Invasion Rate as a Measure of Infectivity of Steinernematid Heterorhabditid Nematodes to Insects’
Received December 6, 1990; accepted April 23, 1991
The invasion rate of three steinernematids and two heterorhabditids into three larval stages of Spodoptera littoralis Boisduval (Lepidoptera: Noctuidae) and into fourth-instar larvae of four Lepidopteran specieswas recorded as a measure of nematode infectivity. The invasion rate was determined by recording the mortality levels of insect larvae exposedto infective juveniles of the various nematode strains for 2,4, and 6 hr. Nematode infectivity was finally expressed as LT,,, the length of exposure required to bring 50% mortality of the insects. In comparison, the LD,, values of all nematode-insect combinations were determined in a dose responseassay. Among the different larval sizes, the fastest invasion rate was recorded with the smallest larvae, for all nematode strains. The LT,, values increase in proportion to S. littoralis larval size, indicating reduction in susceptibility. Among the four insect speciestested Galleria mellonella was found to be the most susceptible with LT,, values two times lower than obtained with the other insects, for each nematode separately. The nematodes Steinernema carpocapsaestrains All and Mexican, S. glueeri, and the IS strain of Heterorhabditis sp. were found to be similarly infectious to the different larval sizes or the various speciestested, with LT,, values of l&3.7 hr, whereas the LT,, values obtained with Heterorhabditis bacteriophora HP88 are 630 times higher than those of the other nematode strains, indicating its poor infectivity. Insect susceptibility and nematode infectivity as measured by the invasion rate assay are compatible with the results obtained in the dosc+response assay. The present study stressesthe use of nematode invasion rate as a mean to evaluate steinernematids’ and heterorhabditids’ infectivity. o lwz Academic PMS. IN. KEY WORDS: Steinernema carpocapsae (strains All and Mexican); Steinernema glaseri; Heterorhabditk bacteriophora (strain HP88); Heterorhabditis sp. (strain IS); Spodoptera littoralis; Heliothis armigem; Earias insulana; Galleria mellonella; bioassay; biological control.
1 Contribution from the Agricultural Research Organization, Dagan, Israel. No. 3113-E, 1990 series.
90 oozz-2011/92 Copyright AI1 rights
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INTRODUCTION The entomopathogenic nematodes from the families Steinernematidae and Heterorhabditidae are known to be biological control agents which serve as alternative measures to chemical control of insect pests (Gaugler, 1981; Kaya, 1985; Poinar, 1986). The symbiotic bacteria Xenorhabdus spp. are carried in the intestines of the third-stage infective juvenile (IJ) (Poinar, 1979; Akhurst, 1983). The IJ invade and release the symbiotic bacteria in the insect’s hemocoel. The insect host dies within 24-48 hr due to septicemia. The nematodes feed upon bacteria and breakdown products, develop, and reproduce within the insect cadaver (Poinar, 1986). Insect mortality depends upon IJ invasion into the hemocoel and the release of the symbiotic bacteria. Therefore, the host range of steinernematids and heterorhabditids is very broad, including several hundred of insect species from 10 orders (Poinar, 1979, 1986). Despite this impressive spectrum of activity, considerable variation was found in the virulence of different nematode strains to specific insect (Bedding et al., 1983; Forschler and Nordin, 1988; Griffin et al., 1989; Simons and van der Schaaf 1986; Wright et al., 1988). Furthermore, host susceptibility differs among insect species (Bedding et al., 1983) or developmental stages (Kaya and Hara, 1980, 1981; Kondo, 1987; Sikora et al., 1979). These differences were determined by comparison of L&o (lethal concentration or LD,, (lethal dose), similarly to tests with chemical pesticides. Commonly, the pathogenicity tests were based on exposing the target pest to IJ of a specific nematode strain on moist filter paper in a petri dish (Beavers, 1984; Belair and Boivin, 1985; Forschler and Nordin, 1988; Kaya and Hara, 1980,198l; Miller, 1989). In other cases the nematodes were forced to pass a physical barrier, such as a sand column, before encountering the target host (Bedding et al., 1983; Molyneux and Bedding, 1984; Toba et al., 1983). In those tests mortality level at a given time was used as the sole criterion, avoiding the
AS A MEASURE
complexity of the factors influencing the mortality rate, i.e., the number of bacteria per IJ (Dunphy et al., 1985), the proportion of juveniles carrying the symbiont (Akhurst, 1983), the invasion rate of the IJ into the host hemocoel, the time it takes to release the bacteria, and finally the virulence of the symbiotic bacterium. With the increasing interest in practical and commercial use of these nematodes as biological control agents, many new populations of steinernematid and heterorhabditid were isolated from various locations in the world. The need for establishing a standard pathogenicity bioassay which will provide a common basis for comparisons of the various nematode isolates is becoming more and more apparent. In a recent study, differences in the invasion rate between various nematode strains into larvae of the Egyptian cotton leafworm, Spodoptera littoralis Boisduval (Lepidoptera: Noctuidae), were recorded (Glazer et al., 1990). The invasion rate was determined by exposing the insect larvae to each nematode strain for extended time periods. The differences were proportional to the mortality level obtained in a common dose response assay, for each nematode strain. It was then suggested that the invasion rate can serve as a criterion to measure the infectivity of different nematodes. The present study was aimed at determining the feasibility of using the invasion rate to measure to the infectivity of five nematode strains to three larval stages of S. littoralis, as well as to larvae of four different species belonging to the family Lepidoptera. MATERIALS
The following nematodes were used throughout this work: Steinernema carpocapsae Weiser, strains All and Mexican, S. glaseri Steiner, and Heterorhabditis bacteriophoru Poinar, strain HP88 all obtained from Biosys, Palo Alto, California, and an undescribed isolate of Heterorhabditis sp., strain IS, which originated in Israel. The nematodes were reared on the greater wax moth (Galleria mellonella) and stored at 8°C according to the method described by Poinar (1979). Larvae of S. littoralis were reared according to Glazer et al. (19901, Heliothis armigera according to Glazer and Navon (1990), Earias insulana according to Klein et al. (1983), and G. mellonella according to Poinar (1979). The invasion rate was elucidated by measuring the effect of exposing the insect larvae to the various nematode species for 2, 4, and 6 hr on the level of insect mortality. The different insect larvae were placed in 5-cm-diam petri dishes padded with filter paper (Whatman No. l), according to Glazer et al. (1990). A concentration of 200 IJ was used per dish. Thirty dishes were used for each nematode/insect combination. Ten insect larvae were removed from the petri dishes after each
exposure period and transferred into nematode-free dishes. Insect mortality was recorded 48 hr after each exposure period. Each experiment was repeated three times. The data obtained in this assay were used to calculate the LT,, values (the time of exposure required to bring about 50% mortality of the insects) for each nematode-insect combination by probit analysis using the Statistical Analysis System @AS Institute, 1982). The overall pathogenicity of the different nematode strains was determined, for comparison with the data obtained from the invasion rate test, using a doseresponse assay. The various insect larvae were exposed to IJs suspended in deionized water at concentration of 25, 50, 100, 200, and 400 nematodes/ml, in 5-cm-diam petri dishes padded with filter paper. Insect mortality was recorded 48 hr after inoculation. Three replicates of 20 insects each for each nematode/insect combination were used in this assay. The experiments were repeated three times. The data obtained in this assay were used to calculate the LD,, values (the dose required to kill 50% of the insects) for each nematodeinsect combination by probit analysis using the Statistical Analysis System @AS Institute, 1982). RESULTS
A comparison of LT,, and LD,, values as a measure of the susceptibility of various S. littoralis larval size is presented in Table 1. For all nematode strains, the lowest LT,, values were obtained with the smallest insects, indicating that this stage is the most susceptible to nematode infection. The LT,, values increase with the development of larvae, indicating that insect susceptibility decreases with increasing size. The LD,, values obtained in the dose-response assay are proportional to those obtained with the invasion rate assay, reconfirming the reduction in larval susceptibility with the increase in size. The data obtained from the invasion rate assay indicate that S. carpocapsae All and Mexican, S. glaseri, and the IS strain of Heterorhabditis sp. are similarly infectious to the insect larvae with LTso values between 1.0 and 3.7 hr (Table l), whereas the LT,, values obtained from the assay with H. bacteriophorcz HP88 are 6-30 times higher than those of the other nematode strains. The data obtained from the dose-response assay reconfirm the previous findings (Table l), but also show that the LD,, values for S. carpocapsae All are 1.4-4 times higher than for S. carpocapsae Mexican, S. glaseri or Heterorhabditis sp. IS, suggesting that the All strain is somewhat less infective. The susceptibility of the various lepidopteran species to the different nematode strains, as determined by the invasion rate assay as well as the dose-response assay, is presented in Table 2. In the invasion rate assay, the LT,, values obtained, for each nematode strain sepa-
of Different Steinernematids
and Heterorhabditids to Stages of Spodoptera LD,, Valuesa
2.1 (1.7-2.8) 2.4 (1.9-3.0) 1.6 (1.1-2.7)
Mexican S. glaseri Heterorhnbditis
(l.t-i.6) 7.6 (4.7-12)
a LT,,, the time of exposure required
31 (24-36) 25 (1731)
Mexican (l.Z.5) 1.8 (M-2.6)
S. glaseri Heterorhubditis
(1.c3.2) H. bacteriophora
(8% 158 (127-211)
& c& 158 (121-211)
(29% 23 (14-29) 257 (221308)
(2X0, 23.5 (18.5-34)
of the insects (hr). LD,,, dose required to kill 50% of the insects.
5-12 times higher than with the other nematodes, indicating the poor infectivity of this strain to the lepidopteran species tested. In the present study, the differences in insect susceptibility and nematode infectivity as measured by the effect of exposure time on insect mortality, i.e., invasion rate, are in general compatible with the differences measured in the dose-response assay. Furthermore, those results are in agreement with previous studies. The reduction in susceptibility of larvae as they increase in size has been demonstrated for S. littoralis by Sikora et al. (1979) and for S. litura by Kondo (1987). It has been well established that insect larvae belonging to the order Lepidoptera are susceptible to infection by steinernematids and heterorhabditids (Poinar, 1979). More specifically, the susceptibility of H. armigera has been shown by Dutky et al. (1956) and Glazer and Navon (1990), of S. littoralis by Sikora et al.
Species Measured Earins insulana
L’L 2.8 (2.2-3.7)
(2.0% 2.5 (m-3.2) 1.5 (1.2-2.3) 49 (29-61)
a LT,,, the time of exposure required to bring about 50% mortality
Lb, 51 (39-67) 35 (27-46)
3.6 (2.844) 3.2 (2.4-3.8)
TABLE 2 and Heterorhabditids LD,, Value8
2.7 w3-3.5) 1.8 (U-2.6) 2.5 (M-3.2) 15.0 (10.3-23)
(7-25) 14 (9-21) (2-11o?i
rately, with larvae of G. mellonella were approximately two times lower in comparison with the LT,, values obtained from the other insects. These data indicate that the larvae of G. mellonella are the most susceptible among the various insects tested. However, in the dose-response assay the results obtained only with H. bacteriophora HP88 also indicated that G. mellonella is the most susceptible among the various insect species with LT,, values at least two times higher than that obtained with other insects, resembling the results obtained in the invasion rate assay. As for the other nematode strains, the results obtained in this assay indicate that all four insects are similarly susceptible to infection by each nematode separately, with LDso values ranging between 12 and 31 IJs/insect. A comparison between the infectivity of the various nematode strains (Table 2) shows that the LT,, and LDsO values obtained with H. bacteriophora HP88 are
to bring about 50% mortality
Larval size (200300 mg)
(30-50 mg) Nematode species
&7) 8.5 (4-14) 177 (lOS259)
LT,o 3.0 (2.34.1) 3.7 (294.5) 2.8 (1.9-3.6) 1.6 (0.9-2.0) (2.L.l)
Galleria mellonella LQw
(5-E) 13 (8-17) (12-& 109 (81-143)
of the insects (hr). LD,,, dose required
L’bo (0.5% 1.0 (0.6-1.7) 1.4 (0.8-2.1, 1.4 (0.8-2.0) (E4.1)
J&io 20 (11-26) 12 (7-18) 10 (7-14)
to kill 50% of the insects.
RATE AS A MEASURE
(1979) and ofE. insuZana by Laumond et al. (1979). The wax moth G. mellonella has been used as a suitable host for rearing and studying steinernematid and heterorhabditid nematodes since Dutky and Hough (1955) demonstrated its high susceptibility to nematode infection. The present finding emphasizes the inferiority of H. bacteriophora HP88 as a biocontrol agent against Lepidoptera as compared with other steinernematids and heterorhabditids. Exposure time as a criterion to evaluate steinernematid and heterorhabditid activity was used in previous studies. Molta and Hominick (1989) measured the infectivity of S. feltiae (= S. carpocapsae) and H. heliothidis (= H. bacteriophoru) against third instars of the yelllowfever mosquito Aedes aegypti larvae by dos+response assay as well as exposure time assay; they found that larval mortality showed a positive linar correlation with both nematode dosage and duration of exposure. Reardon et al. (1986) used the exposure time assay to show that the gypsy moth Lymantria dispar is more susceptible to S. felt&e than S. bibionis ( = S. feltiae). In that study 16 hr of exposure were needed to determine significant differences, while only 4-6 hr were needed in the present study. Those differences suggest that the duration of exposure time should be adjusted according to the specific insect. The various bioassays used so far measured the overall effect of steinernematids and heterorhabditids on the target insect. This effect was expressed by the proportion of dead insects in the total population exposed to the nematodes. The approach presented here is based on the use of a specific component of a pathogenic complex involving the nematode, i.e., invasion rate, rather than the overall effect. ACKNOWLEDGMENT The author thanks Ms. Liora Salame and Ms. Drorit Lapid, of the Department of Nematology, ARO, The Volcani Center, Bet Dagan, Israel, for technical assistance, and Drs. M. Klein and A. Navon of the Department of Entomology, ARO, for supplying the insect larvae. REFERENCES Akhurst, R. J. 1983. Neoaplectana species: Specificity of association with bacteria of the genus Xenorhabdus. Exp. Parasitol. 55, 258263. Beavers, J. B. 1984. Susceptibility of Diaprepes abbeviatus to the parasitic nematode Steinernema glaseri. ICRS Med. Sci. 12, 480. Bedding, R. A., Molyneux, A. S., and Akhurst, R. J. 1983. HeteForhabditis spp., Neoaplectana spp. and Steinernemu kraussei: Interspecific and intraspecific differences in infectivity for insects. Exp.
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