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Fitness of a genetically improved entomopathogenic nematode
JOURNAL
OF INVERTEBRATE
56, 106-l 16 (190)
PATHOLOGY
Fitness of a Genetically RANDYGAUGLER,* *Department
of Entomology
Improved
Entomopathogenic
JAMES F. CAMPBELL,* and TDepartment New Brunswick,
Nematode
AND TERRY R. MCGuIREt
of Biological Sciences, New Jersey 08903
Rutgers
University,
Received August 24, 1989; accepted November 13, 1989 A strain of Steinernema carpocapsae (G-13) selectively bred for improved host-finding was compared against two 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, 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 three correlated responses to selection are discussed. o 1990 Academic press, hc. KEY WORDS: Steinernema carpocapsae; host finding; genetic improvement; entomopathogenic nematode; fitness.
INTRODUCTION
that would quickly initiate host-searching movements. Research on the development of entoGenetic improvement has been used by mopathogenic nematodes for biological biological control workers to select insect control has increased exponentially in re- predators and parasitoids in the laboratory cent years, but implementation remains for traits ranging from improved climatic hampered by questionable field perfortolerance to pesticide resistance. However, mance when compared to chemical insectigenetic improvement remains controvercides (Gaugler, 1988). One factor that may sial, mostly because many workers feel labbe limiting the efficacy of steinemematid oratory selection programs reduce genetic species as biological insecticides is the poor variability and introduce correlated deletehost-finding capabilities of infective-stage rious pleiotropic effects (Roush, 1979). juveniles. Gaugler et al. (I989b) screened That is, selection reduces overall fitness to 21 geographical isolates of Steinernema the point that the “improved” agent’s field ( = Neoaplectana) carpocapsae (see taxoeffectiveness is diminished rather than ennomic revision by Poinar, 1990) and found hanced. Debach (1958) has warned that “it that 8% or less of infective juveniles were must always be borne in mind that in modable to accurately orient to hosts within 1 ifying one character favorably others may hr. This finding may help to explain the ten- go in the opposite direction,” and suggests dency of steinemematid nematodes to re- that suitable laboratory tests be made to main at or near the point of application check for alteration of other traits which (Moyle and Kaya, 1981; Georgis and Poimight preclude field success. Similarly, nar, 1983), which presumably increases Hoy (1979) notes that other desirable atnematode sensitivity to inactivation from tributes may be lost during a selection proenvironmental extremes when they are ap- gram that could affect the success of field plied to exposed surfaces. An approach to releases, and so one component of her resolving this limitation and closing the ef- scheme for genetic improvement is an evalficacy gap between nematodes and chemiuation of fitness. Some fitness parameters, cals would be to develop genetically altered such as dispersal and over-wintering ability strains with enhanced host-finding abilities may be inconsequential for agents such as 106 0022-2011190 $1.50 Copyright Q 1990 by Academic F’ress, Inc. AU rights of reproduction in any form reserved.
FITNESS
OF
S. carpocapsae
entomopathogenic nematodes that are primarily intended for inundative release; others are directly (e.g., pathogenicity) or indirectly (e.g., reproductive potential) relevant, and their impairment would reduce their effective use. A genetic improvement scheme adapted from DeBach (1958) and Hoy (1979) was devised for entomopathogenic nematodes (Gaugler et al., 1989b) and used to select a strain of S. carpocapsae for increased hostfinding (Gaugler et al., 1989a). Key elements of this scheme were provision of adequate genetic variability to reduce the frequency of deleterious alleles and an evaluation of fitness. Selection yielded an improvement in the host-finding abilities of infective juvenile nematodes of more than 20-fold after 13 selections, while the proportion of infectives initiating movement toward the host increased from 32 to 80% and nonmigrating nematodes decreased from 33 to 8% (Gaugler et al., 1989a). We report laboratory studies designed to determine whether fitness parameters important to S. carpocapsae field efficacy as inundative agents have been compromised by our genetic improvement approach. MATERIALS
AND METHODS
Culture sources. Three S. carpocapsae strains were compared: (1) the All strain originated from cultures obtained from Biosys, Palo Alto, California (because this strain is commercially available it was included as a standard for comparison), (2) the Foundation strain which was constructed through round-robin matings of 10 diverse geographical isolates as described by Gaugler et al. (1989b) and which served as the parental strain for (3) a strain (designated G-13) selected for enhanced hostfinding against Galleria larvae for 13 selection rounds (Gaugler et al., 1989a). Procedures were conducted with infective-stage juveniles cultured in last-instar larvae of the greater wax moth, Galleria mellonella, at 25°C according to methods described by
STRAINS
107
Dutky et al. (1964). Only infectives harvested 3-5 days after initial nematode emergence from the host were used in these experiments. Selected G- 13 strain infectives had been reared through four or less Galleria rearing cycles in the absence of selection pressure; previous studies have shown no significant effect on host-finding ability when selection was relaxed for so few rearing cycles (Gaugler et al., 1989a). Pathogenicity. Because pathogenicity is essential to efficacy, a determination of whether nematode ability to cause lethal infections had been affected by selection was made using two different methods: the standard Petri dish (Dutky et al., 1964) and oneon-one (Georgis, 1990) bioassays. The Petri dish assay was performed by placing 10 last-instar Galleria larvae (surface sterilized in 0.1% sodium hypochlorite for 1 min) in a 100 x 15mm Petri dish containing a 9.0-cm filter paper. Infective juveniles suspended in 0.85 ml of deionized water were pipetted onto the paper at concentrations of 10, 20,40, 80, and 160 infectives/dish. Controls were treated with deionized water. The dish was incubated in darkness at 25°C and 100% RH, and larval mortality was recorded at 12-hr intervals over a 96-hr test period. Each dosage for each strain was replicated five times. The one-on-one method uses tissue culture cell wells (24-well dishes) to confine a single infective nematode (suspended in 0.15 ml of deionized water) together with a single surface-sterilized Galleria larvae and a 1.3-cm filter paper. Dishes were incubated as above and mortality was recorded 5 days postexposure. Each nematode strain was replicated 11 times (replicates l-4 had 16 larvae/strain; replicate No. 5 had 32 larvae/strain; replicates 6-11 had 24 larvae/ strain). Sex ratio. Female/male ratios for the three strains were determined by exposing one surface-sterilized Galleria larvae to 50 infective juveniles in a 60 X 15-mm Petri dish with a 5.0-cm filter paper and 0.4 ml of
108
GAUGLER,
CAMPBELL,
deionized water. The dish was incubated as above, cadavers were dissected 5 days later, and the sex of all nematodes was noted. Each strain was replicated 20 times in two blocks of 10 replicates. Host penetration. Experimental procedures were as described above for sex ratio. The total number of nematodes infecting each host was recorded and divided by 50 to determine the proportion of available nematodes initiating successful infections. Reproductive potential. This trait was measured using the standard Petri dish assay described above under pathogenicity except that each dish was inoculated with 500 infective juveniles. Cadavers were transferred to White (1927) nematode traps 5 days post-treatment, and traps were harvested over a 24-day test period. Infective stages were counted and the number of infectives produced per Galleria was determined. There were four replicates for each strain. Storage stability. Preservation of infective stage viability under routine laboratory storage conditions was tested using two approaches: in water and on polyetherpolyurethane sponge. Water storage was evaluated by pipetting 20 ml of a nematode suspension containing 10,000 infective juveniles into a 100 x 15-ml Petri dish and placing the dish in darkness at 4°C. The water level was marked on the side of the dish and adjusted weekly to the original volume. Viability was determined at 0, 2, 6, 10, and 16 weeks by examining 250 infectives from each dish under the stereomicroscope. Criteria for nematode mortality included presence of an opaque esophageal region and absence of movement when touched with a probe. Each nematode strain was replicated five times. Sponge storage was evaluated by pipetting 6 ml of a nematode suspension containing 10,000 infective juveniles onto a piece of polyether-polyurethane sponge (5 mm thickness) fitting the bottom of a 100 x 15mm Petri dish and placing the dish in dark-
AND
MCGUIRE
ness at 4°C. The dish was checked weekly and deionized water (4°C) was added if the sponge appeared to have lost moisture. Viability was determined at 0, 4, 8, and 12 weeks as described above. Each strain was replicated five times. Motility. The Baermann funnel has been used previously to measure the motility of steinernematid nematodes (Gaugler and Boush, 1978). We adapted this method to compare the activity of our three strains. Our Baermann apparatus was constructed by dampening a single 9-cm filter paper (Fisher P8) and placing the paper onto a 60 x 15-mm Petri dish bottom filled with the deionized water so that the paper just contacted the water surface. One thousand infective juveniles suspended in 0.25 ml of water were pipetted onto the center portion of the filter paper and the dish was placed in darkness at 25°C. The dish bottom was replaced with a fresh dish hourly, and the number of infective stages migrating through the filter paper and into the dish was counted over a 4-hr test period. There were four replicates for each nematode strain. A second measure of motility was provided on an agar substrate using the hostfinding assay described by Gaugler et al. (1989a), except that no insect host was provided. Approximately 2000 infective juveniles were brushed onto the center of the assay plate inoculation zone (1 cm wide), held for 1 hr at 25”C, and the number of infective nematodes migrating out of the inoculating zone was recorded. Each strain was replicated with five bioassay plates. Morphology. Adult stages from the three strains were dissected from infected cadavers 5 days postexposure and examined under high magnification for physical anomalies. Five male and five female nematodes were examined from each of 10 cadavers per strain. Infective stages from each of the strains (100 infectives per strain) were also examined for patent defects. Statistical analysis. Probit analysis (SAS
FITNESS
OF S. carpocapsae
Instttute, 1985) was run on log,, transformed data for each replicate by variable time or dose to determine LTS, and LDSo values for the Petri dish pathogenicity assay. Distribution of proportional data from the one-on-one, host penetration, sex ratio, storage stability, and activity assays were normalized using the arcsine (square root) transformation. Balanced data were analyzed using the analysis of variance (ANOVA) procedure and unbalanced data were analyzed using the general linear models (GLM) procedure (SAS Institute, 1985). Duncan’s multiple range test (Duncan, 1955) was used for all multiple comparisons. The error term used for all Duncan’s multiple range tests was the residual mean square, except for the reproductive potential assay which used sample within replicate within strain. The level of significance for all comparisons was 0.05. RESULTS
AND DISCUSSION
Pathogenicity. Entomopathogenic nematodes possessing enhanced host-finding abilities but diminished pathogenicity would be of little use in biological control programs. Fortunately, acquisition of improved host-finding in S. carpocapsae did not involve any reduction in the ability of infective stages to cause lethal infections
COMPAIUSON
OF FITNESS
PARAMETERS
regardless of the bioassay method used to measure pathogenicity (Table 1). The Petri dish assay showed that the three nematode strains possessed an equivalently high degree of pathogenicity to Galleriu larvae, with mean lethal doses (LD,,) ranging from 2 to 3 infective-stage juveniles. The mean lethal time (LT,,) for host death to occur, which provides another measure of pathogenicity, similarly did not differ significantly among the strains at a dose of two nematodes per GalEeria larva (2Q nematodes per dish). At twice this dose, the selected G-13 strain still did not differ significantly from the other two strains, although the Foundation strain inexplicably induced slightly quicker host mortality than the All strain. Because pathogenicity in a population may be obscured by a few highly pathogenic individuals (a single infective juvenile can cause host mortality), an alternative assay that challenges larvae on a one-on-one basis was also conducted. This assay confirmed the results from the Petri dish assay, again showing that the three strains possessed a similar degree of pathogenicity against Galleria (Table 1). In Petri dish assays against larvae of the sweet potato weevil, Cylas formicarius elegantulus, which is also a very susceptible host, Jansson et al. (1990) similarly reported no difference in pathogenicity be-
TABLE 1 FOR THREE STRAINS
OF Steinernema
Pathogenicity Strain G-13 Foundation All
109
STRAINS
LDm”
LT,,” 0 (dose = 2)
LT,” (hr) (dose = 4)
% Host mortalityb
2.9 2 0.4Af 1.8 2 0.3A 1.7 + 0.5A
66.8 + 11.4A 46.8 k 6.5A 51.9 f 8SA
33.8 2 1.4AB 28.2 f 1.5B 40.3 f 3.1A
52 2 6.8A 46.2 + 6.6A 55.4 f 7A
% Host penetrationC 30 f 3.2A 11.2 f 1.4C 16.3 + 1.8B
Sex ratio (F:M)
carpocapsae Reproductive potent& @rogew x 10s)
53~41 t 3A 112.1 k 2.6A 5644 f 6A 100.2 k 6.8B 54% f 5A 85.1 k 3.X
a Determined using standard Petri dish bioassay. * Determined using one-on-one bioassay. c Proportion of infective stages exposed to Galleria larvae penetrating into the hemocoel. d The mean number of infective stages produced per last-instar Galleria larvae. e Movement on an agar substrate in the absence of a host. f Means (&SEM) in each column followed by the same capital letter are not signiticantly different range test [Duncan, 19551).
Motility’ 73.3 2 2.6AB 80.3 + 1.2A 63.1 f 4B
(P > 0.05, Duncan’s
multiple
110
GAUGLER,
CAMPBELL,
tween the All and G-13 strains. But in more realistic (i.e., semi-field) buried root assays, where host-pathogen contact was no longer established and host finding became important, the G-13 significantly outperformed both the All strain and the Hp-88 strain of Heterorhabditis bacteriphora. Host penetration. Host infection in entomopathogenic nematodes is largely a twostage process: host finding followed by host penetration. Selection for host-finding resulted in a significant increase in the proportion of infective stages penetrating to the host hemocoel (Table 1). That is, in a 60 x 15mm Petri dish, where host contact is assured and host-finding would not be expected to play a significant role, one nematode in three from the selected G-13 strain successfully penetrated the host. This was nearly twice that of the All strain and almost three times that of the Foundation strain. This result was unexpected since a higher incidence of penetration should have been reflected in higher pathogenicity as well, particularly in the one-on-one assay. We offer two competing hypotheses to explain these results. First, reliable recovery of the small infective stags from host tissue is difficult, so dissections were performed at 5 days postexposure when penetrating juveniles had developed into adults and were easily found. Because some nematodes initiating infection die before developing into adults (R. Gaugler, unpublished data), the differences observed might reflect differences in maturation success rather than penetration success. A second, and we believe more likely, explanation is that the penetration data are accurate but that the pathogenicity data are flawed. Galleria larvae appear to be so highly susceptible to nematode infection that our bioassays may lack sufficient resolution to discern pathogenicity differences between the strains. The same would hold true for Jansson et al.‘s (1990) Petri dish assays with sweet potato weevil larvae. Moreover, Bedding
AND
MCGUIRE
(1990) does not recommend Petri dish assays because they are too far removed from the natural situation. The lack of concurrence between our pathogenicity and host penetration data, and between Jansson et al.‘s (1990) Petri dish and buried root assays, support Bedding’s argument. The overall rate of penetration into hosts was low, ranging from 11% for the Foundation strain to 30% for the G-13 strain, although this is close to the report by Bednarek and Nowicki (1986) for S. carpocapsae. The inability of most individual infective juveniles to penetrate highly susceptible hosts confined in small dishes over a period of several days is cause for concern to biological control workers. Studies into the mechanism(s) regulating nematode penetration into the host once contact has been established are needed. Sex ratio. Selection for host-finding did not affect sex ratio, as ratios did not differ significantly among the three strains (Table 1). Females were slightly predominant, as the pooled F/M sex ratio for the three strains was 54:46. This is in agreement with the 57:43 sex ratio reported by Bednarek et al. (1986) for a Polish strain of S. carpocapsae. Sex ratio is an important attribute, not because it impacts directly on biological control efficacy, but because a skewed sex ratio would be expected to affect mass production efficiency. Moreover, sex ratio appears to be a trait sensitive to disruption during selection in some natural enemies (Roush and Hoy, 1981; Croft and McMurtry, 1972) and might be indicative of further genetic alterations. The stability of sex ratio in our selected strain is another positive signal that overall fitness has not been critically disrupted. Reproductive potential. There were significant differences between all three strains in the yield of infective stages reared in vivo in Galleria larvae (Table 1). These difference strongly favored the selected G13 strain, which produced 30% more nem-
111
FITNESS OF S. carpocapsae STRAINS
atodes per host than the commercially available All strain, and 12% more than the parental Foundation strain. The Foundation strain outperformed the All strain by 17%. Additionally, several billion infective stages of the G-13 strain have been produced in vitro in liquid monoxenic culture (R. Georgis, personal communication). The test design is too limited to explain the fortuitous increase in reproductive potential. Storage stability. A negative response to host-finding selection was noted in the ability of G-13 infective juveniles to remain viable during storage (Figs. 1, 2). Two different laboratory methods of storing nematodes provided similar results: the All and Foundation strains showed virtually identical storage characteristics, while the selected strain began to show inferior viability after 6-8 weeks. The storage dissimilarities between strains were most pronounced during sponge storage (Fig. l), where 18.3% of selected strain infectives remain viable compared to 56.4 and 52.2% in the All and Foundation strains when the test was ended after 12 weeks; however, this difference was not statistically significant due to considerable data variation.
OALL
s s 5
Less variability was noted in juvenile viability during storage in water (Fig. 2), where significant differences were noted in nematode viability between the selected (37.2%) compared to the All (63.4%) and Foundation (62.1%) strains by the sixth week. Further fluctuations did not occur over the remaining 10 weeks of the experiment. Ironically, in attempting to provide a steinernematid with host-seeking abilities comparable to heterorhabditid nematodes, we may have also provided our G-13 strain with the inferior storage characteristic of heterorhabditids. Reduced storage stability might be a “trade-off’ for increased reproductive potential, as G-13 strain nematodes competing for limited nutrients may produce infective stages possessing less food reserves. These unfavorable results clearly cannot be attributed to increased activity that burns up reserves at an accelerated rate since these nematodes show no evidence of hyperactivity (Gaugler et al., 1989a) and low temperature storage restricts movement. Little research has been reported on factors limiting storage stability.
0 0
40--
FOUN G-13
Z
8z
20-0
I 0
I 2
4
I 6
a
I
I 10
12
STORAGE TIME (WKS) FIG. 1. Storage stability of infective-stage juveniles of three strains of Steinernema carpocapsae held at 4°C on a polyether-polyurethane sponge.
112
GAUGLER,
c 2 2 5 ::w G I
CAMPBELL,
AND
MCGUIRE
80--
60--
1
0
T 40--
2
0
G-13
J-0-J 1
1
1
bQ 20--
STORAGE TIME (WKS) 2. Storage stability of infective-stage juveniles of three strains of Steinernema carpocapsae held at 4°C in water. FIG.
Detrimental effects of selection are important only if they prevent a natural enemy from performing its assigned function (Roush, 1979). If G-13 nematodes are applied soon after production, as is the current practice for all species and strains of entomopathogenic nematodes, their biological control effectiveness would not be hindered. Interestingly, the Kapow strain of S. carpocapsae, which was selected for rapid development (J. Lindegren, personal communication), loses viability rapidly after only 4 weeks of aerated water storage (Capinera et al., 1988), yet remains viable for months when stored on moist plaster of paris (H. Kaya, personal communication). This suggests that the inferior storage stability noted in G-13 nematodes stored in water and on sponge might be overcome or rendered less significant by rapidly improving storage technology (Georgis, 1990). Motility. The motility of G-13 strain infectives in the absence of an insect host appeared little affected by selection. Although All strain infective stages initially passed through the Baermann apparatus at a high rate relative to the other strains, there were no significant differences among the strains
when the test was ended (Fig. 3). In the host-finding assay (Table l), the All strain exhibited significantly lower motility than the Foundation strain, but G-13 strain motility did not differ from either of these strains. These findings lend further support to our earlier visual observations that selection did not result in hyperactive nematodes (Gaugler et al., 1989b) and that enhanced host-finding is not a result of increased undirected activity. Morphology. No morphological anomalies were found in any of the strains, nor were they expected, since large breeding populations were maintained at each rearing cycle. GENERAL DISCUSSION
Acquiring enhanced host-finding abilities does not result in a serious loss of overall fitness, and the potential of the selected G13 strain for inundative biological control appears unlikely to be impaired. We believe these generally favorable results are attributable to having maximized genetic variation in constructing our parental strain through round-robin crosses of 10 different geographical isolates (Gaugler et al.,
FITNESS OF S. carpocapsae
113
STRAINS
70 60 50 40 30 20
/
10
1
0
G-13
0 0
1
2
3
4
TIME (HR) FIG. 3. Vigor of infective-stage juveniles of three strains of Steinernema by migration through a modified Baermann apparatus.
1989b). In addition to maximizing the number of variable loci and providing multiple alleles at a locus, such round-robin crosses can break up strain-specific coadapted gene complexes and/or epistatic combinations that might be responsible for genetic homeostasis (i.e., the tendency for natural selection to resist changes in gene frequency due to artificial selection). White et al. (1970) took a similar approach in crossing four strains and irradiating some lines before imposing selection on Aphytis lingnanensis. In addition, the ease with which entomopathogenic nematodes can be reared and handled assured large populations, reducing the likelihood of inbreeding depression. The fitness of entomopathogenic nematodes depends to a great extent on Xenorhabdus spp. bacteria that are associated with the nematodes. Despite hybridizing 10 isolates of S. carpocupsae to create the parental strain and 13 rounds of selection, there is no evidence that the mutualistic relationship between these two organisms has been unfavorably disturbed. Our data show that those nematode life cycle parameters in which Xenorhabdus spp. bacteria are
carpocapsae
as measured
known to participate, pathogenicity and reproductive potential, were as good or better than the All strain. Selection for any trait can be accompanied by changes in the genome that alter the expression of a second trait; most often these changes produce a decrease in fitness (Falconer, 1981). Beglyarov et al. (1978), for example, reported a loss in biotic potential in phytoseiid mites selected for insectitide resistance and Roush and Hoy (1981) found that resistant mites developed more slowly and were less fecund than susceptible strains. However, there are also examples of selection proceeding without adverse affects on main fitness parameters, as reported by Foumier et al. (1988), again with insecticide resistance in phytoseiid mites. And there are rare instances of favorable alterations in fitness, as in White et al’s (1970) classic work in improving the climatic tolerance of the parasitoid A. lingnanensis and achieving the unexpected side effect of increased general hardiness. Our results also suggest that it is possible to genetically improve a natural enemy without seriously reducing overall fitness. Four of the seven fitness parameters we
114
GAUGLER.
CAMPBELL,
measured remained unchanged in the G-13 line despite 13 rounds of selective breeding. Such a zero response indicates that these traits are independent (genetically uncorrelated) of increased host-finding. Two parameters, host penetration and reproductive potential showed a significant increase in fitness and one parameter, storage stability, showed a significant decrease in fitness in the G-13 strain. These traits are phenotypically correlated with increased hostfinding but they are not necessarily genetically correlated. McGuire (1983) has discussed some of the reasons why two traits might be phenotypically correlated in selected strains: (1) chance, (2) inadvertent selection within the confines of the selection procedure, (3) physical linkage of genes on the chromosomes, or (4) gene pleiotropy (genetic correlation), Any of these reasons might account for our results, but only pleiotropy has the potential to significantly influence the fitness of a genetically improved strain. Two traits within a selected line might become correlated by chance due to sampling error or genetic drift. If only a few pairs of parents are used to establish each selected generation, then it is possible to fix alleles of other independent genes. The phenotype associated with those alleles would then show a positive or negative “correlation” with the selected phenotype. Genetic drift is unlikely to have occurred in our study since hundreds of parents were used in each generation. Inadvertent selection could cause two traits to be correlated if the animals were selected for a second trait as a side effect of the experimental procedure. For example, Murphey (1969) showed that Drosophila melunogaster, which had been selected for negative geotaxis, had also been inadvertently selected for desiccation resistance. That is, flies had to survive for many hours in a dry maze. Inadvertent selection might also account for the inferior storage of the G-13 strain as compared to the wild-type strains. We tested infective stages har-
AND
MCGUIRE
vested directly from White traps for each selection round in the G-13 strain; they were never placed in storage prior to testing (Gaugler et al., 1989a). On the other hand, nematodes from the All and Foundation strains were only irregularly reared in Galleria larvae and were often stored for long periods. Essentially then, the G-13 strain was under no selection for storage stability while the All and Foundation strains were actively, although inadvertently, selected. If the negative association between hostfinding and storage stability in G-13 nematodes is due to inadvertent selection, the further selective breeding should improve storage capability. Inadvertent selection probably does not account for the differences in reproductive potential and host penetration between G-13 and the two wildtype strains as there was no difference in the Galleria infection procedure between these strains. The third possibility is that major genes influencing host-finding behavior are genetically linked with major genes for host penetration, reproductive potential, and storage stability. If genes for all four traits were linked in the Foundation strain then the genes for the three fitness characters could have “hitchhiked” with the genes for superior host-finding and become fixed in the G-13 strain. It is unlikely that major genes for four major traits would occur so close together on a single chromosome; however, the presence or absence of such linkage would have to be determined by careful crosses and genetic analyses. Such analyses would be extremely difficult given our current knowledge of entomopathogenic nematode genetics. Finally, traits might be correlated due to pleiotropy. That is, all or some of the genes associated with the phenotype of improved host-finding would be associated with the phenotypes of increased reproduction, better host penetration, and lower storage stability. If true for storage stability, then we have increased the host-finding ability of the nematode at the expense of infective
FITNESS OF S. carpocapsae
stage longevity. Ultimately, the exact nature of the three observed correlations must be determined by additional genetic analyses. This study enhances the prospects for using laboratory selection as a means of increasing the biological control potential of entomopathogenic nematodes. It is hoped that others will be encouraged to adopt genetic improvement techniques for inundative biological control agents. Our results also suggest that it would be advantageous to apply agricultural quantitative genetics to the genetic improvement of nematodes. Although some types of genetic analyses are not possible due to the breeding system of steinernematid nematodes (e.g., the difficulty of obtaining F-l infective stages), it is still possible to obtain estimates of phenotypic variability of important traits, narrow heritability under specified conditions, and the genetic correlations between traits. With this kind of basic information, it would be possible to design efficient selection procedures that maximize the response to selection of one or more traits and minimize or eliminate any correlated fitness loss. ACKNOWLEDGEMENTS Support from U.S. Department of Agriculture Competitive Research Grant 87-CRCR-l-2298 is gratefully acknowledged. New Jersey Agricultural Experiment Station Publication No. D-08251-18-89, supported by state funds.
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GAUGLER, R., MCGUIRE, T. R., AND CAMPBELL, J. F. 1989b. Genetic variability among strains of entomopathogenic nematode Steinernema feltiae. Z. Nematol.,
21, 247-253.
GEORGIS, R. 1990. Formulation and application technology. In “Entomopathogenic Nematodes for Biological Control” (R. Gaugler and H. K. Kaya, Eds.). CRC Press, Boca Raton, FL, in press. GEORGIS, R., AND POINAR, G. O., JR. 1983. Effect of soil texture on the distribution and infectivity of Neoaplectana carpocapsae (Nematoda: Steinemematidae). Z. Nematol., 15, 308-312. HOY, M. A. 1979. The potential for genetic improvement of predators for pest management programs. In “Genetics in Relation to Insect Management”
116
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(M. A. Hoy and J. J. McKelvey, Jr., Eds.), pp. 106 115. Rockefeller Foundation Press, New York. JANSSON, R., LECRONE, S. H., GAUGLER, R., AND SMART, G. C. 1990. Potential of entomopathogenic nematodes as biological control agents of sweetpotato weevil (Coleoptera: Curculionidae). .Z. .&on. Entomol., in press. MCGUIRE, T. R. 1983. Further evidence for a relationship between central excitatory state (CES) and classical conditioning. Behav. Genetics, 13, 509515. MOYLE, P. L., AND KAYA, H. K. 1981. Dispersal and infectivity of the entomogenous nematode, Neouplectuna carpocupsue Weiser (Rhabditida: Steinemematidae), in sand. J. Nemutol., 13, 295-300. MURPHEY, R. M. 1969. Spatial discrimination performance of Drosophila melanoguster: Some controlled and uncontrolled correlates. Anim. Behuv., 17, 43-56.
POINAR, G. O., JR. 1990. Biology and taxonomy of Steinemematidae and Heterorhabditidae. In “Ento-
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mopathogenic Nematodes for Biological Control” (R. Gaugler and H. K. Kaya, Eds.). CRC Press, Boca Raton, FL, in press. ROUSH, R. T. 1979. Genetic improvement of parasites. In “Genetics in Relation to Insect Management” (M. A. Hoy and J. J. McKelvey, Jr., Eds.), pp. 97105. Rockefeller Foundation Press, New York. ROUSH, R. T., AND HOY, M. A. 1981. Laboratory, glasshouse, and field studies of artificially selected carbaryl resistance in Metuseiulus occidentulis. J. Econ. Entomol., 74, 142-147. SAS Institute 1985. SAS user’s guide: Statistics. SAS Institute, Gary, NC. WHITE, G. F. 1927. A method for obtaining infective nematode larvae from cultures. Science, 66, 302303. WHITE, E. B., DEBACH, P., AND GARBER, M. J. 1970. Artificial selection for genetic adaptation to temperature extremes in Aphytis lingnunensis Compere (Hymenoptera: Aphelinidae). Hilgurdiu, 40, 161191.
OF INVERTEBRATE
56, 106-l 16 (190)
PATHOLOGY
Fitness of a Genetically RANDYGAUGLER,* *Department
of Entomology
Improved
Entomopathogenic
JAMES F. CAMPBELL,* and TDepartment New Brunswick,
Nematode
AND TERRY R. MCGuIREt
of Biological Sciences, New Jersey 08903
Rutgers
University,
Received August 24, 1989; accepted November 13, 1989 A strain of Steinernema carpocapsae (G-13) selectively bred for improved host-finding was compared against two 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, 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 three correlated responses to selection are discussed. o 1990 Academic press, hc. KEY WORDS: Steinernema carpocapsae; host finding; genetic improvement; entomopathogenic nematode; fitness.
INTRODUCTION
that would quickly initiate host-searching movements. Research on the development of entoGenetic improvement has been used by mopathogenic nematodes for biological biological control workers to select insect control has increased exponentially in re- predators and parasitoids in the laboratory cent years, but implementation remains for traits ranging from improved climatic hampered by questionable field perfortolerance to pesticide resistance. However, mance when compared to chemical insectigenetic improvement remains controvercides (Gaugler, 1988). One factor that may sial, mostly because many workers feel labbe limiting the efficacy of steinemematid oratory selection programs reduce genetic species as biological insecticides is the poor variability and introduce correlated deletehost-finding capabilities of infective-stage rious pleiotropic effects (Roush, 1979). juveniles. Gaugler et al. (I989b) screened That is, selection reduces overall fitness to 21 geographical isolates of Steinernema the point that the “improved” agent’s field ( = Neoaplectana) carpocapsae (see taxoeffectiveness is diminished rather than ennomic revision by Poinar, 1990) and found hanced. Debach (1958) has warned that “it that 8% or less of infective juveniles were must always be borne in mind that in modable to accurately orient to hosts within 1 ifying one character favorably others may hr. This finding may help to explain the ten- go in the opposite direction,” and suggests dency of steinemematid nematodes to re- that suitable laboratory tests be made to main at or near the point of application check for alteration of other traits which (Moyle and Kaya, 1981; Georgis and Poimight preclude field success. Similarly, nar, 1983), which presumably increases Hoy (1979) notes that other desirable atnematode sensitivity to inactivation from tributes may be lost during a selection proenvironmental extremes when they are ap- gram that could affect the success of field plied to exposed surfaces. An approach to releases, and so one component of her resolving this limitation and closing the ef- scheme for genetic improvement is an evalficacy gap between nematodes and chemiuation of fitness. Some fitness parameters, cals would be to develop genetically altered such as dispersal and over-wintering ability strains with enhanced host-finding abilities may be inconsequential for agents such as 106 0022-2011190 $1.50 Copyright Q 1990 by Academic F’ress, Inc. AU rights of reproduction in any form reserved.
FITNESS
OF
S. carpocapsae
entomopathogenic nematodes that are primarily intended for inundative release; others are directly (e.g., pathogenicity) or indirectly (e.g., reproductive potential) relevant, and their impairment would reduce their effective use. A genetic improvement scheme adapted from DeBach (1958) and Hoy (1979) was devised for entomopathogenic nematodes (Gaugler et al., 1989b) and used to select a strain of S. carpocapsae for increased hostfinding (Gaugler et al., 1989a). Key elements of this scheme were provision of adequate genetic variability to reduce the frequency of deleterious alleles and an evaluation of fitness. Selection yielded an improvement in the host-finding abilities of infective juvenile nematodes of more than 20-fold after 13 selections, while the proportion of infectives initiating movement toward the host increased from 32 to 80% and nonmigrating nematodes decreased from 33 to 8% (Gaugler et al., 1989a). We report laboratory studies designed to determine whether fitness parameters important to S. carpocapsae field efficacy as inundative agents have been compromised by our genetic improvement approach. MATERIALS
AND METHODS
Culture sources. Three S. carpocapsae strains were compared: (1) the All strain originated from cultures obtained from Biosys, Palo Alto, California (because this strain is commercially available it was included as a standard for comparison), (2) the Foundation strain which was constructed through round-robin matings of 10 diverse geographical isolates as described by Gaugler et al. (1989b) and which served as the parental strain for (3) a strain (designated G-13) selected for enhanced hostfinding against Galleria larvae for 13 selection rounds (Gaugler et al., 1989a). Procedures were conducted with infective-stage juveniles cultured in last-instar larvae of the greater wax moth, Galleria mellonella, at 25°C according to methods described by
STRAINS
107
Dutky et al. (1964). Only infectives harvested 3-5 days after initial nematode emergence from the host were used in these experiments. Selected G- 13 strain infectives had been reared through four or less Galleria rearing cycles in the absence of selection pressure; previous studies have shown no significant effect on host-finding ability when selection was relaxed for so few rearing cycles (Gaugler et al., 1989a). Pathogenicity. Because pathogenicity is essential to efficacy, a determination of whether nematode ability to cause lethal infections had been affected by selection was made using two different methods: the standard Petri dish (Dutky et al., 1964) and oneon-one (Georgis, 1990) bioassays. The Petri dish assay was performed by placing 10 last-instar Galleria larvae (surface sterilized in 0.1% sodium hypochlorite for 1 min) in a 100 x 15mm Petri dish containing a 9.0-cm filter paper. Infective juveniles suspended in 0.85 ml of deionized water were pipetted onto the paper at concentrations of 10, 20,40, 80, and 160 infectives/dish. Controls were treated with deionized water. The dish was incubated in darkness at 25°C and 100% RH, and larval mortality was recorded at 12-hr intervals over a 96-hr test period. Each dosage for each strain was replicated five times. The one-on-one method uses tissue culture cell wells (24-well dishes) to confine a single infective nematode (suspended in 0.15 ml of deionized water) together with a single surface-sterilized Galleria larvae and a 1.3-cm filter paper. Dishes were incubated as above and mortality was recorded 5 days postexposure. Each nematode strain was replicated 11 times (replicates l-4 had 16 larvae/strain; replicate No. 5 had 32 larvae/strain; replicates 6-11 had 24 larvae/ strain). Sex ratio. Female/male ratios for the three strains were determined by exposing one surface-sterilized Galleria larvae to 50 infective juveniles in a 60 X 15-mm Petri dish with a 5.0-cm filter paper and 0.4 ml of
108
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CAMPBELL,
deionized water. The dish was incubated as above, cadavers were dissected 5 days later, and the sex of all nematodes was noted. Each strain was replicated 20 times in two blocks of 10 replicates. Host penetration. Experimental procedures were as described above for sex ratio. The total number of nematodes infecting each host was recorded and divided by 50 to determine the proportion of available nematodes initiating successful infections. Reproductive potential. This trait was measured using the standard Petri dish assay described above under pathogenicity except that each dish was inoculated with 500 infective juveniles. Cadavers were transferred to White (1927) nematode traps 5 days post-treatment, and traps were harvested over a 24-day test period. Infective stages were counted and the number of infectives produced per Galleria was determined. There were four replicates for each strain. Storage stability. Preservation of infective stage viability under routine laboratory storage conditions was tested using two approaches: in water and on polyetherpolyurethane sponge. Water storage was evaluated by pipetting 20 ml of a nematode suspension containing 10,000 infective juveniles into a 100 x 15-ml Petri dish and placing the dish in darkness at 4°C. The water level was marked on the side of the dish and adjusted weekly to the original volume. Viability was determined at 0, 2, 6, 10, and 16 weeks by examining 250 infectives from each dish under the stereomicroscope. Criteria for nematode mortality included presence of an opaque esophageal region and absence of movement when touched with a probe. Each nematode strain was replicated five times. Sponge storage was evaluated by pipetting 6 ml of a nematode suspension containing 10,000 infective juveniles onto a piece of polyether-polyurethane sponge (5 mm thickness) fitting the bottom of a 100 x 15mm Petri dish and placing the dish in dark-
AND
MCGUIRE
ness at 4°C. The dish was checked weekly and deionized water (4°C) was added if the sponge appeared to have lost moisture. Viability was determined at 0, 4, 8, and 12 weeks as described above. Each strain was replicated five times. Motility. The Baermann funnel has been used previously to measure the motility of steinernematid nematodes (Gaugler and Boush, 1978). We adapted this method to compare the activity of our three strains. Our Baermann apparatus was constructed by dampening a single 9-cm filter paper (Fisher P8) and placing the paper onto a 60 x 15-mm Petri dish bottom filled with the deionized water so that the paper just contacted the water surface. One thousand infective juveniles suspended in 0.25 ml of water were pipetted onto the center portion of the filter paper and the dish was placed in darkness at 25°C. The dish bottom was replaced with a fresh dish hourly, and the number of infective stages migrating through the filter paper and into the dish was counted over a 4-hr test period. There were four replicates for each nematode strain. A second measure of motility was provided on an agar substrate using the hostfinding assay described by Gaugler et al. (1989a), except that no insect host was provided. Approximately 2000 infective juveniles were brushed onto the center of the assay plate inoculation zone (1 cm wide), held for 1 hr at 25”C, and the number of infective nematodes migrating out of the inoculating zone was recorded. Each strain was replicated with five bioassay plates. Morphology. Adult stages from the three strains were dissected from infected cadavers 5 days postexposure and examined under high magnification for physical anomalies. Five male and five female nematodes were examined from each of 10 cadavers per strain. Infective stages from each of the strains (100 infectives per strain) were also examined for patent defects. Statistical analysis. Probit analysis (SAS
FITNESS
OF S. carpocapsae
Instttute, 1985) was run on log,, transformed data for each replicate by variable time or dose to determine LTS, and LDSo values for the Petri dish pathogenicity assay. Distribution of proportional data from the one-on-one, host penetration, sex ratio, storage stability, and activity assays were normalized using the arcsine (square root) transformation. Balanced data were analyzed using the analysis of variance (ANOVA) procedure and unbalanced data were analyzed using the general linear models (GLM) procedure (SAS Institute, 1985). Duncan’s multiple range test (Duncan, 1955) was used for all multiple comparisons. The error term used for all Duncan’s multiple range tests was the residual mean square, except for the reproductive potential assay which used sample within replicate within strain. The level of significance for all comparisons was 0.05. RESULTS
AND DISCUSSION
Pathogenicity. Entomopathogenic nematodes possessing enhanced host-finding abilities but diminished pathogenicity would be of little use in biological control programs. Fortunately, acquisition of improved host-finding in S. carpocapsae did not involve any reduction in the ability of infective stages to cause lethal infections
COMPAIUSON
OF FITNESS
PARAMETERS
regardless of the bioassay method used to measure pathogenicity (Table 1). The Petri dish assay showed that the three nematode strains possessed an equivalently high degree of pathogenicity to Galleriu larvae, with mean lethal doses (LD,,) ranging from 2 to 3 infective-stage juveniles. The mean lethal time (LT,,) for host death to occur, which provides another measure of pathogenicity, similarly did not differ significantly among the strains at a dose of two nematodes per GalEeria larva (2Q nematodes per dish). At twice this dose, the selected G-13 strain still did not differ significantly from the other two strains, although the Foundation strain inexplicably induced slightly quicker host mortality than the All strain. Because pathogenicity in a population may be obscured by a few highly pathogenic individuals (a single infective juvenile can cause host mortality), an alternative assay that challenges larvae on a one-on-one basis was also conducted. This assay confirmed the results from the Petri dish assay, again showing that the three strains possessed a similar degree of pathogenicity against Galleria (Table 1). In Petri dish assays against larvae of the sweet potato weevil, Cylas formicarius elegantulus, which is also a very susceptible host, Jansson et al. (1990) similarly reported no difference in pathogenicity be-
TABLE 1 FOR THREE STRAINS
OF Steinernema
Pathogenicity Strain G-13 Foundation All
109
STRAINS
LDm”
LT,,” 0 (dose = 2)
LT,” (hr) (dose = 4)
% Host mortalityb
2.9 2 0.4Af 1.8 2 0.3A 1.7 + 0.5A
66.8 + 11.4A 46.8 k 6.5A 51.9 f 8SA
33.8 2 1.4AB 28.2 f 1.5B 40.3 f 3.1A
52 2 6.8A 46.2 + 6.6A 55.4 f 7A
% Host penetrationC 30 f 3.2A 11.2 f 1.4C 16.3 + 1.8B
Sex ratio (F:M)
carpocapsae Reproductive potent& @rogew x 10s)
53~41 t 3A 112.1 k 2.6A 5644 f 6A 100.2 k 6.8B 54% f 5A 85.1 k 3.X
a Determined using standard Petri dish bioassay. * Determined using one-on-one bioassay. c Proportion of infective stages exposed to Galleria larvae penetrating into the hemocoel. d The mean number of infective stages produced per last-instar Galleria larvae. e Movement on an agar substrate in the absence of a host. f Means (&SEM) in each column followed by the same capital letter are not signiticantly different range test [Duncan, 19551).
Motility’ 73.3 2 2.6AB 80.3 + 1.2A 63.1 f 4B
(P > 0.05, Duncan’s
multiple
110
GAUGLER,
CAMPBELL,
tween the All and G-13 strains. But in more realistic (i.e., semi-field) buried root assays, where host-pathogen contact was no longer established and host finding became important, the G-13 significantly outperformed both the All strain and the Hp-88 strain of Heterorhabditis bacteriphora. Host penetration. Host infection in entomopathogenic nematodes is largely a twostage process: host finding followed by host penetration. Selection for host-finding resulted in a significant increase in the proportion of infective stages penetrating to the host hemocoel (Table 1). That is, in a 60 x 15mm Petri dish, where host contact is assured and host-finding would not be expected to play a significant role, one nematode in three from the selected G-13 strain successfully penetrated the host. This was nearly twice that of the All strain and almost three times that of the Foundation strain. This result was unexpected since a higher incidence of penetration should have been reflected in higher pathogenicity as well, particularly in the one-on-one assay. We offer two competing hypotheses to explain these results. First, reliable recovery of the small infective stags from host tissue is difficult, so dissections were performed at 5 days postexposure when penetrating juveniles had developed into adults and were easily found. Because some nematodes initiating infection die before developing into adults (R. Gaugler, unpublished data), the differences observed might reflect differences in maturation success rather than penetration success. A second, and we believe more likely, explanation is that the penetration data are accurate but that the pathogenicity data are flawed. Galleria larvae appear to be so highly susceptible to nematode infection that our bioassays may lack sufficient resolution to discern pathogenicity differences between the strains. The same would hold true for Jansson et al.‘s (1990) Petri dish assays with sweet potato weevil larvae. Moreover, Bedding
AND
MCGUIRE
(1990) does not recommend Petri dish assays because they are too far removed from the natural situation. The lack of concurrence between our pathogenicity and host penetration data, and between Jansson et al.‘s (1990) Petri dish and buried root assays, support Bedding’s argument. The overall rate of penetration into hosts was low, ranging from 11% for the Foundation strain to 30% for the G-13 strain, although this is close to the report by Bednarek and Nowicki (1986) for S. carpocapsae. The inability of most individual infective juveniles to penetrate highly susceptible hosts confined in small dishes over a period of several days is cause for concern to biological control workers. Studies into the mechanism(s) regulating nematode penetration into the host once contact has been established are needed. Sex ratio. Selection for host-finding did not affect sex ratio, as ratios did not differ significantly among the three strains (Table 1). Females were slightly predominant, as the pooled F/M sex ratio for the three strains was 54:46. This is in agreement with the 57:43 sex ratio reported by Bednarek et al. (1986) for a Polish strain of S. carpocapsae. Sex ratio is an important attribute, not because it impacts directly on biological control efficacy, but because a skewed sex ratio would be expected to affect mass production efficiency. Moreover, sex ratio appears to be a trait sensitive to disruption during selection in some natural enemies (Roush and Hoy, 1981; Croft and McMurtry, 1972) and might be indicative of further genetic alterations. The stability of sex ratio in our selected strain is another positive signal that overall fitness has not been critically disrupted. Reproductive potential. There were significant differences between all three strains in the yield of infective stages reared in vivo in Galleria larvae (Table 1). These difference strongly favored the selected G13 strain, which produced 30% more nem-
111
FITNESS OF S. carpocapsae STRAINS
atodes per host than the commercially available All strain, and 12% more than the parental Foundation strain. The Foundation strain outperformed the All strain by 17%. Additionally, several billion infective stages of the G-13 strain have been produced in vitro in liquid monoxenic culture (R. Georgis, personal communication). The test design is too limited to explain the fortuitous increase in reproductive potential. Storage stability. A negative response to host-finding selection was noted in the ability of G-13 infective juveniles to remain viable during storage (Figs. 1, 2). Two different laboratory methods of storing nematodes provided similar results: the All and Foundation strains showed virtually identical storage characteristics, while the selected strain began to show inferior viability after 6-8 weeks. The storage dissimilarities between strains were most pronounced during sponge storage (Fig. l), where 18.3% of selected strain infectives remain viable compared to 56.4 and 52.2% in the All and Foundation strains when the test was ended after 12 weeks; however, this difference was not statistically significant due to considerable data variation.
OALL
s s 5
Less variability was noted in juvenile viability during storage in water (Fig. 2), where significant differences were noted in nematode viability between the selected (37.2%) compared to the All (63.4%) and Foundation (62.1%) strains by the sixth week. Further fluctuations did not occur over the remaining 10 weeks of the experiment. Ironically, in attempting to provide a steinernematid with host-seeking abilities comparable to heterorhabditid nematodes, we may have also provided our G-13 strain with the inferior storage characteristic of heterorhabditids. Reduced storage stability might be a “trade-off’ for increased reproductive potential, as G-13 strain nematodes competing for limited nutrients may produce infective stages possessing less food reserves. These unfavorable results clearly cannot be attributed to increased activity that burns up reserves at an accelerated rate since these nematodes show no evidence of hyperactivity (Gaugler et al., 1989a) and low temperature storage restricts movement. Little research has been reported on factors limiting storage stability.
0 0
40--
FOUN G-13
Z
8z
20-0
I 0
I 2
4
I 6
a
I
I 10
12
STORAGE TIME (WKS) FIG. 1. Storage stability of infective-stage juveniles of three strains of Steinernema carpocapsae held at 4°C on a polyether-polyurethane sponge.
112
GAUGLER,
c 2 2 5 ::w G I
CAMPBELL,
AND
MCGUIRE
80--
60--
1
0
T 40--
2
0
G-13
J-0-J 1
1
1
bQ 20--
STORAGE TIME (WKS) 2. Storage stability of infective-stage juveniles of three strains of Steinernema carpocapsae held at 4°C in water. FIG.
Detrimental effects of selection are important only if they prevent a natural enemy from performing its assigned function (Roush, 1979). If G-13 nematodes are applied soon after production, as is the current practice for all species and strains of entomopathogenic nematodes, their biological control effectiveness would not be hindered. Interestingly, the Kapow strain of S. carpocapsae, which was selected for rapid development (J. Lindegren, personal communication), loses viability rapidly after only 4 weeks of aerated water storage (Capinera et al., 1988), yet remains viable for months when stored on moist plaster of paris (H. Kaya, personal communication). This suggests that the inferior storage stability noted in G-13 nematodes stored in water and on sponge might be overcome or rendered less significant by rapidly improving storage technology (Georgis, 1990). Motility. The motility of G-13 strain infectives in the absence of an insect host appeared little affected by selection. Although All strain infective stages initially passed through the Baermann apparatus at a high rate relative to the other strains, there were no significant differences among the strains
when the test was ended (Fig. 3). In the host-finding assay (Table l), the All strain exhibited significantly lower motility than the Foundation strain, but G-13 strain motility did not differ from either of these strains. These findings lend further support to our earlier visual observations that selection did not result in hyperactive nematodes (Gaugler et al., 1989b) and that enhanced host-finding is not a result of increased undirected activity. Morphology. No morphological anomalies were found in any of the strains, nor were they expected, since large breeding populations were maintained at each rearing cycle. GENERAL DISCUSSION
Acquiring enhanced host-finding abilities does not result in a serious loss of overall fitness, and the potential of the selected G13 strain for inundative biological control appears unlikely to be impaired. We believe these generally favorable results are attributable to having maximized genetic variation in constructing our parental strain through round-robin crosses of 10 different geographical isolates (Gaugler et al.,
FITNESS OF S. carpocapsae
113
STRAINS
70 60 50 40 30 20
/
10
1
0
G-13
0 0
1
2
3
4
TIME (HR) FIG. 3. Vigor of infective-stage juveniles of three strains of Steinernema by migration through a modified Baermann apparatus.
1989b). In addition to maximizing the number of variable loci and providing multiple alleles at a locus, such round-robin crosses can break up strain-specific coadapted gene complexes and/or epistatic combinations that might be responsible for genetic homeostasis (i.e., the tendency for natural selection to resist changes in gene frequency due to artificial selection). White et al. (1970) took a similar approach in crossing four strains and irradiating some lines before imposing selection on Aphytis lingnanensis. In addition, the ease with which entomopathogenic nematodes can be reared and handled assured large populations, reducing the likelihood of inbreeding depression. The fitness of entomopathogenic nematodes depends to a great extent on Xenorhabdus spp. bacteria that are associated with the nematodes. Despite hybridizing 10 isolates of S. carpocupsae to create the parental strain and 13 rounds of selection, there is no evidence that the mutualistic relationship between these two organisms has been unfavorably disturbed. Our data show that those nematode life cycle parameters in which Xenorhabdus spp. bacteria are
carpocapsae
as measured
known to participate, pathogenicity and reproductive potential, were as good or better than the All strain. Selection for any trait can be accompanied by changes in the genome that alter the expression of a second trait; most often these changes produce a decrease in fitness (Falconer, 1981). Beglyarov et al. (1978), for example, reported a loss in biotic potential in phytoseiid mites selected for insectitide resistance and Roush and Hoy (1981) found that resistant mites developed more slowly and were less fecund than susceptible strains. However, there are also examples of selection proceeding without adverse affects on main fitness parameters, as reported by Foumier et al. (1988), again with insecticide resistance in phytoseiid mites. And there are rare instances of favorable alterations in fitness, as in White et al’s (1970) classic work in improving the climatic tolerance of the parasitoid A. lingnanensis and achieving the unexpected side effect of increased general hardiness. Our results also suggest that it is possible to genetically improve a natural enemy without seriously reducing overall fitness. Four of the seven fitness parameters we
114
GAUGLER.
CAMPBELL,
measured remained unchanged in the G-13 line despite 13 rounds of selective breeding. Such a zero response indicates that these traits are independent (genetically uncorrelated) of increased host-finding. Two parameters, host penetration and reproductive potential showed a significant increase in fitness and one parameter, storage stability, showed a significant decrease in fitness in the G-13 strain. These traits are phenotypically correlated with increased hostfinding but they are not necessarily genetically correlated. McGuire (1983) has discussed some of the reasons why two traits might be phenotypically correlated in selected strains: (1) chance, (2) inadvertent selection within the confines of the selection procedure, (3) physical linkage of genes on the chromosomes, or (4) gene pleiotropy (genetic correlation), Any of these reasons might account for our results, but only pleiotropy has the potential to significantly influence the fitness of a genetically improved strain. Two traits within a selected line might become correlated by chance due to sampling error or genetic drift. If only a few pairs of parents are used to establish each selected generation, then it is possible to fix alleles of other independent genes. The phenotype associated with those alleles would then show a positive or negative “correlation” with the selected phenotype. Genetic drift is unlikely to have occurred in our study since hundreds of parents were used in each generation. Inadvertent selection could cause two traits to be correlated if the animals were selected for a second trait as a side effect of the experimental procedure. For example, Murphey (1969) showed that Drosophila melunogaster, which had been selected for negative geotaxis, had also been inadvertently selected for desiccation resistance. That is, flies had to survive for many hours in a dry maze. Inadvertent selection might also account for the inferior storage of the G-13 strain as compared to the wild-type strains. We tested infective stages har-
AND
MCGUIRE
vested directly from White traps for each selection round in the G-13 strain; they were never placed in storage prior to testing (Gaugler et al., 1989a). On the other hand, nematodes from the All and Foundation strains were only irregularly reared in Galleria larvae and were often stored for long periods. Essentially then, the G-13 strain was under no selection for storage stability while the All and Foundation strains were actively, although inadvertently, selected. If the negative association between hostfinding and storage stability in G-13 nematodes is due to inadvertent selection, the further selective breeding should improve storage capability. Inadvertent selection probably does not account for the differences in reproductive potential and host penetration between G-13 and the two wildtype strains as there was no difference in the Galleria infection procedure between these strains. The third possibility is that major genes influencing host-finding behavior are genetically linked with major genes for host penetration, reproductive potential, and storage stability. If genes for all four traits were linked in the Foundation strain then the genes for the three fitness characters could have “hitchhiked” with the genes for superior host-finding and become fixed in the G-13 strain. It is unlikely that major genes for four major traits would occur so close together on a single chromosome; however, the presence or absence of such linkage would have to be determined by careful crosses and genetic analyses. Such analyses would be extremely difficult given our current knowledge of entomopathogenic nematode genetics. Finally, traits might be correlated due to pleiotropy. That is, all or some of the genes associated with the phenotype of improved host-finding would be associated with the phenotypes of increased reproduction, better host penetration, and lower storage stability. If true for storage stability, then we have increased the host-finding ability of the nematode at the expense of infective
FITNESS OF S. carpocapsae
stage longevity. Ultimately, the exact nature of the three observed correlations must be determined by additional genetic analyses. This study enhances the prospects for using laboratory selection as a means of increasing the biological control potential of entomopathogenic nematodes. It is hoped that others will be encouraged to adopt genetic improvement techniques for inundative biological control agents. Our results also suggest that it would be advantageous to apply agricultural quantitative genetics to the genetic improvement of nematodes. Although some types of genetic analyses are not possible due to the breeding system of steinernematid nematodes (e.g., the difficulty of obtaining F-l infective stages), it is still possible to obtain estimates of phenotypic variability of important traits, narrow heritability under specified conditions, and the genetic correlations between traits. With this kind of basic information, it would be possible to design efficient selection procedures that maximize the response to selection of one or more traits and minimize or eliminate any correlated fitness loss. ACKNOWLEDGEMENTS Support from U.S. Department of Agriculture Competitive Research Grant 87-CRCR-l-2298 is gratefully acknowledged. New Jersey Agricultural Experiment Station Publication No. D-08251-18-89, supported by state funds.
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