite performance in such conditions a systematic selective breeding programme considering a proper choice of nematode species, the target insect and the insects environment was undertaken. In the designed selection procedure only individuals with the greatest ability to search effectively for the target insect larvae in their natural habitat, and to infect them shortly after application and to reproduce in their haemocoel were allowed to contribute their progeny to the next generation of the selected parasite strain. The selection conducted for 34 generations significantly improved the nematode ability to find and infect L. solani larvae in the mushroom casing. The nematodes infected and killed the insects' shortly after application and due to recycling in the host cadavers they provided good control of the pest population over the entire mushroom-growing cycle.

Peters-A; Ehlers-RU; Simoes-N (ed.); Boemare-N (ed.); Ehlers-R-U. 1998. Evaluation and selection for enhanced nematode pathogenicity against Tipula spp. SO:Entomopathogenic nematodes. Pathogenicity of entomopathogenic nematodes versus insect defence mechanisms: Impact on selection of virulent strains. Proceedings held at Universidade dos Acres, Ponta Delgada, Acores, Portugal, 17 to 20 March 1996. 1998, 225-241; 33 ref.
The infection process of Steinernema feltiae in Tipula oleracea and, partly, T. paludosa, was investigated to establish traits limiting nematode pathogenicity. Dauer larvae (DL) of S. feltiae react to host cues of T. oleracea and are arrested near the larvae. Steinernema feltiae does not show significant differences in host finding of Galleria mellonella and Tipula spp.. However, nematode invasion is significantly higher in G. mellonella. Pathogenicity to T. oleracea and nematode invasion was correlated among different nematode strains and species examined. The invasion activity is considered to be an important trait limiting nematode pathogenicity. Encapsulation of S. feltiae in T. oleracea may limit the nematodes pathogenicity, especially when low nematode doses are applied. The encapsulation response is highly variable between individual tipulid larvae which restricts improvement by selective breeding. The pathogenicity of the nematodes symbiotic bacteria, Xenorhabdus bovienii, is low. In the nematode/bacteria complex, however, pathogenicity is synergistically increased. Selective breeding for improved invasion of nematodes into T. oleracea has been applied to S. feltiae and H. bacteriophora using different techniques. Both nematode species responded to selection. The response of H. bacteriophora was significantly higher than that of S. feltiae.

Sulistyanto-D; Ehlers-RU; Simoes-N (ed.); Boemare-N (ed.); Ehlers-R -U 1998. Host invasion and genetic selection for enhanced penetration activity. Entomopathogenic nematodes. Pathogenicity of entomopathogenic nematodes versus insect defence mechanisms: Impact on selection of virulent strains. Proceedings held at Universidade dos Acres, Ponta Delgada, Acores, Portugal, 17 to 20 March 1996. 1998, 61-70; 7 ref.
Nematode invasion was investigated with a penetration bioassay and the results were compared with nematode invasion into living host insects. In contrast to in vivo conditions, in the assay all tested nematode species (Steinernema feltiae, S. glaseri, S. riobravis [S. riobrave], H. bacteriophora and H. megidis) also penetrated the thick walled insect integuments. Cues able to induce cuticle penetration were insect surface washings, 0.2% haemolymph, faeces or suspensions of symbiotic bacteria. Nematodes never penetrated through the cuticle when water without a stimulus was offered, indicating that the cues are not located on the surface of the cuticle. Once holes were made nematodes used these ports for invasion. When two alternative hosts were offered the response in the assay differed significantly from nematode invasion obtained with the living insect. Galleria mellonella were much more attractive and more nematodes invaded compared with data obtained from Tipula oleracea and Phyllopertha horticola. The contrary was found in the bioassay in which T. oleracea was most attractive. Genetic selection was applied to enhance the penetration activity. After 6 selection cycles no drastic increase was recorded for H. megidis, possibly due to its narrow genetic diversity. Results with H. bacteriophora indicate that selection using the natural host result in a higher increase in the penetration activity.